Prof.ssa Laura Calabresi

Pubblicazioni su PubMed

• In Silico Description of the Direct Inhibition Mechanism of Endothelial Lipase by ANGPTL3.
  Int J Mol Sci

  2024 Mar;25(6):. doi: 3555.
  
  Montavoci Linda, Ben Mariem Omar, Saporiti Simona, Laurenzi Tommaso, Palazzolo Luca, Ossoli Alice Federica, Guerrini Uliano, Calabresi Laura, Eberini Ivano
  
  Abstract
  
  Angiopoietin-like protein 3 (ANGPTL3) is a plasmatic protein that plays a crucial role in lipoprotein metabolism by inhibiting the lipoprotein lipase (LPL) and the endothelial lipase (EL) responsible for the hydrolysis of phospholipids on high-density lipoprotein (HDL). Interest in developing new pharmacological therapies aimed at inhibiting ANGPTL3 has been growing due to the hypolipidemic and antiatherogenic profile observed in its absence. The goal of this study was the in silico characterization of the interaction between ANGPTL3 and EL. Because of the lack of any structural information on both the trimeric coiled-coil N-terminal domain of ANGPTL3 and the EL homodimer as well as data regarding their interactions, the first step was to obtain the three-dimensional model of these two proteins. The models were then refined via molecular dynamics (MD) simulations and used to investigate the interaction mechanism. The analysis of interactions in different docking poses and their refinement via MD allowed the identification of three specific glutamates of ANGPTL3 that recognize a positively charged patch on the surface of EL. These ANGPTL3 key residues, i.e., Glu154, Glu157, and Glu160, could form a putative molecular recognition site for EL. This study paves the way for future investigations aimed at confirming the recognition site and at designing novel inhibitors of ANGPTL3.
  
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• Exploiting routine laboratory test to identify primary severe hypertriglyceridaemic patients in a large Italian hospital.
  Eur J Prev Cardiol

  2024 Feb;():. doi: zwae056.
  
  Pavanello Chiara, Pazzucconi Franco, Parolini Marina, Turri Marta, Mombelli Giuliana Germana, Castiglione Sofia, Alberti Antonia, De Maria Renata, Calabresi Laura
  
  
  
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• Ropeginterferon phase 2 randomized study in low-risk polycythemia vera: 5-year drug survival and efficacy outcomes.
  Ann Hematol

  2024 Feb;103(2):437-442. doi: 10.1007/s00277-023-05577-9.
  
  Barbui Tiziano, Carobbio Alessandra, De Stefano Valerio, Alvarez-Larran Alberto, Ghirardi Arianna, Carioli Greta, Fenili Francesca, Rossi Elena, Ciceri Fabio, Bonifacio Massimiliano, Iurlo Alessandra, Palandri Francesca, Benevolo Giulia, Pane Fabrizio, Ricco Alessandra, Carli Giuseppe, Caramella Marianna, Rapezzi Davide, Musolino Caterina, Siragusa Sergio, Rumi Elisa, Patriarca Andrea, Cascavilla Nicola, Mora Barbara, Cacciola Emma, Calabresi Laura, Loscocco Giuseppe Gaetano, Guglielmelli Paola, Gesullo Francesca, Betti Silvia, Ramundo Francesco, Lunghi Francesca, Scaffidi Luigi, Bucelli Cristina, Cattaneo Daniele, Vianelli Nicola, Bellini Marta, Finazzi Maria Chiara, Tognoni Gianni, Rambaldi Alessandro, Vannucchi Alessandro Maria
  
  Abstract
  
  In patients with low-risk polycythemia vera, exposure to low-dose Ropeginterferon alfa-2b (Ropeg) 100 µg every 2 weeks for 2 years was more effective than the standard treatment of therapeutic phlebotomy in maintaining target hematocrit (HCT) (45%) with a reduction in the need for phlebotomy without disease progression. In the present paper, we analyzed drug survival, defined as a surrogate measure of the efficacy, safety, adherence, and tolerability of Ropeg in patients followed up to 5 years. During the first 2 years, Ropeg and phlebotomy-only (Phl-O) were discontinued in 33% and 70% of patients, respectively, for lack of response (12 in the Ropeg arm vs. 34 in the Phl-O arm) or adverse events (6 vs. 0) and withdrawal of consent in (3 vs. 10). Thirty-six Ropeg responders continued the drug for up to 3 years, and the probability of drug survival after a median of 3.15 years was 59%. Notably, the primary composite endpoint was maintained in 97%, 94%, and 94% of patients still on drug at 3, 4, and 5 years, respectively, and 60% of cases were phlebotomy-free. Twenty-three of 63 Phl-O patients (37%) failed the primary endpoint and were crossed over to Ropeg; among the risk factors for this failure, the need for more than three bloodletting procedures in the first 6 months emerged as the most important determinant. In conclusion, to improve the effectiveness of Ropeg, we suggest increasing the dose and using it earlier driven by high phlebotomy need in the first 6 months post-diagnosis.
  
  © 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
  
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• Flipped C-Terminal Ends of APOA1 Promote ABCA1-Dependent Cholesterol Efflux by Small HDLs.
  Circulation

  2024 Mar;149(10):774-787. doi: 10.1161/CIRCULATIONAHA.123.065959.
  
  He Yi, Pavanello Chiara, Hutchins Patrick M, Tang Chongren, Pourmousa Mohsen, Vaisar Tomas, Song Hyun D, Pastor Richard W, Remaley Alan T, Goldberg Ira J, Costacou Tina, Sean Davidson W, Bornfeldt Karin E, Calabresi Laura, Segrest Jere P, Heinecke Jay W
  
  Abstract
  
  BACKGROUND:
  Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear.
  
  METHODS:
  We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs.
  
  RESULTS:
  We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC.
  
  CONCLUSIONS:
  We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.
  
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• Flipped C-Terminal Ends of APOA1 Promote ABCA1-dependent Cholesterol Efflux by Small HDLs.
  medRxiv

  2023 Nov;():. doi: 2023.11.03.23297986.
  
  He Yi, Pavanello Chiara, Hutchins Patrick M, Tang Chongren, Pourmousa Mohsen, Vaisar Tomas, Song Hyun D, Pastor Richard W, Remaley Alan T, Goldberg Ira J, Costacou Tina, Davidson W Sean, Bornfeldt Karin E, Calabresi Laura, Segrest Jere P, Heinecke Jay W
  
  Abstract
  
  BACKGROUND:
  Cholesterol efflux capacity (CEC) predicts cardiovascular disease (CVD) independently of HDL cholesterol (HDL-C) levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 pathway, but the underlying mechanisms are unclear.
  
  METHODS:
  We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 in the different particles, and the CECs of plasma and isolated HDLs.
  
  RESULTS:
  We quantified macrophage and ABCA1 CEC of four distinct sizes of reconstituted HDL (r-HDL). CEC increased as particle size decreased. MS/MS analysis of chemically crosslinked peptides and molecular dynamics simulations of APOA1 (HDL's major protein) indicated that the mobility of that protein's C-terminus was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs-like r-HDLs-are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3-5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC.
  
  CONCLUSIONS:
  We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the two antiparallel molecules of APOA1 are flipped off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased CVD risk. Thus, extra-small and small HDLs may be key mediators and indicators of HDL's cardioprotective effects.
  
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• Dapagliflozin-Induced Myocardial Flow Reserve Improvement is not Associated with HDL Ability to Stimulate Endothelial Nitric Oxide Production.
  Diabetes Ther

  2024 Jan;15(1):257-268. doi: 10.1007/s13300-023-01491-5.
  
  Capece Umberto, Pavanello Chiara, Cinti Francesca, Leccisotti Lucia, Mezza Teresa, Ciccarelli Gea, Moffa Simona, Di Giuseppe Gianfranco, Soldovieri Laura, Brunetti Michela, Giordano Alessandro, Giaccari Andrea, Calabresi Laura, Ossoli Alice
  
  Abstract
  
  BACKGROUND:
  Sodium-glucose cotransporter-2 (SGLT2) inhibitors have shown controversial results in modulating plasma lipids in clinical trials. Most studies found slight increases in high-density lipoprotein (HDL) cholesterol but few have provided evidence on HDL functionality with disappointing results. However, there is broad agreement that these drugs provide cardiovascular protection through several mechanisms. Our group demonstrated that dapagliflozin improves myocardial flow reserve (MFR) in patients with type 2 diabetes (T2D) with coronary artery disease (CAD). The underlying mechanisms are still unknown, although in vitro studies have suggested the involvement of nitric oxide (NO).
  
  AIM:
  To investigate changes in HDL-mediated modulation of NO production with dapagliflozin and whether there is an association with MFR.
  
  METHODS:
  Sixteen patients with CAD-T2D were enrolled and randomized 1:1 to dapagliflozin or placebo for 4 weeks. Blood samples were collected before and after treatment for each group. The ability of HDL to stimulate NO production in endothelial cells was tested in vitro by incubating human umbilical vein endothelial cells (HUVEC) with apoB-depleted (apoB-D) serum of these patients. The production of NO was assessed by fluorescent assay, and results were expressed as fold versus untreated cells.
  
  RESULTS:
  Change in HDL-mediated NO production remained similar in dapagliflozin and placebo group, even after adjustment for confounders. There were no significant correlations between HDL-mediated NO production and MFR either at baseline or after treatment. No changes were found in HDL cholesterol in either group, while low-density lipoprotein cholesterol (LDL cholesterol) significantly decreased compared to baseline only in treatment group (p?=?0.043).
  
  CONCLUSIONS:
  In patients with T2D-CAD, beneficial effects of dapagliflozin on coronary microcirculation seem to be unrelated to HDL functions. However, HDL capacity to stimulate NO production is not impaired at baseline; thus, the effect of drug treatments would be negligible. To conclude, we can assume that HDL-independent molecular pathways are involved in the improvement of MFR in this population.
  
  TRIAL REGISTRATION:
  EudraCT No. 2016-003614-27; ClinicalTrials.gov Identifier: NCT03313752.
  
  © 2023. The Author(s).
  
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• Generation and validation of a classification model to diagnose familial hypercholesterolaemia in adults.
  Atherosclerosis

  2023 Oct;383():117314. doi: 10.1016/j.atherosclerosis.2023.117314.
  
  Albuquerque João, Medeiros Ana Margarida, Alves Ana Catarina, Jannes Cinthia Elim, Mancina Rosellina M, Pavanello Chiara, Chora Joana Rita, Mombelli Giuliana, Calabresi Laura, Pereira Alexandre da Costa, Krieger José Eduardo, Romeo Stefano, Bourbon Mafalda, Antunes Marília
  
  Abstract
  
  BACKGROUND AND AIMS:
  The early diagnosis of familial hypercholesterolaemia is associated with a significant reduction in cardiovascular disease (CVD) risk. While the recent use of statistical and machine learning algorithms has shown promising results in comparison with traditional clinical criteria, when applied to screening of potential FH cases in large cohorts, most studies in this field are developed using a single cohort of patients, which may hamper the application of such algorithms to other populations. In the current study, a logistic regression (LR) based algorithm was developed combining observations from three different national FH cohorts, from Portugal, Brazil and Sweden. Independent samples from these cohorts were then used to test the model, as well as an external dataset from Italy.
  
  METHODS:
  The area under the receiver operating characteristics (AUROC) and precision-recall (AUPRC) curves was used to assess the discriminatory ability among the different samples. Comparisons between the LR model and Dutch Lipid Clinic Network (DLCN) clinical criteria were performed by means of McNemar tests, and by the calculation of several operating characteristics.
  
  RESULTS:
  AUROC and AUPRC values were generally higher for all testing sets when compared to the training set. Compared with DLCN criteria, a significantly higher number of correctly classified observations were identified for the Brazilian (p 
  CONCLUSIONS:
  Compared to DLCN criteria, the LR model revealed improved ability to correctly classify observations, and was able to retain a similar number of FH cases, with less false positive retention. Generalization of the LR model was very good across all testing samples, suggesting it can be an effective screening tool if applied to different populations.
  
  Copyright © 2023 Elsevier B.V. All rights reserved.
  
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• Apolipoprotein E isoforms differentially affect LCAT-dependent cholesterol esterification.
  Atherosclerosis

  2023 Oct;382():117266. doi: 10.1016/j.atherosclerosis.2023.117266.
  
  Vitali Cecilia, Pavanello Chiara, Turri Marta, Lund-Katz Sissel, Phillips Michael C, Catapano Alberico Luigi, Baragetti Andrea, Norata Giuseppe Danilo, Veglia Fabrizio, Calabresi Laura
  
  Abstract
  
  BACKGROUND AND AIMS:
  LCAT esterifies cholesterol in both HDL (?-activity) and apoB-containing lipoproteins (?-activity). The main activator of LCAT ?-activity is apoE, which in humans exists in 3 main different isoforms (E2, E3 and E4). Here, to gather insights into the potential role of LCAT in apoB-containing lipoprotein metabolism, we investigated the ability of apoE isoforms to promote LCAT-mediated cholesterol esterification.
  
  METHODS:
  We evaluated the plasma cholesterol esterification rate (CER) in 311 individuals who express functional LCAT and either apoE2, apoE3, or apoE4 and in 28 individuals who also carried LCAT mutations causing selective loss of LCAT ?-activity (Fish-Eye Disease (FED)-causing mutations). The association of carrier status with CER was determined using an adjusted linear regression model. The kinetic of LCAT activity towards reconstituted HDLs (rHDLs) containing each apoE isoform was determined using the Michaelis-Menten model.
  
  RESULTS:
  Plasma CER was ?20% higher in apoE2 carriers compared to apoE3 carriers, and ?30% higher in apoE2 carriers compared to apoE4 carriers. After adjusting for age, sex, total cholesterol, HDL-C, apoA-I, apoB, chronic kidney disease diagnosis, zygosity, and LCAT concentration, CER remained significantly different among carriers of the three apoE isoforms. The same trend was observed in carriers of FED-causing mutations. rHDLs containing apoE2 were associated with a lower affinity but higher maximal esterification rate, compared to particles containing apoE3 or apoE4.
  
  CONCLUSION:
  The present results suggest that the apoE2 isoform is associated with a higher LCAT-mediated cholesterol esterification. This observation may contribute to the characterization of the peculiar functional properties of apoE2.
  
  Copyright © 2023 Elsevier B.V. All rights reserved.
  
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• Abnormal Lipoproteins Trigger Oxidative Stress-Mediated Apoptosis of Renal Cells in LCAT Deficiency.
  Antioxidants (Basel)

  2023 Jul;12(8):. doi: 1498.
  
  Gomaraschi Monica, Turri Marta, Strazzella Arianna, Lhomme Marie, Pavanello Chiara, Le Goff Wilfried, Kontush Anatol, Calabresi Laura, Ossoli Alice
  
  Abstract
  
  Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease caused by the loss of function mutations in the gene. LCAT deficiency is characterized by an abnormal lipoprotein profile with severe reduction in plasma levels of high-density lipoprotein (HDL) cholesterol and the accumulation of lipoprotein X (LpX). Renal failure is the major cause of morbidity and mortality in FLD patients; the pathogenesis of renal disease is only partly understood, but abnormalities in the lipoprotein profile could play a role in disease onset and progression. Serum and lipoprotein fractions from LCAT deficient carriers and controls were tested for renal toxicity on podocytes and tubular cells, and the underlying mechanisms were investigated at the cellular level. Both LpX and HDL from LCAT-deficient carriers triggered oxidative stress in renal cells, which culminated in cell apoptosis. These effects are partly explained by lipoprotein enrichment in unesterified cholesterol and ceramides, especially in the HDL fraction. Thus, alterations in lipoprotein composition could explain some of the nephrotoxic effects of LCAT deficient lipoproteins on podocytes and tubular cells.
  
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• Evaluation of high-density lipoprotein-bound long non-coding RNAs in subjects with familial hypercholesterolaemia.
  Eur J Clin Invest

  2024 Jan;54(1):e14083. doi: 10.1111/eci.14083.
  
  Scicali Roberto, Bosco Giosiana, Scamporrino Alessandra, Di Mauro Stefania, Filippello Agnese, Di Giacomo Barbagallo Francesco, Spampinato Salvatore, Pavanello Chiara, Ossoli Alice, Di Pino Antonino, Calabresi Laura, Purrello Francesco, Piro Salvatore
  
  Abstract
  
  BACKGROUND:
  Long non-coding RNAs (lncRNAs) could be attractive circulating biomarkers for cardiovascular risk stratification in subjects at high atherosclerotic cardiovascular disease risk such as familial hypercholesterolaemia (FH). Our aim was to investigate the presence of lncRNAs carried by high-density lipoprotein (HDL) in FH subjects and to evaluate the associations of HDL-lncRNAs with lipoproteins and mechanical vascular impairment assessed by pulse wave velocity (PWV).
  
  METHODS:
  This was a retrospective observational study involving 94 FH subjects on statin treatment. Biochemical assays, HDL purification, lncRNA and PWV analyses were performed in all subjects.
  
  RESULTS:
  LncRNA HIF1A-AS2, LASER and LEXIS were transported by HDL; moreover, HDL-lncRNA LEXIS was associated with Lp(a) plasma levels (p?.01). In a secondary analysis, the study population was stratified into two groups based on the Lp(a) median value. The high-Lp(a) group exhibited a significant increase of PWV compared to the low-Lp(a) group (9.23?±?.61 vs. 7.67?±?.56, p?.01). While HDL-lncRNA HIF1A-AS2 and LASER were similar in the two groups, the high-Lp(a) group exhibited a significant downregulation of HDL-lncRNA LEXIS compared to the low-Lp(a) group (fold change -4.4, p?.0001). Finally, Lp(a) and HDL-lncRNA LEXIS were associated with PWV (for Lp(a) p?.01; for HDL-lncRNA LEXIS p?.05).
  
  CONCLUSIONS:
  LncRNA HIF1A-AS2, LASER and LEXIS were transported by HDL; moreover, significant relationships of HDL-lncRNA LEXIS with Lp(a) levels and PWV were found. Our study suggests that HDL-lncRNA LEXIS may be useful to better identify FH subjects with more pronounced vascular damage.
  
  © 2023 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.
  
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• Clonal dynamics and copy number variants by single-cell analysis in leukemic evolution of myeloproliferative neoplasms.
  Am J Hematol

  2023 Oct;98(10):1520-1531. doi: 10.1002/ajh.27013.
  
  Calabresi Laura, Carretta Chiara, Romagnoli Simone, Rotunno Giada, Parenti Sandra, Bertesi Matteo, Bartalucci Niccolò, Rontauroli Sebastiano, Chiereghin Chiara, Castellano Sara, Gentili Giulia, Maccari Chiara, Vanderwert Fiorenza, Mannelli Francesco, Della Porta Matteo, Manfredini Rossella, Vannucchi Alessandro Maria, Guglielmelli Paola
  
  Abstract
  
  Transformation from chronic (CP) to blast phase (BP) in myeloproliferative neoplasm (MPN) remains poorly characterized, and no specific mutation pattern has been highlighted. BP-MPN represents an unmet need, due to its refractoriness to treatment and dismal outcome. Taking advantage of the granularity provided by single-cell sequencing (SCS), we analyzed paired samples of CP and BP in 10 patients to map clonal trajectories and interrogate target copy number variants (CNVs). Already at diagnosis, MPN present as oligoclonal diseases with varying ratio of mutated and wild-type cells, including cases where normal hematopoiesis was entirely surmised by mutated clones. BP originated from increasing clonal complexity, either on top or independent of a driver mutation, through acquisition of novel mutations as well as accumulation of clones harboring multiple mutations, that were detected at CP by SCS but were missed by bulk sequencing. There were progressive copy-number imbalances from CP to BP, that configured distinct clonal profiles and identified recurrences in genes including NF1, TET2, and BCOR, suggesting an additional level of complexity and contribution to leukemic transformation. EZH2 emerged as the gene most frequently affected by single nucleotide and CNVs, that might result in EZH2/PRC2-mediated transcriptional deregulation, as supported by combined scATAC-seq and snRNA-seq analysis of the leukemic clone in a representative case. Overall, findings provided insights into the pathogenesis of MPN-BP, identified CNVs as a hitherto poorly characterized mechanism and point to EZH2 dysregulation as target. Serial assessment of clonal dynamics might potentially allow early detection of impending disease transformation, with therapeutic implications.
  
  © 2023 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.
  
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• Association of Intima-Media Thickness Measured at the Common Carotid Artery With Incident Carotid Plaque: Individual Participant Data Meta-Analysis of 20 Prospective Studies.
  J Am Heart Assoc

  2023 Jun;12(12):e027657. doi: e027657.
  
  Tschiderer Lena, Seekircher Lisa, Izzo Raffaele, Mancusi Costantino, Manzi Maria V, Baldassarre Damiano, Amato Mauro, Tremoli Elena, Veglia Fabrizio, Tuomainen Tomi-Pekka, Kauhanen Jussi, Voutilainen Ari, Iglseder Bernhard, Lind Lars, Rundek Tatjana, Desvarieux Moise, Kato Akihiko, de Groot Eric, A?çi Gülay, Ok Ercan, Agewall Stefan, Beulens Joline W J, Byrne Christopher D, Calder Philip C, Gerstein Hertzel C, Gresele Paolo, Klingenschmid Gerhard, Nagai Michiaki, Olsen Michael H, Parraga Grace, Safarova Maya S, Sattar Naveed, Skilton Michael, Stehouwer Coen D A, Uthoff Heiko, van Agtmael Michiel A, van der Heijden Amber A, Zozuli?ska-Zió?kiewicz Dorota A, Park Hyun-Woong, Lee Moo-Sik, Bae Jang-Ho, Beloqui Oscar, Landecho Manuel F, Plichart Matthieu, Ducimetiere Pierre, Empana Jean Philippe, Bokemark Lena, Bergström Göran, Schmidt Caroline, Castelnuovo Samuela, Calabresi Laura, Norata Giuseppe D, Grigore Liliana, Catapano Alberico, Zhao Dong, Wang Miao, Liu Jing, Ikram M Arfan, Kavousi Maryam, Bots Michiel L, Sweeting Michael J, Lorenz Matthias W, Willeit Peter,
  
  Abstract
  
  Background The association between common carotid artery intima-media thickness (CCA-IMT) and incident carotid plaque has not been characterized fully. We therefore aimed to precisely quantify the relationship between CCA-IMT and carotid plaque development. Methods and Results We undertook an individual participant data meta-analysis of 20 prospective studies from the Proof-ATHERO (Prospective Studies of Atherosclerosis) consortium that recorded baseline CCA-IMT and incident carotid plaque involving 21?494 individuals without a history of cardiovascular disease and without preexisting carotid plaque at baseline. Mean baseline age was 56?years (SD, 9?years), 55% were women, and mean baseline CCA-IMT was 0.71?mm (SD, 0.17?mm). Over a median follow-up of 5.9?years (5th-95th percentile, 1.9-19.0?years), 8278 individuals developed first-ever carotid plaque. We combined study-specific odds ratios (ORs) for incident carotid plaque using random-effects meta-analysis. Baseline CCA-IMT was approximately log-linearly associated with the odds of developing carotid plaque. The age-, sex-, and trial arm-adjusted OR for carotid plaque per SD higher baseline CCA-IMT was 1.40 (95% CI, 1.31-1.50; =63.9%). The corresponding OR that was further adjusted for ethnicity, smoking, diabetes, body mass index, systolic blood pressure, low- and high-density lipoprotein cholesterol, and lipid-lowering and antihypertensive medication was 1.34 (95% CI, 1.24-1.45; =59.4%; 14 studies; 16?297 participants; 6381 incident plaques). We observed no significant effect modification across clinically relevant subgroups. Sensitivity analysis restricted to studies defining plaque as focal thickening yielded a comparable OR (1.38 [95% CI, 1.29-1.47]; =57.1%; 14 studies; 17?352 participants; 6991 incident plaques). Conclusions Our large-scale individual participant data meta-analysis demonstrated that CCA-IMT is associated with the long-term risk of developing first-ever carotid plaque, independent of traditional cardiovascular risk factors.
  
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• Plasma and cerebrospinal fluid cholesterol esterification is hampered in Alzheimer's disease.
  Alzheimers Res Ther

  2023 May;15(1):95. doi: 95.
  
  Turri Marta, Conti Elisa, Pavanello Chiara, Gastoldi Francesco, Palumbo Marcella, Bernini Franco, Aprea Vittoria, Re Francesca, Barbiroli Alberto, Emide Davide, Galimberti Daniela, Tremolizzo Lucio, Zimetti Francesca, Calabresi Laura,
  
  Abstract
  
  OBJECTIVE:
  The purpose of this study was to evaluate cholesterol esterification and HDL subclasses in plasma and cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients.
  
  METHODS:
  The study enrolled 70 AD patients and 74 cognitively normal controls comparable for age and sex. Lipoprotein profile, cholesterol esterification, and cholesterol efflux capacity (CEC) were evaluated in plasma and CSF.
  
  RESULTS:
  AD patients have normal plasma lipids but significantly reduced unesterified cholesterol and unesterified/total cholesterol ratio. Lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate (CER), two measures of the efficiency of the esterification process, were reduced by 29% and 16%, respectively, in the plasma of AD patients. Plasma HDL subclass distribution in AD patients was comparable to that of controls but the content of small discoidal pre?-HDL particles was significantly reduced. In agreement with the reduced pre?-HDL particles, cholesterol efflux capacity mediated by the transporters ABCA1 and ABCG1 was reduced in AD patients' plasma. The CSF unesterified to total cholesterol ratio was increased in AD patients, and CSF CER and CEC from astrocytes were significantly reduced in AD patients. In the AD group, a significant positive correlation was observed between plasma unesterified cholesterol and unesterified/total cholesterol ratio with A? CSF content.
  
  CONCLUSION:
  Taken together our data indicate that cholesterol esterification is hampered in plasma and CSF of AD patients and that plasma cholesterol esterification biomarkers (unesterified cholesterol and unesterified/total cholesterol ratio) are significantly associated to disease biomarkers (i.e., CSF A?).
  
  © 2023. The Author(s).
  
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• HDL particles and infection-related death: when size matters.
  Cardiovasc Res

  2023 May;119(4):883-885. doi: 10.1093/cvr/cvad043.
  
  Calabresi Laura, Norata Giuseppe Danilo
  
  
  
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• Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model.
  Leukemia

  2023 May;37(5):1068-1079. doi: 10.1038/s41375-023-01867-3.
  
  Bianchi Elisa, Rontauroli Sebastiano, Tavernari Lara, Mirabile Margherita, Pedrazzi Francesca, Genovese Elena, Sartini Stefano, Dall'Ora Massimiliano, Grisendi Giulia, Fabbiani Luca, Maccaferri Monica, Carretta Chiara, Parenti Sandra, Fantini Sebastian, Bartalucci Niccolò, Calabresi Laura, Balliu Manjola, Guglielmelli Paola, Potenza Leonardo, Tagliafico Enrico, Losi Lorena, Dominici Massimo, Luppi Mario, Vannucchi Alessandro Maria, Manfredini Rossella
  
  Abstract
  
  Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.
  
  © 2023. The Author(s).
  
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• Two novel variants in the lecithin:cholesterol acyltransferase gene resulted in classic LCAT deficiency.
  Atheroscler Plus

  2022 Aug;49():28-31. doi: 10.1016/j.athplu.2022.05.005.
  
  Fistrek Prlic Margareta, Coric Marijana, Calabresi Laura, Pavanello Chiara, Mosca Lorena, Cavallari Ugo, Vukovic Brinar Ivana, Karanovic Sandra, Laganovic Mario, Jelakovic Bojan
  
  Abstract
  
  BACKGROUND AND AIMS:
  We report the first two cases of familial lecithin:cholesterol acyltransferase (LCAT) deficiency in Croatia with classical clinical and biochemical features.
  
  PATIENTS AND METHODS:
  A 30-year-old man with nephrotic syndrome, corneal opacities, hepatosplenomegaly, anemia, low high-density lipoprotein (HDL)-cholesterol levels and arterial hypertension (blood pressure >200/100 mmHg) was admitted to our department. At admission, he had an elevated creatinine serum level (233 ?mol/L), proteinuria of 12 g in 24-h urine (g/24 h), 3-7 erythrocytes in urine sediment and notable anemia (hemoglobin level 90 g/l). His HDL-cholesterol was significantly low (0.42 mmol/L). Besides chronic kidney disease (CKD), other secondary causes of hypertension were ruled out. The patient was previously diagnosed with membranous nephropathy and treated unsuccessfully with immunosuppressive agents (steroids, cyclosporine, cyclophosphamide). Re-evaluation of histopathological findings of kidney biopsy revealed massive deposition of lipid material in the glomerular basal membrane and in the mesangial region. His 4-year younger brother was also evaluated due to corneal opacities and new-onset arterial hypertension. Nephrotic range proteinuria with preserved global renal function was determined. He also had very low HDL-cholesterol levels.
  
  RESULTS:
  Kidney biopsies from both patients were consistent with LCAT deficiency. The disease was confirmed by measurement of LCAT enzyme activity, plasma cholesterol esterification rate, and genetic testing. Two novel missense variants in the gene (c.496G > A and c.1138T  >  C) were found.
  
  CONCLUSIONS:
  To our knowledge, the presented cases are the first reported cases of genetic LCAT deficiency in Croatia. Given the clinical presentation, the complete lack of LCAT activity and cholesterol esterification rate, diagnosis of familial LCAT deficiency was made.
  
  © 2022 The Authors.
  
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• Genetically determined deficiency of ANGPTL3 does not alter HDL ability to preserve endothelial homeostasis.
  Biochim Biophys Acta Mol Cell Biol Lipids

  2023 Mar;1868(3):159263. doi: 10.1016/j.bbalip.2022.159263.
  
  Ossoli Alice, Minicocci Ilenia, Turri Marta, Di Costanzo Alessia, D'Erasmo Laura, Bini Simone, Montavoci Linda, Veglia Fabrizio, Calabresi Laura, Arca Marcello
  
  Abstract
  
  Individuals with loss-of-function mutations in the ANGPTL3 gene express a rare lipid phenotype called Familial Combined Hypolipidemia (FHBL2). FHBL2 individuals show reduced plasma concentrations of total cholesterol and triglycerides as well as of lipoprotein particles, including HDL. This feature is particularly remarkable in homozygotes in whom ANGPTL3 in blood is completely absent. ANGPTL3 acts as a circulating inhibitor of LPL and EL and it is thought that EL hyperactivity is the cause of plasma HDL reduction in FHBL2. Nevertheless, the consequences of ANGTPL3 deficiency on HDL functionality have been poorly explored. In this report, HDL isolated from homozygous and heterozygous FHBL2 individuals were evaluated for their ability to preserve endothelial homeostasis as compared to control HDL. It was found that only the complete absence of ANGPTL3 alters HDL subclass distribution, as homozygous, but not heterozygous, carriers have reduced content of large and increased content of small HDL with no alterations in HDL2 and HDL3 size. The plasma content of pre?-HDL was reduced in carriers and showed a positive correlation with plasma ANGPTL3 levels. Changes in composition did not however alter the functionality of FHBL2 HDL, as particles isolated from carriers retained their capacity to promote NO production and to inhibit VCAM-1 expression in endothelial cells. Furthermore, no significant changes in circulating levels of soluble ICAM-1 and E-selectin were detected in carriers. These results indicate that changes in HDL composition associated with the partial or complete absence of ANGPTL3 did not alter some of the potentially anti-atherogenic functions of these lipoproteins.
  
  Copyright © 2022 Elsevier B.V. All rights reserved.
  
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• Plasma HDL pattern, cholesterol efflux and cholesterol loading capacity of serum in carriers of a novel missense variant (Gly176Trp) of endothelial lipase.
  J Clin Lipidol

  2022 ;16(5):694-703. doi: 10.1016/j.jacl.2022.08.002.
  
  Pisciotta Livia, Ossoli Alice, Ronca Annalisa, Garuti Anna, Fresa Raffaele, Favari Elda, Calabresi Laura, Calandra Sebastiano, Bertolini Stefano
  
  Abstract
  
  BACKGROUND:
  Loss of function variants of LIPG gene encoding endothelial lipase (EL) are associated with primary hyperalphalipoproteinemia (HALP), a lipid disorder characterized by elevated plasma levels of high density lipoprotein cholesterol (HDL-C).
  
  OBJECTIVE:
  Aim of the study was the phenotypic and genotypic characterization of a family with primary HALP.
  
  METHODS:
  HDL subclasses distribution was determined by polyacrylamide gradient gel electrophoresis. Serum content of pre?-HDL was assessed by (2D)-electrophoresis. Cholesterol efflux capacity (CEC) of serum mediated by ABCA1, ABCG1 or SR-BI was assessed using cells expressing these proteins. Cholesterol loading capacity (CLC) of serum was assayed using cultured human macrophages. Next generation sequencing was used for DNA analysis. Plasma EL mass was determined by ELISA.
  
  RESULTS:
  Three family members had elevated plasma HDL-C, apoA-I and total phospholipids, as well as a reduced content of pre?-HDL. These subjects were heterozygous carriers of a novel variant of LIPG gene [c.526 G>T, p.(Gly176Trp)] found to be deleterious in silico. Plasma EL mass in carriers was lower than in controls. CEC of sera mediated by ABCA1 and ABCG1 transporters was substantially reduced in the carriers. This effect was maintained after correction for serum HDL concentration. The sera of carriers were found to have a higher CLC in cultured human macrophages than control sera.
  
  CONCLUSION:
  The novel p.(Gly176Trp) variant of endothelial lipase is associated with changes in HDL composition and subclass distribution as well as with functional changes affecting cholesterol efflux capacity of serum which suggest a defect in the early steps of revere cholesterol transport.
  
  Copyright © 2022. Published by Elsevier Inc.
  
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• Case report: Unusual and extremely severe lipoprotein X-mediated hypercholesterolemia in extrahepatic pediatric cholestasis.
  Front Pediatr

  2022 ;10():969081. doi: 969081.
  
  Colantuono Rossella, Pavanello Chiara, Pietrobattista Andrea, Turri Marta, Francalanci Paola, Spada Marco, Vajro Pietro, Calabresi Laura, Mandato Claudia
  
  Abstract
  
  BACKGROUND:
  Lipoprotein X (LpX) - mediated extremely severe hyperlipidemia is a possible feature detectable in children with syndromic paucity of intralobular bile ducts (Alagille syndrome) but rarely in other types of intra- and/or extrahepatic infantile cholestasis.
  
  CASE PRESENTATION:
  Here we report on a previously well 18-month child admitted for cholestatic jaundice and moderate hepatomegaly. Laboratory tests at entry showed conjugated hyperbilirubinemia, elevated values of serum aminotransferases, gamma-glutamyl transpeptidase (GGT) and bile acids (100 folds upper normal values). Extremely severe and ever-increasing hypercholesterolemia (total cholesterol up to 1,730 mg/dl) prompted an extensive search for causes of high GGT and/or hyperlipidemic cholestasis, including an extensive genetic liver panel (negative) and a liver biopsy showing a picture of obstructive cholangitis, biliary fibrosis, and bile duct proliferation with normal MDR3 protein expression. Results of a lipid study showed elevated values of unesterified cholesterol, phospholipids, and borderline/low apolipoprotein B, and low high-density lipoprotein-cholesterol. Chromatographic analysis of plasma lipoproteins fractions isolated by analytical ultracentrifugation revealed the presence of the anomalous lipoprotein (LpX). Magnetic resonance cholangiopancreatography and percutaneous transhepatic cholangiography showed stenosis of the confluence of the bile ducts with dilation of the intrahepatic biliary tract and failure to visualize the extrahepatic biliary tract. Surgery revealed focal fibroinflammatory stenosis of the left and right bile ducts confluence, treated with resection and bilioenteric anastomosis, followed by the rapid disappearance of LpX, paralleling the normalization of serum lipids, bilirubin, and bile acids, with a progressive reduction of hepatobiliary enzymes.
  
  CONCLUSION:
  We have described a unique case of focal non-neoplastic extrahepatic biliary stenosis of uncertain etiology, presenting with unusual extremely high levels of LpX-mediated hypercholesterolemia, a condition which is frequently mistaken for LDL on routine clinical tests.
  
  Copyright © 2022 Colantuono, Pavanello, Pietrobattista, Turri, Francalanci, Spada, Vajro, Calabresi and Mandato.
  
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• Immunoblotting-assisted assessment of JAK/STAT and PI3K/Akt/mTOR signaling in myeloproliferative neoplasms CD34+ stem cells.
  Methods Cell Biol

  2022 ;171():81-109. doi: 10.1016/bs.mcb.2022.04.005.
  
  Calabresi Laura, Balliu Manjola, Bartalucci Niccolò
  
  Abstract
  
  Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.
  
  Copyright © 2022 Elsevier Inc. All rights reserved.
  
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• Effects of PCSK9 inhibitors on HDL cholesterol efflux and serum cholesterol loading capacity in familial hypercholesterolemia subjects: a multi-lipid-center real-world evaluation.
  Front Mol Biosci

  2022 ;9():925587. doi: 925587.
  
  Palumbo Marcella, Giammanco Antonina, Purrello Francesco, Pavanello Chiara, Mombelli Giuliana, Di Pino Antonino, Piro Salvatore, Cefalù Angelo Baldassare, Calabresi Laura, Averna Maurizio, Bernini Franco, Zimetti Francesca, Adorni Maria Pia, Scicali Roberto
  
  Abstract
  
  Proprotein convertase subtilisin/kexin type 9 (PCSK9), beyond regulating LDL cholesterol (LDL-c) plasma levels, exerts several pleiotropic effects by modulating lipid metabolism in extrahepatic cells such as macrophages. Macrophage cholesterol homeostasis depends on serum lipoprotein functions, including the HDL capacity to promote cell cholesterol efflux (CEC) and the serum capacity to promote cell cholesterol loading (CLC). The aim of this observational study was to investigate the effect of PCSK9 inhibitors (PCSK9-i) treatment on HDL-CEC and serum CLC in patients with familial hypercholesterolemia (FH). 31 genetically confirmed FH patients were recruited. Blood was collected and serum isolated at baseline and after 6 months of PCSK9-i treatment. HDL-CEC was evaluated through the main pathways with a radioisotopic cell-based assay. Serum CLC was assessed fluorimetrically in human THP-1 monocyte-derived macrophages. After treatment with PCSK9-i, total cholesterol and LDL-c significantly decreased (-41.6%,
  Copyright © 2022 Palumbo, Giammanco, Purrello, Pavanello, Mombelli, Di Pino, Piro, Cefalù, Calabresi, Averna, Bernini, Zimetti, Adorni and Scicali.
  
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• Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans.
  J Lipid Res

  2022 Jul;63(7):100232. doi: 100232.
  
  Pavanello Chiara, Ossoli Alice, Strazzella Arianna, Risè Patrizia, Veglia Fabrizio, Lhomme Marie, Parini Paolo, Calabresi Laura
  
  Abstract
  
  Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0-37.8] mg/dl) and slightly reduced in heterozygotes (218 [153-234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.
  
  Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.
  
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• Emerging role of HDL in brain cholesterol metabolism and neurodegenerative disorders.
  Biochim Biophys Acta Mol Cell Biol Lipids

  2022 May;1867(5):159123. doi: 10.1016/j.bbalip.2022.159123.
  
  Turri Marta, Marchi Cinzia, Adorni Maria Pia, Calabresi Laura, Zimetti Francesca
  
  Abstract
  
  High-density lipoproteins (HDL) play a key role in cholesterol homeostasis maintenance in the central nervous system (CNS), by carrying newly synthesized cholesterol from astrocytes to neurons, to support their lipid-related physiological functions. As occurs for plasma HDL, brain lipoproteins are assembled through the activity of membrane cholesterol transporters, undergo remodeling mediated by specific enzymes and transport proteins, and finally deliver cholesterol to neurons by a receptor-mediated internalization process. A growing number of evidences indicates a strong association between alterations of CNS cholesterol homeostasis and neurodegenerative disorders, in particular Alzheimer's disease (AD), and a possible role in this relationship may be played by defects in brain HDL metabolism. In the present review, we summarize and critically examine the current state of knowledge on major modifications of HDL and HDL-mediated brain cholesterol transport in AD, by taking into consideration the individual steps of this process. We also describe potential and encouraging HDL-based therapies that could represent new therapeutic strategies for AD treatment. Finally, we revise the main plasma and brain HDL modifications in other neurodegenerative disorders including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal dementia (FTD).
  
  Copyright © 2022 Elsevier B.V. All rights reserved.
  
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• Reduced Levels of ABCA1 Transporter Are Responsible for the Cholesterol Efflux Impairment in ?-Amyloid-Induced Reactive Astrocytes: Potential Rescue from Biomimetic HDLs.
  Int J Mol Sci

  2021 Dec;23(1):. doi: 102.
  
  Sierri Giulia, Dal Magro Roberta, Vergani Barbara, Leone Biagio Eugenio, Formicola Beatrice, Taiarol Lorenzo, Fagioli Stefano, Kravicz Marcelo, Tremolizzo Lucio, Calabresi Laura, Re Francesca
  
  Abstract
  
  The cerebral synthesis of cholesterol is mainly handled by astrocytes, which are also responsible for apoproteins' synthesis and lipoproteins' assembly required for the cholesterol transport in the brain parenchyma. In Alzheimer disease (AD), these processes are impaired, likely because of the astrogliosis, a process characterized by morphological and functional changes in astrocytes. Several ATP-binding cassette transporters expressed by brain cells are involved in the formation of nascent discoidal lipoproteins, but the effect of beta-amyloid (A?) assemblies on this process is not fully understood. In this study, we investigated how of A?-induced astrogliosis affects the metabolism of cholesterol in vitro. We detected an impairment in the cholesterol efflux of reactive astrocytes attributable to reduced levels of ABCA1 transporters that could explain the decreased lipoproteins' levels detected in AD patients. To approach this issue, we designed biomimetic HDLs and evaluated their performance as cholesterol acceptors. The results demonstrated the ability of apoA-I nanodiscs to cross the blood-brain barrier in vitro and to promote the cholesterol efflux from astrocytes, making them suitable as a potential supportive treatment for AD to compensate the depletion of cerebral HDLs.
  
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• Abdominal obesity negatively influences key metrics of reverse cholesterol transport.
  Biochim Biophys Acta Mol Cell Biol Lipids

  2022 Feb;1867(2):159087. doi: 10.1016/j.bbalip.2021.159087.
  
  Härdfeldt Jennifer, Cariello Marica, Simonelli Sara, Ossoli Alice, Scialpi Natasha, Piglionica Marilidia, Pasculli Emanuela, Noia Alessia, Berardi Elsa, Suppressa Patrizia, Piazzolla Giuseppina, Sabbà Carlo, Calabresi Laura, Moschetta Antonio
  
  Abstract
  
  Cardiometabolic risk factors increase the risk of atherosclerotic cardiovascular disease (ASCVD), but whether these metabolic anomalies affect the anti-atherogenic function of reverse cholesterol transport (RCT) is not yet clearly known. The present study aimed to delineate if the function and maturation of high density lipoprotein (HDL) particles cross-sectionally associate with surrogate markers of ASCVD in a population comprising of different degree of cardiometabolic risk. We enrolled 131 subjects and characterized cardiometabolic risk based on the IDF criteria's for metabolic syndrome (MS). In this population, cholesterol efflux capacity (CEC), Lecithin-cholesterol acyltransferase (LCAT) and ApoA-1 glycation was associated with waist circumference, abdominal visceral fat (VFA) and abdominal subcutaneous fat. In multivariate analyses, VFA was identified as a critical contributor for low CEC and LCAT. When stratified into groups based on the presence of cardiometabolic risk factors, we found a prominent reduction in CEC and LCAT as a function of the progressive increase of cardiometabolic risk from 0-2, 0-3 to 0-4/5, whereas an increase in Pre-?-HDL and ApoA-1 glycation was observed between the lowest and highest risk groups. These findings confirm the connection between MS and its predisposing conditions to an impairment of atheroprotective efflux-promoting function of HDLs. Furthermore, we have identified the bona fide pathogenically contribution of abdominal obesity to profound alterations of key metrics of RCT.
  
  Copyright © 2021 Elsevier B.V. All rights reserved.
  
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• The HDL mimetic CER-001 remodels plasma lipoproteins and reduces kidney lipid deposits in inherited lecithin:cholesterol acyltransferase deficiency.
  J Intern Med

  2022 Mar;291(3):364-370. doi: 10.1111/joim.13404.
  
  Pavanello Chiara, Turri Marta, Strazzella Arianna, Tulissi Patrizia, Pizzolitto Stefano, De Maglio Giovanna, Nappi Riccardo, Calabresi Laura, Boscutti Giuliano
  
  Abstract
  
  BACKGROUND:
  Kidney failure is the major cause of morbidity and mortality in familial lecithin:cholesterol acyltransferase deficiency (FLD), a rare inherited lipid disorder with no cure. Lipoprotein X (LpX), an abnormal lipoprotein, is primarily accountable for nephrotoxicity.
  
  METHODS:
  CER-001 was tested in an FLD patient with dramatic kidney disease for 12 weeks.
  
  RESULTS:
  Infusions of CER-001 normalized the lipoprotein profile, with a disappearance of the abnormal LpX in favour of normal-sized LDL. The worsening of kidney function was slowed by the treatment, and kidney biopsy showed a slight reduction of lipid deposits and a stabilization of the disease. In vitro experiments demonstrate that CER-001 progressively reverts lipid accumulation in podocytes by a dual effect: remodelling plasma lipoproteins and removing LpX-induced lipid deposit.
  
  CONCLUSION:
  This study demonstrates that CER-001 may represent a therapeutic option in FLD patients. It also has the potential to be beneficial in other renal diseases characterized by kidney lipid deposits.
  
  © 2021 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.
  
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• The MPL mutation.
  Int Rev Cell Mol Biol

  2021 ;365():163-178. doi: 10.1016/bs.ircmb.2021.09.003.
  
  Guglielmelli Paola, Calabresi Laura
  
  Abstract
  
  Myeloproliferative neoplasms (MPN) patients share driver mutations in JAK2, MPL or CALR genes leading to the activation of the thrombopoietin receptor (TPOR) and downstream signaling pathways. JAK2 mutation drives all the three major entities of MPN (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis) through the constitutive activation of TPOR, erythropoietin (EPOR) and colony stimulating factor 3 receptor (CSF3R) signaling. MPL is a proto-oncogene encoding for TPOR, the hematopoietic growth factor receptor of myeloid stem cells. MPL mutants induce the stable dimerization of TPOR that in turn activate JAK2 and the thrombopoietin pathway. The thrombopoietin pathway plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of hematopoietic stem cells. Little wonder therefore that mutations of MPL result in thrombocytosis, leading to an abnormal MPL trafficking or receptor activation. Finally, some extremely rare germline genetic variants in MPL can induce MPN-like hereditary disease. Against this molecular background, TPOR is a key actor in the MPN development and MPL mutations are of major relevance to fully elucidate the molecular mechanisms underlying the clinical manifestations of MPN and to arrange novel therapeutic strategies aiming to disrupt the dysegulated signaling cascade. This chapter will focus on the role MPL in the pathogenesis of MPN and in familial thrombocytosis and will review these different subtypes of somatic and germline genetic variants by dissecting how they impact clinical phenotype.
  
  Copyright © 2021 Elsevier Inc. All rights reserved.
  
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• Editor's message.
  Atheroscler Plus

  2021 Sep;43():1. doi: 10.1016/j.athplu.2021.09.002.
  
  Calabresi Laura
  
  
  
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• Long-term efficacy of lipoprotein apheresis and lomitapide in the treatment of homozygous familial hypercholesterolemia (HoFH): a cross-national retrospective survey.
  Orphanet J Rare Dis

  2021 Sep;16(1):381. doi: 381.
  
  D'Erasmo Laura, Gallo Antonio, Cefalù Angelo Baldassare, Di Costanzo Alessia, Saheb Samir, Giammanco Antonina, Averna Maurizio, Buonaiuto Alessio, Iannuzzo Gabriella, Fortunato Giuliana, Puja Arturo, Montalcini Tiziana, Pavanello Chiara, Calabresi Laura, Vigna Giovanni Battista, Bucci Marco, Bonomo Katia, Nota Fabio, Sampietro Tiziana, Sbrana Francesco, Suppressa Patrizia, Sabbà Carlo, Fimiani Fabio, Cesaro Arturo, Calabrò Paolo, Palmisano Silvia, D'Addato Sergio, Pisciotta Livia, Bertolini Stefano, Bittar Randa, Kalmykova Olga, Béliard Sophie, Carrié Alain, Arca Marcello, Bruckert Eric
  
  Abstract
  
  BACKGROUND:
  Homozygous familial hypercholesterolemia (HoFH) is a rare life-threatening condition that represents a therapeutic challenge. The vast majority of HoFH patients fail to achieve LDL-C targets when treated with the standard protocol, which associates maximally tolerated dose of lipid-lowering medications with lipoprotein apheresis (LA). Lomitapide is an emerging therapy in HoFH, but its place in the treatment algorithm is disputed because a comparison of its long-term efficacy versus LA in reducing LDL-C burden is not available. We assessed changes in long-term LDL-C burden and goals achievement in two independent HoFH patients' cohorts, one treated with lomitapide in Italy (n?=?30) and the other with LA in France (n?=?29).
  
  RESULTS:
  The two cohorts differed significantly for genotype (p?=?0.004), baseline lipid profile (p?0.001), age of treatment initiation (p?0.001), occurrence of cardiovascular disease (p?=?0.003) as well as follow-up duration (p?0.001). The adjunct of lomitapide to conventional lipid-lowering therapies determined an additional 58.0% reduction of last visit LDL-C levels, compared to 37.1% when LA was added (p?=?0.004). Yearly on-treatment LDL-C?70 mg/dl and?55 mg/dl goals were only achieved in 45.5% and 13.5% of HoFH patients treated with lomitapide. The long-term exposure to LDL-C burden was found to be higher in LA than in Lomitapide cohort (13,236.1?±?5492.1 vs. 11,656.6?±?4730.9 mg/dL-year respectively, p?=?0.002). A trend towards fewer total cardiovascular events was observed in the Lomitapide than in the LA cohort.
  
  CONCLUSIONS:
  In comparison with LA, lomitapide appears to provide a better control of LDL-C in HoFH. Further studies are needed to confirm this data and establish whether this translates into a reduction of cardiovascular risk.
  
  © 2021. The Author(s).
  
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• Activated IL-6 signaling contributes to the pathogenesis of, and is a novel therapeutic target for, CALR-mutated MPNs.
  Blood Adv

  2021 Apr;5(8):2184-2195. doi: 10.1182/bloodadvances.2020003291.
  
  Balliu Manjola, Calabresi Laura, Bartalucci Niccolò, Romagnoli Simone, Maggi Laura, Manfredini Rossella, Lulli Matteo, Guglielmelli Paola, Vannucchi Alessandro Maria
  
  Abstract
  
  Calreticulin (CALR), an endoplasmic reticulum-associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1-like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD34+ cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit-Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD34+ cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.
  
  © 2021 by The American Society of Hematology.
  
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• High-Density Lipoproteins and the Kidney.
  Cells

  2021 Mar;10(4):. doi: 764.
  
  Strazzella Arianna, Ossoli Alice, Calabresi Laura
  
  Abstract
  
  Dyslipidemia is a typical trait of patients with chronic kidney disease (CKD) and it is typically characterized by reduced high-density lipoprotein (HDL)-cholesterol(c) levels. The low HDL-c concentration is the only lipid alteration associated with the progression of renal disease in mild-to-moderate CKD patients. Plasma HDL levels are not only reduced but also characterized by alterations in composition and structure, which are responsible for the loss of atheroprotective functions, like the ability to promote cholesterol efflux from peripheral cells and antioxidant and anti-inflammatory proprieties. The interconnection between HDL and renal function is confirmed by the fact that genetic HDL defects can lead to kidney disease; in fact, mutations in apoA-I, apoE, apoL, and lecithin-cholesterol acyltransferase (LCAT) are associated with the development of renal damage. Genetic LCAT deficiency is the most emblematic case and represents a unique tool to evaluate the impact of alterations in the HDL system on the progression of renal disease. Lipid abnormalities detected in LCAT-deficient carriers mirror the ones observed in CKD patients, which indeed present an acquired LCAT deficiency. In this context, circulating LCAT levels predict CKD progression in individuals at early stages of renal dysfunction and in the general population. This review summarizes the main alterations of HDL in CKD, focusing on the latest update of acquired and genetic LCAT defects associated with the progression of renal disease.
  
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• The Association between HDL-C and Subclinical Atherosclerosis Depends on CETP Plasma Concentration: Insights from the IMPROVE Study.
  Biomedicines

  2021 Mar;9(3):. doi: 286.
  
  Colombo Gualtiero I, Bianconi Vanessa, Bonomi Alice, Simonelli Sara, Amato Mauro, Frigerio Beatrice, Ravani Alessio, Vitali Cecilia, Sansaro Daniela, Coggi Daniela, Mannarino Massimo R, Savonen Kai P, Kurl Sudhir, Gigante Bruna, Smit Andries J, Giral Philippe, Tremoli Elena, Calabresi Laura, Veglia Fabrizio, Pirro Matteo, Baldassarre Damiano, On Behalf Of The Improve Study Group
  
  Abstract
  
  The impact of cholesteryl ester transfer protein (CETP) on atherosclerosis is highly debated. This study aimed to investigate the associations between plasma CETP or CETP genotypes and carotid intima-media thickness (cIMT) and the influence of high-density lipoprotein cholesterol (HDL-C) on these associations. Plasma CETP and HDL-C concentrations were measured in 552 subjects free of any pharmacological treatment from the IMPROVE cohort, which includes 3711 European subjects at high cardiovascular risk. CETP single-nucleotide polymorphisms (SNPs) and cIMT measures (cIMT; cIMT of bifurcations, common and internal carotids; plaque-free common carotid [PF CC]-IMT) were available for the full cohort. In drug-free subjects, plasma CETP correlated with HDL-C levels (r = 0.19,
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• Vasculoprotective properties of plasma lipoproteins from brown bears (Ursus arctos).
  J Lipid Res

  2021 ;62():100065. doi: 100065.
  
  Pedrelli Matteo, Parini Paolo, Kindberg Jonas, Arnemo Jon M, Bjorkhem Ingemar, Aasa Ulrika, Westerståhl Maria, Walentinsson Anna, Pavanello Chiara, Turri Marta, Calabresi Laura, Öörni Katariina, Camejo Gérman, Fröbert Ole, Hurt-Camejo Eva
  
  Abstract
  
  Plasma cholesterol and triglyceride (TG) levels are twice as high in hibernating brown bears (Ursus arctos) than healthy humans. Yet, bears display no signs of early stage atherosclerosis development when adult. To explore this apparent paradox, we analyzed plasma lipoproteins from the same 10 bears in winter (hibernation) and summer using size exclusion chromatography, ultracentrifugation, and electrophoresis. LDL binding to arterial proteoglycans (PGs) and plasma cholesterol efflux capacity (CEC) were also evaluated. The data collected and analyzed from bears were also compared with those from healthy humans. In bears, the cholesterol ester, unesterified cholesterol, TG, and phospholipid contents of VLDL and LDL were higher in winter than in summer. The percentage lipid composition of LDL differed between bears and humans but did not change seasonally in bears. Bear LDL was larger, richer in TGs, showed prebeta electrophoretic mobility, and had 5-10 times lower binding to arterial PGs than human LDL. Finally, plasma CEC was higher in bears than in humans, especially the HDL fraction when mediated by ABCA1. These results suggest that in brown bears the absence of early atherogenesis is likely associated with a lower affinity of LDL for arterial PGs and an elevated CEC of bear plasma.
  
  Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
  
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• Intracoronary monocyte expression pattern and HDL subfractions after non-ST elevation myocardial infarction.
  Biochim Biophys Acta Mol Basis Dis

  2021 Jun;1867(6):166116. doi: 10.1016/j.bbadis.2021.166116.
  
  Cariello Marica, Salvia Roberto, Härdfeldt Jennifer, Piglionica Marilidia, Rutigliano David, Caldarola Pasquale, Ossoli Alice, Vacca Michele, Graziano Giusi, Battaglia Stefano, Zerlotin Roberta, Arconzo Maria, Crudele Lucilla, Sabbà Carlo, Calabresi Laura, Moschetta Antonio
  
  Abstract
  
  AIMS:
  Coronary artery disease (CAD) is described as a range of clinical conditions including myocardial infarction (MI) and unstable angina. Lipid and apolipoprotein profiles together with the study of cholesterol deposit and efflux serve to identify novel pre and post infarct scenarios for the treatment of these patients. In (non-ST elevation myocardial infarction) NSTEMI patients, we analysed both systemic and intracoronary serum ability to accept cholesterol as well as cholesterol efflux capacity (CEC) of monocytes in terms of expression of genes involved in the reverse cholesterol transport (RCT).
  
  METHODS AND RESULTS:
  While HDL-C quantity was similar between systemic and coronary arterial blood, in 21 NSTEMI patients we observed a significant reduction of the pre?-HDL fraction and the levels of Apolipoproteins AI, AII, B and E in coronary versus systemic serum. These data are complemented with the observed reduction of CEC. On the contrary, compared to systemic arterial monocytes, in coronary microenvironment of NSTEMI patients after myocardial infarction, the monocytes exhibited a higher mRNA expression of nuclear receptor LXR? and its targets ABCA1 and APOE, which drive cholesterol efflux capacity.
  
  CONCLUSION:
  In this cross-sectional study we observe that in the immediate post infarction period, there is a spontaneous bona fide ligand-induced activation of the LXR driven cholesterol efflux capacity of intracoronary monocytes to overcome the reduced serum ability to accept cholesterol and to inhibit the post-infarction pro-inflammatory local microenvironment.
  
  Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.
  
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• Interactions of Oxysterols with Atherosclerosis Biomarkers in Subjects with Moderate Hypercholesterolemia and Effects of a Nutraceutical Combination ( BB536, Red Yeast Rice Extract) (Randomized, Double-Blind, Placebo-Controlled Study).
  Nutrients

  2021 Jan;13(2):. doi: 427.
  
  Cicolari Stefania, Pavanello Chiara, Olmastroni Elena, Puppo Marina Del, Bertolotti Marco, Mombelli Giuliana, Catapano Alberico L, Calabresi Laura, Magni Paolo
  
  Abstract
  
  BACKGROUND:
  Oxysterol relationship with cardiovascular (CV) risk factors is poorly explored, especially in moderately hypercholesterolaemic subjects. Moreover, the impact of nutraceuticals controlling hypercholesterolaemia on plasma levels of 24-, 25- and 27-hydroxycholesterol (24-OHC, 25-OHC, 27-OHC) is unknown.
  
  METHODS:
  Subjects ( = 33; 18-70 years) with moderate hypercholesterolaemia (low-density lipoprotein cholesterol (LDL-C:): 130-200 mg/dL), in primary CV prevention as well as low CV risk were studied cross-sectionally. Moreover, they were evaluated after treatment with a nutraceutical combination ( BB536, red yeast rice extract (10 mg/dose monacolin K)), following a double-blind, randomized, placebo-controlled design. We evaluated 24-OHC, 25-OHC and 27-OHC levels by gas chromatography/mass spectrometry analysis.
  
  RESULTS:
  24-OHC and 25-OHC were significantly correlated, 24-OHC was correlated with apoB. 27-OHC and 27-OHC/total cholesterol (TC) were higher in men (median 209 ng/mL and 77 ng/mg, respectively) vs. women (median 168 ng/mL and 56 ng/mg, respectively); 27-OHC/TC was significantly correlated with abdominal circumference, visceral fat and, negatively, with high-density lipoprotein cholesterol (HDL-C). Triglycerides were significantly correlated with 24-OHC, 25-OHC and 27-OHC and with 24-OHC/TC and 25-OHC/TC. After intervention, 27-OHC levels were significantly reduced by 10.4% in the nutraceutical group Levels of 24-OHC, 24-OHC/TC, 25-OHC, 25-OHC/TC and 27-OHC/TC were unchanged.
  
  CONCLUSIONS:
  In this study, conducted in moderate hypercholesterolemic subjects, we observed novel relationships between 24-OHC, 25-OHC and 27-OHC and CV risk biomarkers. In addition, no adverse changes of OHC levels upon nutraceutical treatment were found.
  
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• rHDL modeling and the anchoring mechanism of LCAT activation.
  J Lipid Res

  2021 ;62():100006. doi: 100006.
  
  Laurenzi Tommaso, Parravicini Chiara, Palazzolo Luca, Guerrini Uliano, Gianazza Elisabetta, Calabresi Laura, Eberini Ivano
  
  Abstract
  
  Lecithin:cholesterol-acyl transferase (LCAT) plays a major role in cholesterol metabolism as it is the only extracellular enzyme able to esterify cholesterol. LCAT activity is required for lipoprotein remodeling and, most specifically, for the growth and maturation of HDLs. In fact, genetic alterations affecting LCAT functionality may cause a severe reduction in plasma levels of HDL-cholesterol with important clinical consequences. Although several hypotheses were formulated, the exact molecular recognition mechanism between LCAT and HDLs is still unknown. We employed a combination of structural bioinformatics procedures to deepen the insights into the HDL-LCAT interplay that promotes LCAT activation and cholesterol esterification. We have generated a data-driven model of reconstituted HDL (rHDL) and studied the dynamics of an assembled rHDL::LCAT supramolecular complex, pinpointing the conformational changes originating from the interaction between LCAT and apolipoprotein A-I (apoA-I) that are necessary for LCAT activation. Specifically, we propose a mechanism in which the anchoring of LCAT lid to apoA-I helices allows the formation of a hydrophobic hood that expands the LCAT active site and shields it from the solvent, allowing the enzyme to process large hydrophobic substrates.
  
  Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
  
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• HDL-Mediated Cholesterol Efflux and Plasma Loading Capacities Are Altered in Subjects with Metabolically- but Not Genetically Driven Non-Alcoholic Fatty Liver Disease (NAFLD).
  Biomedicines

  2020 Dec;8(12):. doi: 625.
  
  Di Costanzo Alessia, Ronca Annalisa, D'Erasmo Laura, Manfredini Matteo, Baratta Francesco, Pastori Daniele, Di Martino Michele, Ceci Fabrizio, Angelico Francesco, Del Ben Maria, Pavanello Chiara, Turri Marta, Calabresi Laura, Favari Elda, Arca Marcello
  
  Abstract
  
  . Non-alcoholic fatty liver disease (NAFLD) increases the risk of atherosclerosis but this risk may differ between metabolically- vs. genetically-driven NAFLD. High-density lipoprotein (HDL)-mediated cholesterol efflux (CEC) and plasma loading capacity (CLC) are key factors in atherogenesis. . To test whether CEC and CLC differ between metabolically- vs. genetically-determined NAFLD. : CEC and CLC were measured in 19 patients with metabolic NAFLD and wild-type genotype (Group M), 10 patients with genetic NAFLD carrying M148M genotype (Group G), and 10 controls wild-types and without NAFLD. CEC and CLC were measured ex vivo by isotopic and fluorimetric techniques using cellular models. : Compared with Group G, Group M showed reduced total CEC (-18.6%;
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• CER-001 ameliorates lipid profile and kidney disease in a mouse model of familial LCAT deficiency.
  Metabolism

  2021 Mar;116():154464. doi: 10.1016/j.metabol.2020.154464.
  
  Ossoli Alice, Strazzella Arianna, Rottoli Daniela, Zanchi Cristina, Locatelli Monica, Zoja Carlamaria, Simonelli Sara, Veglia Fabrizio, Barbaras Ronald, Tupin Cyrille, Dasseux Jean-Louis, Calabresi Laura
  
  Abstract
  
  OBJECTIVE:
  CER-001 is an HDL mimetic that has been tested in different pathological conditions, but never with LCAT deficiency. This study was designed to investigate whether the absence of LCAT affects the catabolic fate of CER-001, and to evaluate the effects of CER-001 on kidney disease associated with LCAT deficiency.
  
  METHODS:
  Lcat and wild-type mice received CER-001 (2.5, 5, 10?mg/kg) intravenously for 2?weeks. The plasma lipid/ lipoprotein profile and HDL subclasses were analyzed. In a second set of experiments, Lcat mice were injected with LpX to induce renal disease and treated with CER-001 and then the plasma lipid profile, lipid accumulation in the kidney, albuminuria and glomerular podocyte markers were evaluated.
  
  RESULTS:
  In Lcat mice a decrease in total cholesterol and triglycerides, and an increase in HDL-c was observed after CER-001 treatment. While in wild-type mice CER-001 entered the classical HDL remodeling pathway, in the absence of LCAT it disappeared from the plasma shortly after injection and ended up in the kidney. In a mouse model of renal disease in LCAT deficiency, treatment with CER-001 at 10?mg/kg for one month had beneficial effects not only on the lipid profile, but also on renal disease, by limiting albuminuria and podocyte dysfunction.
  
  CONCLUSIONS:
  Treatment with CER-001 ameliorates the dyslipidemia typically associated with LCAT deficiency and more importantly limits renal damage in a mouse model of renal disease in LCAT deficiency. The present results provide a rationale for using CER-001 in FLD patients.
  
  Copyright © 2020 Elsevier Inc. All rights reserved.
  
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• Progression of chronic kidney disease in familial LCAT deficiency: a follow-up of the Italian cohort.
  J Lipid Res

  2020 Dec;61(12):1784-1788. doi: 10.1194/jlr.P120000976.
  
  Pavanello Chiara, Ossoli Alice, Arca Marcello, D'Erasmo Laura, Boscutti Giuliano, Gesualdo Loreto, Lucchi Tiziano, Sampietro Tiziana, Veglia Fabrizio, Calabresi Laura
  
  Abstract
  
  Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.
  
  Copyright © 2020 Pavanello et al.
  
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• Activation of Naturally Occurring Lecithin:Cholesterol Acyltransferase Mutants by a Novel Activator Compound.
  J Pharmacol Exp Ther

  2020 Dec;375(3):463-468. doi: 10.1124/jpet.120.000159.
  
  Pavanello Chiara, Ossoli Alice, Turri Marta, Strazzella Arianna, Simonelli Sara, Laurenzi Tommaso, Kono Keita, Yamada Keisuke, Kiyosawa Naoki, Eberini Ivano, Calabresi Laura
  
  Abstract
  
  Lecithin:cholesterol acyltransferase (LCAT) is a unique plasma enzyme able to esterify cholesterol, and it plays an important role in HDL maturation and promotion of reverse cholesterol transport. Familial LCAT deficiency (FLD; OMIM number 245900) is a rare recessive disease that results from loss-of-function mutations in the gene and has no cure. In this study, we assessed the in vitro efficacy of a novel small-molecule LCAT activator. Cholesterol esterification rate (CER) and LCAT activity were tested in plasma from six controls and five FLD homozygous carriers of various mutations at different doses of the compound (0.1, 1, and 10 µg/ml). In control plasma, the compound significantly increased both CER (
  Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.
  
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• Low Plasma Lecithin: Cholesterol Acyltransferase (LCAT) Concentration Predicts Chronic Kidney Disease.
  J Clin Med

  2020 Jul;9(7):. doi: 2289.
  
  Baragetti Andrea, Ossoli Alice, Strazzella Arianna, Simonelli Sara, Baragetti Ivano, Grigore Liliana, Pellegatta Fabio, Catapano Alberico L, Norata Giuseppe Danilo, Calabresi Laura
  
  Abstract
  
  Low high-density lipoprotein-cholesterol (HDL-c) is the most remarkable lipid trait both in mild-to-moderate chronic kidney disease (CKD) patients as well as in advanced renal disease stages, and we have previously shown that reduced lecithin:cholesterol acyltransferase (LCAT) concentration is a major determinant of the low HDL phenotype. In the present study, we test the hypothesis that reduced LCAT concentration in CKD contributes to the progression of renal damage. The study includes two cohorts of subjects selected from the PLIC study: a cohort of 164 patients with CKD (NefroPLIC cohort) and a cohort of 164 subjects selected from the PLIC participants with a basal estimated glomerular filtration rate (eGFR) > 60 mL/min/1.73 m (PLIC cohort). When the NefroPLIC patients were categorized according to the LCAT concentration, patients in the 1st tertile showed the highest event rate at follow-up with an event hazard ratio significantly higher compared to the 3rd LCAT tertile. Moreover, in the PLIC cohort, subjects in the 1st LCAT tertile showed a significantly faster impairment of kidney function compared to subjects in the 3rd LCAT tertile. Serum from subjects in the 1st LCAT tertile promoted a higher reactive oxygen species (ROS) production in renal cells compared to serum from subjects in the third LCAT tertile, and this effect was contrasted by pre-incubation with recombinant human LCAT (rhLCAT). The present study shows that reduced plasma LCAT concentration predicts CKD progression over time in patients with renal dysfunction, and, even more striking, it predicts the impairment of kidney function in the general population.
  
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• Genetic, biochemical, and clinical features of LCAT deficiency: update for 2020.
  Curr Opin Lipidol

  2020 Aug;31(4):232-237. doi: 10.1097/MOL.0000000000000697.
  
  Pavanello Chiara, Calabresi Laura
  
  Abstract
  
  PURPOSE OF REVIEW:
  Genetic LCAT deficiency is a rare metabolic disorder characterized by low-plasma HDL cholesterol levels. Clinical manifestations of the disease include corneal opacification, anemia, and renal disease, which represents the major cause of morbidity and mortality in carriers.
  
  RECENT FINDINGS:
  Biochemical and clinical manifestations of the disease are very heterogeneous among carriers. The collection of large series of affected individuals is needed to answer various open questions on this rare disorder of lipid metabolism, such as the cause of renal damage in patients with complete LCAT deficiency and the cardiovascular risk in carriers of different LCAT gene mutations.
  
  SUMMARY:
  Familial LCAT deficiency is a rare disease, with serious clinical manifestations, which can occur in the first decades of life, and presently with no cure. The timely diagnosis in carriers, together with the identification of disease biomarkers able to predict the evolution of clinical manifestations, would be of great help in the identification of carriers to address to future available therapies.
  
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• The Prospective Studies of Atherosclerosis (Proof-ATHERO) Consortium: Design and Rationale.
  Gerontology

  2020 ;66(5):447-459. doi: 10.1159/000508498.
  
  Tschiderer Lena, Seekircher Lisa, Klingenschmid Gerhard, Izzo Raffaele, Baldassarre Damiano, Iglseder Bernhard, Calabresi Laura, Liu Jing, Price Jackie F, Bae Jang-Ho, Brouwers Frank P, de Groot Eric, Schmidt Caroline, Bergström Göran, A?çi Gülay, Gresele Paolo, Okazaki Shuhei, Kapellas Kostas, Landecho Manuel F, Sattar Naveed, Agewall Stefan, Zou Zhi-Yong, Byrne Christopher D, Nanayakkara Prabath W B, Papagianni Aikaterini, Witham Miles D, Bernal Enrique, Ekart Robert, van Agtmael Michiel A, Neves Mario F, Sato Eiichi, Ezhov Marat, Walters Matthew, Olsen Michael H, Stoli? Radojica, Zozuli?ska-Zió?kiewicz Dorota A, Hanefeld Markolf, Staub Daniel, Nagai Michiaki, Nieuwkerk Pythia T, Huisman Menno V, Kato Akihiko, Honda Hirokazu, Parraga Grace, Magliano Dianna, Gabriel Rafael, Rundek Tatjana, Espeland Mark A, Kiechl Stefan, Willeit Johann, Lind Lars, Empana Jean Philippe, Lonn Eva, Tuomainen Tomi-Pekka, Catapano Alberico, Chien Kuo-Liong, Sander Dirk, Kavousi Maryam, Beulens Joline W J, Bots Michiel L, Sweeting Michael J, Lorenz Matthias W, Willeit Peter,
  
  Abstract
  
  Atherosclerosis - the pathophysiological mechanism shared by most cardiovascular diseases - can be directly or indirectly assessed by a variety of clinical tests including measurement of carotid intima-media thickness, carotid plaque, -ankle-brachial index, pulse wave velocity, and coronary -artery calcium. The Prospective Studies of Atherosclerosis -(Proof-ATHERO) consortium (https://clinicalepi.i-med.ac.at/research/proof-athero/) collates de-identified individual-participant data of studies with information on atherosclerosis measures, risk factors for cardiovascular disease, and incidence of cardiovascular diseases. It currently comprises 74 studies that involve 106,846 participants from 25 countries and over 40 cities. In summary, 21 studies recruited participants from the general population (n = 67,784), 16 from high-risk populations (n = 22,677), and 37 as part of clinical trials (n = 16,385). Baseline years of contributing studies range from April 1980 to July 2014; the latest follow-up was until June 2019. Mean age at baseline was 59 years (standard deviation: 10) and 50% were female. Over a total of 830,619 person-years of follow-up, 17,270 incident cardiovascular events (including coronary heart disease and stroke) and 13,270 deaths were recorded, corresponding to cumulative incidences of 2.1% and 1.6% per annum, respectively. The consortium is coordinated by the Clinical Epidemiology Team at the Medical University of Innsbruck, Austria. Contributing studies undergo a detailed data cleaning and harmonisation procedure before being incorporated in the Proof-ATHERO central database. Statistical analyses are being conducted according to pre-defined analysis plans and use established methods for individual-participant data meta-analysis. Capitalising on its large sample size, the multi-institutional collaborative Proof-ATHERO consortium aims to better characterise, understand, and predict the development of atherosclerosis and its clinical consequences.
  
  © 2020 S. Karger AG, Basel.
  
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• Enhanced engraftment of human myelofibrosis stem and progenitor cells in MISTRG mice.
  Blood Adv

  2020 Jun;4(11):2477-2488. doi: 10.1182/bloodadvances.2019001364.
  
  Lysenko Veronika, Wildner-Verhey van Wijk Nicole, Zimmermann Kathrin, Weller Marie-Christine, Bühler Marco, Wildschut Mattheus H E, Schürch Patrick, Fritz Christine, Wagner Ulrich, Calabresi Laura, Psaila Bethan, Flavell Richard A, Vannucchi Alessandro M, Mead Adam J, Wild Peter J, Dirnhofer Stefan, Manz Markus G, Theocharides Alexandre P A
  
  Abstract
  
  The engraftment potential of myeloproliferative neoplasms in immunodeficient mice is low. We hypothesized that the physiological expression of human cytokines (macrophage colony-stimulating factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) combined with human signal regulatory protein ? expression in Rag2-/-Il2r?-/- (MISTRG) mice might provide a supportive microenvironment for the development and maintenance of hematopoietic stem and progenitor cells (HSPC) from patients with primary, post-polycythemia or post-essential thrombocythemia myelofibrosis (MF). We show that MISTRG mice, in contrast to standard immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ and Rag2-/-Il2r?-/- mice, supported engraftment of all patient samples investigated independent of MF disease stage or risk category. Moreover, MISTRG mice exhibited significantly higher human MF engraftment levels in the bone marrow, peripheral blood, and spleen and supported secondary repopulation. Bone marrow fibrosis development was limited to 3 of 14 patient samples investigated in MISTRG mice. Disease-driving mutations were identified in all xenografts, and targeted sequencing revealed maintenance of the primary patient sample clonal composition in 7 of 8 cases. Treatment of engrafted mice with the current standard-of-care Janus kinase inhibitor ruxolitinib led to a reduction in human chimerism. In conclusion, the established MF patient-derived xenograft model supports robust engraftment of MF HSPCs and maintains the genetic complexity observed in patients. The model is suited for further testing of novel therapeutic agents to expedite their transition into clinical trials.
  
  © 2020 by The American Society of Hematology.
  
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• Familial LCAT deficiency and cardiovascular disease: the game is not over. A case of dramatic multivessel atherosclerosis.
  Minerva Med

  2023 Aug;114(4):535-537. doi: 10.23736/S0026-4806.20.06633-1.
  
  Bigazzi Federico, Dal Pino Beatrice, Pavanello Chiara, Sbrana Francesco, Aquaro Giovanni D, Napoli Vinicio, Palmieri Cataldo, Barison Andrea, Calabresi Laura, Sampietro Tiziana
  
  
  
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• Lecithin:cholesterol acyltransferase: symposium on 50 years of biomedical research from its discovery to latest findings.
  J Lipid Res

  2020 Aug;61(8):1142-1149. doi: 10.1194/jlr.S120000720.
  
  Norum Kaare R, Remaley Alan T, Miettinen Helena E, Strøm Erik H, Balbo Bruno E P, Sampaio Carlos A T L, Wiig Ingrid, Kuivenhoven Jan Albert, Calabresi Laura, Tesmer John J, Zhou Mingyue, Ng Dominic S, Skeie Bjørn, Karathanasis Sotirios K, Manthei Kelly A, Retterstøl Kjetil
  
  Abstract
  
  LCAT converts free cholesterol to cholesteryl esters in the process of reverse cholesterol transport. Familial LCAT deficiency (FLD) is a genetic disease that was first described by Kaare R. Norum and Egil Gjone in 1967. This report is a summary from a 2017 symposium where Dr. Norum recounted the history of FLD and leading experts on LCAT shared their results. The Tesmer laboratory shared structural findings on LCAT and the close homolog, lysosomal phospholipase A2. Results from studies of FLD patients in Finland, Brazil, Norway, and Italy were presented, as well as the status of a patient registry. Drs. Kuivenhoven and Calabresi presented data from carriers of genetic mutations suggesting that FLD does not necessarily accelerate atherosclerosis. Dr. Ng shared that LCAT-null mice were protected from diet-induced obesity, insulin resistance, and nonalcoholic fatty liver disease. Dr. Zhou presented multiple innovations for increasing LCAT activity for therapeutic purposes, whereas Dr. Remaley showed results from treatment of an FLD patient with recombinant human LCAT (rhLCAT). Dr. Karathanasis showed that rhLCAT infusion in mice stimulates cholesterol efflux and suggested that it could also enhance cholesterol efflux from macrophages. While the role of LCAT in atherosclerosis remains elusive, the consensus is that a continued study of both the enzyme and disease will lead toward better treatments for patients with heart disease and FLD.
  
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• Extramedullary blastic transformation of primary myelofibrosis in the form of disseminated myeloid sarcoma: a case report and review of the literature.
  Clin Exp Med

  2020 May;20(2):313-320. doi: 10.1007/s10238-020-00616-5.
  
  Coltro Giacomo, Mannelli Francesco, Vergoni Federica, Santi Raffaella, Massi Daniela, Siliani Luisa Margherita, Marzullo Antonella, Bonifacio Stefania, Pelo Elisabetta, Pacilli Annalisa, Paoli Chiara, Franci Annalisa, Calabresi Laura, Bosi Alberto, Vannucchi Alessandro Maria, Guglielmelli Paola
  
  Abstract
  
  Splenomegaly is a key clinical manifestation of myelofibrosis, and splenectomy is currently indicated in patients with drug refractory, symptomatic splenomegaly or with the aim of improving refractory cytopenias. Transformation to acute myeloid leukemia occurs in up to 20% of patients with myelofibrosis, while cases of myeloid sarcoma have been reported very unfrequently. In this manuscript, we report the case of a 60-year-old man with a history of primary myelofibrosis who underwent splenectomy because of drug-refractory massive splenomegaly, systemic symptoms and anemia. At the opening of the peritoneal cavity, the spleen resulted massively enlarged and tenaciously entrapped by a pervasive neoplastic-like tissue. The extensive involvement of the abdomen fatally complicated the surgical procedure. At postmortem examination, the spleen showed a diffuse infiltration of immature cells that were also found in the peritoneum, bowel, liver, lungs and myocardium. After immunohistochemical, cytogenetic, flow cytometric and molecular characterization of neoplastic population, a diagnosis of disseminated myeloid sarcoma of the spleen was made. This case report highlights a very unusual case of myeloid sarcoma originating from the spleen in a patient with myelofibrosis who had no evidence of bone marrow or peripheral blood involvement by leukemic cells. Molecular characterization showed that leukemic cells originated from the founding clone of the chronic phase. The sarcoma could not be suspected based on clinical findings and was diagnosed only at the time of surgical procedure and autopsy. This case suggests that leukemic transformation of myelofibrosis can originate outside the bone marrow and, presumably rarely, present as a granulocytic sarcoma.
  
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• LIPA gene mutations affect the composition of lipoproteins: Enrichment in ACAT-derived cholesteryl esters.
  Atherosclerosis

  2020 Mar;297():8-15. doi: 10.1016/j.atherosclerosis.2020.01.026.
  
  Arnaboldi Lorenzo, Ossoli Alice, Giorgio Eleonora, Pisciotta Livia, Lucchi Tiziano, Grigore Liliana, Pavanello Chiara, Granata Agnese, Pasta Andrea, Arosio Beatrice, Azzolino Domenico, Baragetti Andrea, Castelnuovo Samuela, Corsini Alberto, Catapano Alberico L, Calabresi Laura, Gomaraschi Monica
  
  Abstract
  
  BACKGROUND AND AIMS:
  Cholesteryl ester storage disease (CESD) due to LIPA gene mutations is characterized by hepatic steatosis, hypercholesterolemia and hypoalphalipoproteinemia, exposing affected patients to an increased cardiovascular risk. Further insights into the impact of LIPA gene mutations on lipid/lipoprotein metabolism are limited. Aim of the study was to investigate the effect of carrying one or two mutant LIPA alleles on lipoprotein composition and function.
  
  METHODS:
  Lipoproteins were isolated from 6 adult CESD patients, 5 relatives carrying one mutant LIPA allele (carriers) and 12 sex/age matched controls. Lipid profile, lipoprotein mass composition and the fatty acid distribution of cholesteryl esters (CEs) were assessed. HDL function was evaluated as the ability to promote nitric oxide release by endothelial cells.
  
  RESULTS:
  Despite the lipid-lowering therapy, total cholesterol, LDL-cholesterol and triglycerides were increased in CESD patients compared to controls, while HDL-cholesterol was reduced. Carriers also displayed elevated total and LDL-cholesterol. Very low and intermediate density lipoproteins from CESD patients and carriers were enriched in CEs compared to the control ones, with a concomitant reduction of triglycerides. Fatty acid composition of CEs in serum and lipoproteins showed a depletion of linoleate content in CESD patients, due to the reduced LCAT activity. In CESD HDL, fatty acid distribution of CEs was shifted towards saturated ones, if compared to control HDL. The changes in HDL composition did not affect HDL ability to promote nitric oxide release by endothelial cells.
  
  CONCLUSIONS:
  LIPA gene mutations significantly affected plasma levels and lipid composition of lipoproteins, likely contributing to the increased cardiovascular risk of affected patients.
  
  Copyright © 2020 Elsevier B.V. All rights reserved.
  
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• Virtual genetic diagnosis for familial hypercholesterolemia powered by machine learning.
  Eur J Prev Cardiol

  2020 Oct;27(15):1639-1646. doi: 10.1177/2047487319898951.
  
  Pina Ana, Helgadottir Saga, Mancina Rosellina Margherita, Pavanello Chiara, Pirazzi Carlo, Montalcini Tiziana, Henriques Roberto, Calabresi Laura, Wiklund Olov, Macedo M Paula, Valenti Luca, Volpe Giovanni, Romeo Stefano
  
  Abstract
  
  AIMS:
  Familial hypercholesterolemia (FH) is the most common genetic disorder of lipid metabolism. The gold standard for FH diagnosis is genetic testing, available, however, only in selected university hospitals. Clinical scores - for example, the Dutch Lipid Score - are often employed as alternative, more accessible, albeit less accurate FH diagnostic tools. The aim of this study is to obtain a more reliable approach to FH diagnosis by a "virtual" genetic test using machine-learning approaches.
  
  METHODS AND RESULTS:
  We used three machine-learning algorithms (a classification tree (CT), a gradient boosting machine (GBM), a neural network (NN)) to predict the presence of FH-causative genetic mutations in two independent FH cohorts: the FH Gothenburg cohort (split into training data (?=?174) and internal test (?=?74)) and the FH-CEGP Milan cohort (external test, ?=?364). By evaluating their area under the receiver operating characteristic (AUROC) curves, we found that the three machine-learning algorithms performed better (AUROC 0.79 (CT), 0.83 (GBM), and 0.83 (NN) on the Gothenburg cohort, and 0.70 (CT), 0.78 (GBM), and 0.76 (NN) on the Milan cohort) than the clinical Dutch Lipid Score (AUROC 0.68 and 0.64 on the Gothenburg and Milan cohorts, respectively) in predicting carriers of FH-causative mutations.
  
  CONCLUSION:
  In the diagnosis of FH-causative genetic mutations, all three machine-learning approaches we have tested outperform the Dutch Lipid Score, which is the clinical standard. We expect these machine-learning algorithms to provide the tools to implement a virtual genetic test of FH. These tools might prove particularly important for lipid clinics without access to genetic testing.
  
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• Analysis of HDL-microRNA panel in heterozygous familial hypercholesterolemia subjects with LDL receptor null or defective mutation.
  Sci Rep

  2019 Dec;9(1):20354. doi: 20354.
  
  Scicali Roberto, Di Pino Antonino, Pavanello Chiara, Ossoli Alice, Strazzella Arianna, Alberti Antonia, Di Mauro Stefania, Scamporrino Alessandra, Urbano Francesca, Filippello Agnese, Piro Salvatore, Rabuazzo Agata Maria, Calabresi Laura, Purrello Francesco
  
  Abstract
  
  In the last years increasing attention has been given to the connection between genotype/phenotype and cardiovascular events in subjects with familial hypercholesterolemia (FH). MicroRNAs (miRs) bound to high-density lipoprotein (HDL) may contribute to better discriminate the cardiovascular risk of FH subjects. Our aim was to evaluate the HDL-miR panel in heterozygous FH (HeFH) patients with an LDLR null or defective mutation and its association with pulse wave velocity (PWV). We evaluated lipid panel, HDL-miR panel and PWV in 32 LDLR null mutation (LDLR-null group) and 35 LDLR defective variant (LDLR-defective group) HeFH patients. HDL-miR-486 and HDL-miR-92a levels were more expressed in the LDLR-null group than the LDLR-defective group. When we further stratified the study population into three groups according to both the LDLR genotype and history of ASCVD (LDLR-null/not-ASCVD, LDLR-defective/not-ASCVD and LDLR/ASCVD groups), both the LDLR/ASCVD and the LDLR-null/not-ASCVD groups had a higher expression of HDL-miR-486 and HDL-miR-92a than the LDLR-defective/not-ASCVD group. Finally, HDL-miR-486 and HDL-miR-92a were independently associated with PWV. In conclusion, the LDLR-null group exhibited HDL-miR-486 and HDL-miR-92a levels more expressed than the LDLR-defective group. Further studies are needed to evaluate these HDL-miRs as predictive biomarkers of cardiovascular events in FH.
  
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• Rare dyslipidaemias, from phenotype to genotype to management: a European Atherosclerosis Society task force consensus statement.
  Lancet Diabetes Endocrinol

  2020 Jan;8(1):50-67. doi: 10.1016/S2213-8587(19)30264-5.
  
  Hegele Robert A, Borén Jan, Ginsberg Henry N, Arca Marcello, Averna Maurizio, Binder Christoph J, Calabresi Laura, Chapman M John, Cuchel Marina, von Eckardstein Arnold, Frikke-Schmidt Ruth, Gaudet Daniel, Hovingh G Kees, Kronenberg Florian, Lütjohann Dieter, Parhofer Klaus G, Raal Frederick J, Ray Kausik K, Remaley Alan T, Stock Jane K, Stroes Erik S, Tokgözo?lu Lale, Catapano Alberico L
  
  Abstract
  
  Genome sequencing and gene-based therapies appear poised to advance the management of rare lipoprotein disorders and associated dyslipidaemias. However, in practice, underdiagnosis and undertreatment of these disorders are common, in large part due to interindividual variability in the genetic causes and phenotypic presentation of these conditions. To address these challenges, the European Atherosclerosis Society formed a task force to provide practical clinical guidance focusing on patients with extreme concentrations (either low or high) of plasma low-density lipoprotein cholesterol, triglycerides, or high-density lipoprotein cholesterol. The task force also recognises the scarcity of quality information regarding the prevalence and outcomes of these conditions. Collaborative registries are needed to improve health policy for the care of patients with rare dyslipidaemias.
  
  Copyright © 2020 Elsevier Ltd. All rights reserved.
  
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• Lipid accumulation impairs lysosomal acid lipase activity in hepatocytes: Evidence in NAFLD patients and cell cultures.
  Biochim Biophys Acta Mol Cell Biol Lipids

  2019 Dec;1864(12):158523. doi: 10.1016/j.bbalip.2019.158523.
  
  Gomaraschi Monica, Fracanzani Anna Ludovica, Dongiovanni Paola, Pavanello Chiara, Giorgio Eleonora, Da Dalt Lorenzo, Norata Giuseppe Danilo, Calabresi Laura, Consonni Dario, Lombardi Rosa, Branchi Adriana, Fargion Silvia
  
  Abstract
  
  AIMS:
  It has been hypothesized that the activity of lysosomal acid lipase (LAL), a key enzyme involved in lipid metabolism, is involved in the NAFLD phenotype. To clarify the role of LAL in NAFLD, we studied 164 consecutive patients with biopsy-proven NAFLD and fat-loaded HepG2 cells.
  
  METHODS:
  LAL activity was measured (i) on dried blood spots (DBS) from NAFLD patients and dyslipidemic subjects without fatty liver and (ii) on liver biopsies from NAFLD patients. LAL activity and expression were evaluated in HepG2 cells cultured in the presence of free fatty acids (FAs), with or without a PPAR-alpha agonist.
  
  RESULTS:
  LAL activity was significantly reduced in patients with NAFLD compared to dyslipidemic subjects. LAL activity measured in liver biopsies from NAFLD patients was highly correlated to that measured on DBS and was independent of LAL expression in the liver. In a fully adjusted model, LAL activity on DBS was associated only with platelets and, when normalized by platelet count, it did not differ according to fibrosis stage. In vitro, FA loading of HepG2 fully replicated the impairment of LAL activity observed in NALFD patients. In these cells, the activation of PPAR-alpha receptors prevented and corrected FA-induced LAL impairment, by stimulating FA oxidation and LAL expression.
  
  CONCLUSIONS:
  LAL activity is reduced in NAFLD patients, independently from disease progression. In vitro, impaired LAL activity induced by FA loading was rescued by PPAR-alpha activation. These data suggest that the pharmacological modulation of LAL should be explored in the management of NAFLD patients.
  
  Copyright © 2019 Elsevier B.V. All rights reserved.
  
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• Correction to: Nutraceutical approach for the management of cardiovascular risk - a combination containing the probiotic Bifidobacterium longum BB536 and red yeast rice extract: results from a randomized, double-blind, placebo-controlled study.
  Nutr J

  2019 Sep;18(1):54. doi: 54.
  
  Ruscica Massimiliano, Pavanello Chiara, Gandini Sara, Macchi Chiara, Botta Margherita, Dall'Orto Daria, Del Puppo Marina, Bertolotti Marco, Bosisio Raffaella, Mombelli Giuliana, Sirtori Cesare R, Calabresi Laura, Magni Paolo
  
  Abstract
  
  Following publication of the original article [1], the authors reported an error in the affiliation of the third author, Sara Gandini. The correct affiliation should read: Division of Epidemiology and Biostatistics, IEO, European Institute of Oncology IRCCS, Milan, Italy.
  
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• Individuals with familial hypercholesterolemia and cardiovascular events have higher circulating Lp(a) levels.
  J Clin Lipidol

  2019 ;13(5):778-787.e6. doi: 10.1016/j.jacl.2019.06.011.
  
  Pavanello Chiara, Pirazzi Carlo, Bjorkman Kristina, Sandstedt Joakim, Tarlarini Claudia, Mosca Lorena, Romeo Stefano, Calabresi Laura, Mancina Rosellina Margherita
  
  Abstract
  
  BACKGROUND:
  Cardiovascular disease (CVD) is a major cause of mortality and morbidity. Increased low-density lipoprotein cholesterol (LDL-C) level is its major risk factor. Familial hypercholesterolemia (FH) is a genetic disorder characterized by elevated LDL-C since birth and subsequent premature CVD. There is a heterogeneity in the CVD onset in patients with FH. This is potentially due to the presence of other independent risk factors. Lipoprotein(a) [Lp(a)] is an LDL-like particle and represents a strong risk factor for CVD.
  
  OBJECTIVE:
  Our objective was to understand the contribution of Lp(a) in the susceptibility to CVD in individuals with genetic diagnosis of FH.
  
  METHODS:
  We measured Lp(a) levels in 2 independent and well-characterized genetic-FH cohorts: the FH-Gothenburg cohort (n = 190) and the FH-CEGP Milan cohort (n = 160). The genetic diagnosis was performed by targeted next-generation sequencing (FH-Gothenburg and part of the FH-CEGP Milan cohort), or by Sanger sequencing.
  
  RESULTS:
  We show that among individuals with genetic diagnosis of FH, those with previous CVD had higher Lp(a) levels. In addition, analyzing the response to the lipid-lowering therapies, we have also shown that statins had the same LDL-C-lowering effect irrespective of the type of FH-causative mutation. However, when we examined the lipid-lowering effect of proprotein convertase subtilisin/kexin type 9 inhibition by antibodies, we observed a trend in a better reduction of the LDL-C level in carriers of nonsense mutations.
  
  CONCLUSION:
  In conclusion, our results suggest that Lp(a) contributes to CVD onset in individuals with genetic diagnosis of FH. Our finding supports the importance to identify an efficacious therapy to lower Lp(a) in patients with FH to prevent CVD onset or recurrence.
  
  Copyright © 2019 National Lipid Association. Published by Elsevier Inc. All rights reserved.
  
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• Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants.
  Sci Rep

  2019 Jul;9(1):10558. doi: 10558.
  
  Salati Simona, Genovese Elena, Carretta Chiara, Zini Roberta, Bartalucci Niccolò, Prudente Zelia, Pennucci Valentina, Ruberti Samantha, Rossi Chiara, Rontauroli Sebastiano, Enzo Elena, Calabresi Laura, Balliu Manjola, Mannarelli Carmela, Bianchi Elisa, Guglielmelli Paola, Tagliafico Enrico, Vannucchi Alessandro M, Manfredini Rossella
  
  Abstract
  
  Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.
  
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• Treatment with fibrates is associated with higher LAL activity in dyslipidemic patients.
  Pharmacol Res

  2019 Sep;147():104362. doi: 10.1016/j.phrs.2019.104362.
  
  Pavanello Chiara, Baragetti Andrea, Branchi Adriana, Grigore Liliana, Castelnuovo Samuela, Giorgio Eleonora, Catapano Alberico L, Calabresi Laura, Gomaraschi Monica
  
  Abstract
  
  Lysosomal acid lipase (LAL) is responsible for the hydrolysis of cholesteryl esters (CE) and triglycerides (TG) within the lysosomes; generated cholesterol and free fatty acids (FFA) are released in the cytosol where they can regulate their own synthesis and metabolism. When LAL is not active, as in case of genetic mutations, CE and TG accumulate in the lysosomal compartment, while the lack of release of cholesterol and FFA in the cytosol leads to an upregulation of their synthesis. Thus, LAL plays a central role in the intracellular homeostasis of lipids. Since there are no indications about the effect of different lipid-lowering agents on LAL activity, aim of the study was to address the relationship between LAL activity and the type of lipid-lowering therapy in a cohort of dyslipidemic patients. LAL activity was measured on dried blood spot from 120 patients with hypercholesterolemia or mixed dyslipidemia and was negatively correlated to LDL-cholesterol levels. Among enrolled patients, ninety-one were taking one or more lipid-lowering drugs, as statins, fibrates, ezetimibe and omega-3 polyunsaturated fatty acids. When patients were stratified according to the type of lipid-lowering treatment, i.e. untreated, taking statins or taking fibrates, LAL activity was significantly higher in those with fibrates, even after adjustment for sex, age, BMI, lipid parameters, liver function, metabolic syndrome, diabetes and statin use. In a subset of patients tested after 3 months of treatment with micronized fenofibrate, LAL activity raised by 21%; the increase was negatively correlated with baseline LAL activity. Thus, the use of fibrates is independently associated with higher LAL activity in dyslipidemic patients, suggesting that the positive effects of PPAR-? activation on cellular and systemic lipid homeostasis can also include an improved LAL activity.
  
  Copyright © 2019 Elsevier Ltd. All rights reserved.
  
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• The Extent of Human Apolipoprotein A-I Lipidation Strongly Affects the ?-Amyloid Efflux Across the Blood-Brain Barrier .
  Front Neurosci

  2019 ;13():419. doi: 419.
  
  Dal Magro Roberta, Simonelli Sara, Cox Alysia, Formicola Beatrice, Corti Roberta, Cassina Valeria, Nardo Luca, Mantegazza Francesco, Salerno Domenico, Grasso Gianvito, Deriu Marco Agostino, Danani Andrea, Calabresi Laura, Re Francesca
  
  Abstract
  
  Much evidence suggests a protective role of high-density lipoprotein (HDL) and its major apolipoprotein apoA-I, in Alzheimer's disease (AD). The biogenesis of nascent HDL derived from a first lipidation of apoA-I, which is synthesized by the liver and intestine but not in the brain, in a process mediated by ABCA1. The maturation of nascent HDL in mature spherical HDL is due to a subsequent lipidation step, LCAT-mediated cholesterol esterification, and the change of apoA-I conformation. Therefore, different subclasses of apoA-I-HDL simultaneously exist in the blood circulation. Here, we investigated if and how the lipidation state affects the ability of apoA-I-HDL to target and modulate the cerebral ?-amyloid (A?) content from the periphery, that is thus far unclear. In particular, different subclasses of HDL, each with different apoA-I lipidation state, were purified from human plasma and their ability to cross the blood-brain barrier (BBB), to interact with A? aggregates, and to affect A? efflux across the BBB was assessed using a transwell system. The results showed that discoidal HDL displayed a superior capability to promote A? efflux (9 × 10 cm/min), when compared to apoA-I in other lipidation states. In particular, no effect on A? efflux was detected when apoA-I was in mature spherical HDL, suggesting that apoA-I conformation, and lipidation could play a role in A? clearance from the brain. Finally, when apoA-I folded its structure in discoidal HDL, rather than in spherical ones, it was able to cross the BBB and strongly destabilize the conformation of A? fibrils by decreasing the order of the fibril structure (-24%) and the ?-sheet content (-14%). These data suggest that the extent of apoA-I lipidation, and consequently its conformation, may represent crucial features that could exert their protective role in AD pathogenesis.
  
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• Recombinant LCAT (Lecithin:Cholesterol Acyltransferase) Rescues Defective HDL (High-Density Lipoprotein)-Mediated Endothelial Protection in Acute Coronary Syndrome.
  Arterioscler Thromb Vasc Biol

  2019 May;39(5):915-924. doi: 10.1161/ATVBAHA.118.311987.
  
  Ossoli Alice, Simonelli Sara, Varrenti Marisa, Morici Nuccia, Oliva Fabrizio, Stucchi Miriam, Gomaraschi Monica, Strazzella Arianna, Arnaboldi Lorenzo, Thomas Michael J, Sorci-Thomas Mary G, Corsini Alberto, Veglia Fabrizio, Franceschini Guido, Karathanasis Sotirios K, Calabresi Laura
  
  Abstract
  
  Objective- Aim of this study was to evaluate changes in LCAT (lecithin:cholesterol acyltransferase) concentration and activity in patients with an acute coronary syndrome, to investigate if these changes are related to the compromised capacity of HDL (high-density lipoprotein) to promote endothelial nitric oxide (NO) production, and to assess if rhLCAT (recombinant human LCAT) can rescue the defective vasoprotective HDL function. Approach and Results- Thirty ST-segment-elevation myocardial infarction (STEMI) patients were enrolled, and plasma was collected at hospital admission, 48 and 72 hours thereafter, at hospital discharge, and at 30-day follow-up. Plasma LCAT concentration and activity were measured and related to the capacity of HDL to promote NO production in cultured endothelial cells. In vitro studies were performed in which STEMI patients' plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in endothelial cells. The plasma concentration of the LCAT enzyme significantly decreases during STEMI with a parallel significant reduction in LCAT activity. HDL isolated from STEMI patients progressively lose the capacity to promote NO production by endothelial cells, and the reduction is related to decreased LCAT concentration. In vitro incubation of STEMI patients' plasma with rhLCAT restores HDL ability to promote endothelial NO production, possibly related to significant modification in HDL phospholipid classes. Conclusions- Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during acute coronary syndrome. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in acute coronary syndrome.
  
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• Nutraceutical approach for the management of cardiovascular risk - a combination containing the probiotic Bifidobacterium longum BB536 and red yeast rice extract: results from a randomized, double-blind, placebo-controlled study.
  Nutr J

  2019 Feb;18(1):13. doi: 13.
  
  Ruscica Massimiliano, Pavanello Chiara, Gandini Sara, Macchi Chiara, Botta Margherita, Dall'Orto Daria, Del Puppo Marina, Bertolotti Marco, Bosisio Raffaella, Mombelli Giuliana, Sirtori Cesare R, Calabresi Laura, Magni Paolo
  
  Abstract
  
  BACKGROUND:
  Probiotics incorporated into dairy products have been shown to reduce total (TC) and LDL cholesterolemia (LDL-C) in subjects with moderate hypercholesterolemia. More specifically, probiotics with high biliary salt hydrolase activity, e.g. Bifidobacterium longum BB536, may decrease TC and LDL-C by lowering intestinal cholesterol reabsorption and, combined with other nutraceuticals, may be useful to manage hypercholesterolemia in subjects with low cardiovascular (CV) risk. This study was conducted to evaluate the efficacy and safety of a nutraceutical combination containing Bifidobacterium longum BB536, red yeast rice (RYR) extract (10?mg/day monacolin K), niacin, coenzyme Q10 (Lactoflorene Colesterolo®). The end-points were changes of lipid CV risk markers (LDL-C, TC, non-HDL-cholesterol (HDL-C), triglycerides (TG), apolipoprotein B (ApoB), HDL-C, apolipoprotein AI (ApoAI), lipoprotein(a) (Lp(a), proprotein convertase subtilisin/kexin type 9 (PCSK9)), and of markers of cholesterol synthesis/absorption.
  
  METHODS:
  A 12-week randomized, parallel, double-blind, placebo-controlled study. Thirty-three subjects (18-70?years) in primary CV prevention and low CV risk (SCORE: 0-1% in 24 and 2-4% in 9 subjects; LDL-C: 130-200?mg/dL) were randomly allocated to either nutraceutical (N?=?16) or placebo (N?=?17).
  
  RESULTS:
  Twelve-week treatment with the nutraceutical combination, compared to placebo, significantly reduced TC (-?16.7%), LDL-C (-?25.7%), non-HDL-C (-?24%) (all p?0.0001), apoB (-?17%, p?=?0.003). TG, HDL-C, apoAI, Lp(a), PCSK9 were unchanged. Lathosterol:TC ratio was significantly reduced by the nutraceutical combination, while campesterol:TC ratio and sitosterol:TC ratio did not change, suggesting reduction of synthesis without increased absorption of cholesterol. No adverse effects and a 97% compliance were observed.
  
  CONCLUSIONS:
  A 12-week treatment with a nutraceutical combination containing the probiotic Bifidobacterium longum BB536 and RYR extract significantly improved the atherogenic lipid profile and was well tolerated by low CV risk subjects.
  
  TRIAL REGISTRATION:
  NCT02689934 .
  
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• A proteomic approach to identify novel disease biomarkers in LCAT deficiency.
  J Proteomics

  2019 Apr;198():113-118. doi: 10.1016/j.jprot.2018.12.005.
  
  Simonelli Sara, Ossoli Alice, Banfi Cristina, Pavanello Chiara, Calabresi Laura, Gianazza Elisabetta
  
  Abstract
  
  Genetic LCAT deficiency is a rare recessive autosomal disease due to loss-of-function mutations in the gene coding for the enzyme lecithin:cholesterol acyltransferase (LCAT). Homozygous carriers are characterized by corneal opacity, haemolytic anaemia and renal disease, which represent the first cause of morbidity and mortality in these subjects. Diagnostic and prognostic markers capable of early detecting declining kidney function in these subjects are not available, and the specific serum or urine proteomic signature of LCAT deficient carriers has never been assessed. Taking advantage of a proteomic approach, we performed 2-DE analysis of carriers' plasma and identified proteins present at different concentration in samples from homozygous carriers. Our data confirm the well-known alterations in the concentration of circulating apolipoproteins, with a statistically significant decrease of both apoA-I and apoA-II and a statistically significant increase of apoC-III. Furthermore, we observed increased level of alpha-1-antitrypsin, zinc-alpha-2-glycoprotein and retinol-binding protein 4, and reduced level of clusterin and haptoglobin. Interestingly, only beta but not alpha subunit of haptoglobin is significant reduced in homozygous subjects. Despite the limited sample size, our findings set the basis for assessing the identified protein in a larger population and for correlating their levels with clinical markers of renal function and anaemia. SIGNIFICANCE: This investigation defines the effects of LCAT deficiency on the level of the major plasma proteins in homozygous and heterozygous carriers. Increase for some proteins, with different function, together with a drop for haptoglobin, and specifically for haptoglobin beta chains, are reported for the first time as part of a coherent signature. We are glad to have the opportunity to report our findings on this subject, which is one of the main interests for our research group, when Journal of Proteomics celebrates its 10th anniversary. With its various sections devoted to different areas of research, this journal is a privileged forum for publishing proteomic investigations without restrictions either in sample type or in technical approach. It is as well a privileged forum for reviewing literature data on various topics related to proteomics investigation, as colleagues in our research group have done over the years; by the way, a good share of the reviewed papers were as well reports published in Journal of Proteomics itself. The journal also offers opportunities for focused surveys through thematic issues devoted to a variety of subjects, timely selected for their current relevance in research; it was an honour for colleagues in our group to recently act as editors of one of those. Out of this diverse experience, we express our appreciation for the endeavour of Journal of Proteomics in its first 10?years of life - and wish identical and possibly greater success for the time to come.
  
  Copyright © 2018 Elsevier B.V. All rights reserved.
  
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• Systematic Lab Knowledge Integration for Management of Lipid Excess in High-Risk Patients: Rationale and Design of the SKIM LEAN Project.
  Front Big Data

  2018 ;1():4. doi: 4.
  
  Pavanello Chiara, Parolini Marina, Alberti Antonia, Carenini Michele, Maino Paolo, Mombelli Giuliana, Pazzucconi Franco, Origgi Gianni, Orsi Federica, Trivella Maria Giovanna, Calabresi Laura, De Maria Renata
  
  Abstract
  
  SKIM LEAN aims at exploiting Electronic Health Records (EHRs) to integrate knowledge derived from routine laboratory tests with background analysis of clinical databases, for the identification and early referral to specialist care, where appropriate, of patients with hypercholesterolemia, who may be inadequately controlled according to their cardiovascular (CV) risk level. SKIM LEAN addresses gaps in care that may occur through the lack of coordination between primary and specialist care, incomplete adherence to clinical guidelines, or poor patient's compliance to the physician's prescriptions because of comorbidities or drug side effects. Key project objectives include: (1) improved health professionals' competence and patient empowerment through a two-tiered educational website for general practitioners (GPs) and patients, and (2) implementation of a hospital-community shared care pathway to increase the proportion of patients at high/very-high CV risk (Familial Hypercholesterolemia, previous CV events) who achieve target LDL cholesterol (LDL-C) levels. Thanks to a close collaboration between clinical and information technology partners, SKIM LEAN will fully exploit the value of big data deriving from EHRs, and filter such knowledge using clinically-derived algorithms to risk-stratify patients. Alerts for GPs will be generated with interpreted test results. GPs will be able to refer patients with uncontrolled LDL-C within the shared pathway to the lipid or secondary prevention outpatient clinics of NIG hospital. Metrics to verify the project achievements include web-site visits, the number of alerts generated, numbers of patients referred by GPs, the proportion of secondary prevention patients who achieve LDL-C 50% decrease from baseline.
  
  Copyright © 2018 Pavanello, Parolini, Alberti, Carenini, Maino, Mombelli, Pazzucconi, Origgi, Orsi, Trivella, Calabresi and De Maria.
  
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• Plasma PCSK9 levels and lipoprotein distribution are preserved in carriers of genetic HDL disorders.
  Biochim Biophys Acta Mol Cell Biol Lipids

  2018 Sep;1863(9):991-997. doi: 10.1016/j.bbalip.2018.05.015.
  
  Ruscica Massimiliano, Simonelli Sara, Botta Margherita, Ossoli Alice, Lupo Maria Giovanna, Magni Paolo, Corsini Alberto, Arca Marcello, Pisciotta Livia, Veglia Fabrizio, Franceschini Guido, Ferri Nicola, Calabresi Laura
  
  Abstract
  
  Proprotein convertase subtilisin/kexin 9 (PCSK9), a protein regulating the number of cell-surface LDL receptors (LDLR), circulates partially associated to plasma lipoproteins. How this interaction alters PCSK9 plasma levels is still unclear. In the present study, we took advantage of the availability of a large cohort of carriers of genetic HDL disorders to evaluate how HDL defects affect plasma PCSK9 levels and its distribution among lipoproteins. Plasma PCSK9 concentrations were determined by ELISA in carriers of mutations in LCAT, ABCA1, or APOAI genes, and lipoprotein distribution was analyzed by FPLC. Carriers of one or two mutations in the LCAT gene show plasma PCSK9 levels comparable to that of unaffected family controls (homozygotes, 159.4?ng/mL (124.9;243.3); heterozygotes, 180.3?ng/mL (127.6;251.5) and controls, 190.4?ng/mL (146.7;264.4); P for trend?=?0.33). Measurement of PCSK9 in plasma of subjects carrying mutations in ABCA1 or APOAI genes confirmed normal values. When fractionated by FPLC, PCSK9 peaked in a region between LDL and HDL in control subjects. In carriers of all HDL defects, lipoprotein profile shows a strong reduction of HDL, but the distribution of PCSK9 was superimposable to that of controls. In conclusion, the present study demonstrates that in genetically determined low HDL states plasma PCSK9 concentrations and lipoprotein distribution are preserved, thus suggesting that HDL may not be involved in PCSK9 transport in plasma.
  
  Copyright © 2018 Elsevier B.V. All rights reserved.
  
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• Complete and Partial Lecithin:Cholesterol Acyltransferase Deficiency Is Differentially Associated With Atherosclerosis.
  Circulation

  2018 Sep;138(10):1000-1007. doi: 10.1161/CIRCULATIONAHA.118.034706.
  
  Oldoni Federico, Baldassarre Damiano, Castelnuovo Samuela, Ossoli Alice, Amato Mauro, van Capelleveen Julian, Hovingh G Kees, De Groot Eric, Bochem Andrea, Simonelli Sara, Barbieri Simone, Veglia Fabrizio, Franceschini Guido, Kuivenhoven Jan Albert, Holleboom Adriaan G, Calabresi Laura
  
  Abstract
  
  BACKGROUND:
  Lecithin:cholesterol acyltransferase (LCAT) is the sole enzyme that esterifies cholesterol in plasma. Its role in the supposed protection from atherogenesis remains unclear because mutations in LCAT causing fish-eye disease (FED) or familial LCAT deficiency (FLD) have been reported to be associated with more or instead less carotid atherosclerosis, respectively. This discrepancy may be associated with the loss of cholesterol esterification on only apolipoprotein AI (FED) or on both apolipoprotein AI- and apolipoprotein B-containing lipoproteins (FLD), an aspect that has thus far not been investigated.
  
  METHODS:
  Seventy-four heterozygotes for LCAT mutations recruited from Italy and the Netherlands were assigned to FLD (n=33) or FED (n=41) groups and compared with 280 control subjects. Subclinical atherosclerosis was assessed with carotid intima-media thickness.
  
  RESULTS:
  Compared with control subjects, total cholesterol was lower by 16% (-32.9 mg/dL) and 7% (-14.9 mg/dL) and high-density lipoprotein cholesterol was lower by 29% (-16.7 mg/dL) and 36% (-20.7 mg/dL) in the FLD and FED groups, respectively. Subjects with FLD displayed a significant 18% lower low-density lipoprotein cholesterol compared with subjects with FED (101.9±35.0 versus 123.6±47.4 mg/dL; P=0.047) and control subjects (122.6±35.0 mg/dL; P=0.003). Remarkably, all 3 intima-media thickness parameters were lower in subjects with FLD compared with FED and control subjects (accounting for age, sex, body mass index, smoking, hypertension, family history of cardiovascular disease, and plasma lipids). After additional correction for nationality and ultrasonographic methods, average and maximum intima-media thickness remained significantly lower when subjects with FLD were compared with those with FED (0.59 versus 0.73 mm, P=0.003; and 0.87 versus 1.24 mm, P
  CONCLUSIONS:
  In this head-to-head comparison, FLD and FED mutations were shown to be associated with decreased and increased atherosclerosis, respectively. We propose that this discrepancy is related to the capacity of LCAT to generate cholesterol esters on apolipoprotein B-containing lipoproteins. Although this capacity is lost in FLD, it is unaffected in FED. These results are important when considering LCAT as a target to decrease atherosclerosis.
  
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• Dysfunctional HDL as a Therapeutic Target for Atherosclerosis Prevention.
  Curr Med Chem

  2019 ;26(9):1610-1630. doi: 10.2174/0929867325666180316115726.
  
  Ossoli Alice, Pavanello Chiara, Giorgio Eleonora, Calabresi Laura, Gomaraschi Monica
  
  Abstract
  
  Hypercholesterolemia is one of the main risk factors for the development of atherosclerosis. Among the various lipoprotein classes, however, high density lipoproteins (HDL) are inversely associated with the incidence of atherosclerosis, since they are able to exert a series of atheroprotective functions. The central role of HDL within the reverse cholesterol transport, their antioxidant and anti-inflammatory properties and their ability to preserve endothelial homeostasis are likely responsible for HDL-mediated atheroprotection. However, drugs that effectively raise HDL-C failed to result in a decreased incidence of cardiovascular event, suggesting that plasma levels of HDL-C and HDL function are not always related. Several evidences are showing that different pathologic conditions, especially those associated with an inflammatory response, can cause dramatic alterations of HDL protein and lipid cargo resulting in HDL dysfunction. Established and investigational drugs designed to affect lipid metabolism and to increase HDL-C are only partly effective in correcting HDL dysfunction.
  
  Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.
  
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• High Density Lipoproteins Inhibit Oxidative Stress-Induced Prostate Cancer Cell Proliferation.
  Sci Rep

  2018 Feb;8(1):2236. doi: 2236.
  
  Ruscica Massimiliano, Botta Margherita, Ferri Nicola, Giorgio Eleonora, Macchi Chiara, Franceschini Guido, Magni Paolo, Calabresi Laura, Gomaraschi Monica
  
  Abstract
  
  Recent evidence suggests that oxidative stress can play a role in the pathogenesis and the progression of prostate cancer (PCa). Reactive oxygen species (ROS) generation is higher in PCa cells compared to normal prostate epithelial cells and this increase is proportional to the aggressiveness of the phenotype. Since high density lipoproteins (HDL) are known to exert antioxidant activities, their ability to reduce ROS levels and the consequent impact on cell proliferation was tested in normal and PCa cell lines. HDL significantly reduced basal and HO-induced oxidative stress in normal, androgen receptor (AR)-positive and AR-null PCa cell lines. AR, scavenger receptor BI and ATP binding cassette G1 transporter were not involved. In addition, HDL completely blunted HO-induced increase of cell proliferation, through their capacity to prevent the HO-induced shift of cell cycle distribution from G0/G1 towards G2/M phase. Synthetic HDL, made of the two main components of plasma-derived HDL (apoA-I and phosphatidylcholine) and which are under clinical development as anti-atherosclerotic agents, retained the ability of HDL to inhibit ROS production in PCa cells. Collectively, HDL antioxidant activity limits cell proliferation induced by ROS in AR-positive and AR-null PCa cell lines, thus supporting a possible role of HDL against PCa progression.
  
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• Autosomal Recessive Hypercholesterolemia: Long-Term Cardiovascular Outcomes.
  J Am Coll Cardiol

  2018 Jan;71(3):279-288. doi: 10.1016/j.jacc.2017.11.028.
  
  D'Erasmo Laura, Minicocci Ilenia, Nicolucci Antonio, Pintus Paolo, Roeters Van Lennep Janine E, Masana Luis, Mata Pedro, Sánchez-Hernández Rosa Maria, Prieto-Matos Pablo, Real Josè T, Ascaso Juan F, Lafuente Eduardo Esteve, Pocovi Miguel, Fuentes Francisco J, Muntoni Sandro, Bertolini Stefano, Sirtori Cesare, Calabresi Laura, Pavanello Chiara, Averna Maurizio, Cefalu Angelo Baldassare, Noto Davide, Pacifico Adolfo Arturo, Pes Giovanni Mario, Harada-Shiba Mariko, Manzato Enzo, Zambon Sabina, Zambon Alberto, Vogt Anja, Scardapane Marco, Sjouke Barbara, Fellin Renato, Arca Marcello
  
  Abstract
  
  BACKGROUND:
  Autosomal recessive hypercholesterolemia (ARH) is a rare lipid disorder characterized by premature atherosclerotic cardiovascular disease (ASCVD). There are sparse data for clinical management and cardiovascular outcomes in ARH.
  
  OBJECTIVES:
  Evaluation of changes in lipid management, achievement of low-density lipoprotein cholesterol (LDL-C) goals and cardiovascular outcomes in ARH.
  
  METHODS:
  Published ARH cases were identified by electronic search. All corresponding authors and physicians known to treat these patients were asked to provide follow-up information, using a standardized protocol.
  
  RESULTS:
  We collected data for 52 patients (28 females, 24 males; 31.1 ± 17.1 years of age; baseline LDL-C: 571.9 ± 171.7 mg/dl). During a mean follow-up of 14.1 ± 7.3 years, there was a significant increase in the use of high-intensity statin and ezetimibe in combination with lipoprotein apheresis; in 6 patients, lomitapide was also added. Mean LDL-C achieved at nadir was 164.0 ± 85.1 mg/dl (-69.6% from baseline), with a better response in patients taking lomitapide (-88.3%). Overall, 23.1% of ARH patients reached LDL-C of 
  CONCLUSIONS:
  Despite intensive treatment, LDL-C in ARH patients remains far from targets, and this translates into a poor long-term cardiovascular prognosis. Our data highlight the importance of an early diagnosis and treatment and confirm the fact that an effective treatment protocol for ARH is still lacking.
  
  Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
  
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• Inhibitors of the PI3K/mTOR pathway prevent STAT5 phosphorylation in mutated cells through PP2A/CIP2A axis.
  Oncotarget

  2017 Nov;8(57):96710-96724. doi: 10.18632/oncotarget.18073.
  
  Bartalucci Niccolò, Calabresi Laura, Balliu Manjola, Martinelli Serena, Rossi Maria Caterina, Villeval Jean Luc, Annunziato Francesco, Guglielmelli Paola, Vannucchi Alessandro M
  
  Abstract
  
  Inhibition of the constitutively activated JAK/STAT pathway in JAK2V617F mutated cells by the JAK1/JAK2 inhibitor ruxolitinib resulted in clinical benefits in patients with myeloproliferative neoplasms. However, evidence of disease-modifying effects remains scanty; furthermore, some patients do not respond adequately to ruxolitinib, or have transient responses, thus novel treatment strategies are needed. Here we demonstrate that ruxolitinib causes incomplete inhibition of STAT5 in mutated cells due to persistence of phosphorylated serine residues of STAT5b, that conversely are targeted by PI3K and mTORC1 inhibitors. We found that PI3K/mTOR-dependent phosphorylation of STAT5b serine residues involves Protein Phosphatase 2A and its repressor CIP2A. The levels of CIP2A were found increased in cells harboring the JAK2V617F mutation, and we provide evidence of a correlation between clinical responses and the extent of CIP2A downregulation in myelofibrosis patients receiving the mTOR inhibitor RAD001 in a phase II clinical trial. To achieve maximal inhibition of STAT5 phosphorylation, we combined ruxolitinib with BKM120, a PI3K inhibitor, and RAD001, an mTOR inhibitor, obtaining improved efficacy in mutated cell lines, primary patients' cells, and knock-in mice. These findings contribute to understanding the effectiveness of PI3K/mTOR inhibitors in MPN and argue for the rationale to develop combination clinical trials.
  
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• CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles.
  Blood Cancer J

  2017 Dec;7(12):638. doi: 638.
  
  Zini Roberta, Guglielmelli Paola, Pietra Daniela, Rumi Elisa, Rossi Chiara, Rontauroli Sebastiano, Genovese Elena, Fanelli Tiziana, Calabresi Laura, Bianchi Elisa, Salati Simona, Cazzola Mario, Tagliafico Enrico, Vannucchi Alessandro M, Manfredini Rossella,
  
  Abstract
  
  Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50-70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20-30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.
  
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• ABCA1 and HDL are required to modulate smooth muscle cells phenotypic switch after cholesterol loading.
  Atherosclerosis

  2017 Nov;266():8-15. doi: 10.1016/j.atherosclerosis.2017.09.012.
  
  Castiglioni Silvia, Monti Matteo, Arnaboldi Lorenzo, Canavesi Monica, Ainis Buscherini Giuditta, Calabresi Laura, Corsini Alberto, Bellosta Stefano
  
  Abstract
  
  BACKGROUND AND AIMS:
  Cholesterol-loaded smooth muscle cells (SMCs) modify their phenotypic behavior becoming foam cells. To characterize the role of ABCA1 and HDL in this process, we evaluated HDL effects on cholesterol-induced phenotypic changes in SMCs expressing or not ABCA1.
  
  METHODS:
  SMCs, isolated from the aortae of wild-type (WT) and Abca1 knock-out (KO) mice, were cholesterol-loaded using a "water-soluble cholesterol''.
  
  RESULTS:
  Cholesterol loading downregulates the expression of Acta2 and calponin (SMC markers), and increases the expression of Mac-2, CD11b and MHCII (inflammation-related genes and surface antigens) and Abca1, Abcg1. HDL normalizes SMC marker expression and reduces the expression of inflammation-related genes/proteins in WT cells, an effect not observed with free apoA-I. The effect of HDL is almost lost in Abca1 KO cells, as well as when Abca1 is silenced in WT SMC. HDL does not differently affect cholesterol downloading in WT or KO cells and stimulates phospholipids removal in WT cells. Similarly, the expression of myocardin and its modulators, such as miR-143/145, is reduced by cholesterol loading in WT and Abca1 KO SMCs; HDL normalizes their levels in WT cells but not in KO cells. On the contrary, cholesterol loading induces Klf4 expression while HDL restores Klf4 to basal levels in WT cells, but again this effect is not observed in KO cells.
  
  CONCLUSIONS:
  Our results indicate that HDL, by interacting with ABCA1, modulates the miR143/145-myocardin axis and prevents the cholesterol-induced gene expression modification in SMCs regardless of its cholesterol unloading capacity.
  
  Copyright © 2017 Elsevier B.V. All rights reserved.
  
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• Plasma cholesterol homeostasis, HDL remodeling and function during the acute phase reaction.
  J Lipid Res

  2017 Oct;58(10):2051-2060. doi: 10.1194/jlr.P076463.
  
  Zimetti Francesca, De Vuono Stefano, Gomaraschi Monica, Adorni Maria Pia, Favari Elda, Ronda Nicoletta, Ricci Maria Anastasia, Veglia Fabrizio, Calabresi Laura, Lupattelli Graziana
  
  Abstract
  
  Acute phase reaction (APR) is a systemic inflammation triggered by several conditions associated with lipid profile alterations. We evaluated whether APR also associates with changes in cholesterol synthesis and absorption, HDL structure, composition, and cholesterol efflux capacity (CEC). We analyzed 59 subjects with APR related to infections, oncologic causes, or autoimmune diseases and 39 controls. We detected no difference in markers of cholesterol synthesis and absorption. Conversely, a significant reduction of LpA-I- and LpAI:AII-containing HDL (-28% and -44.8%, respectively) and of medium-sized HDL (-10.5%) occurred in APR. Total HDL CEC was impaired in APR subjects (-18%). Evaluating specific CEC pathways, we found significant reductions in CEC by aqueous diffusion and by the transporters scavenger receptor B-I and ABCG1 (-25.5, -41.1 and -30.4%, respectively). ABCA1-mediated CEC was not affected. Analyses adjusted for age and gender provided similar results. In addition, correcting for HDL-cholesterol (HDL-C) levels, the differences in aqueous diffusion total and ABCG1-CEC remained significant. APR subjects displayed higher levels of HDL serum amyloid A (+20-folds; = 0.003). In conclusion, APR does not associate with cholesterol synthesis and absorption changes but with alterations of HDL composition and a marked impairment of HDL CEC, partly independent of HDL-C serum level reduction.
  
  Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.
  
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• Nutraceutical approaches to metabolic syndrome.
  Ann Med

  2017 Dec;49(8):678-697. doi: 10.1080/07853890.2017.1366042.
  
  Sirtori Cesare R, Pavanello Chiara, Calabresi Laura, Ruscica Massimiliano
  
  Abstract
  
  Metabolic Syndrome (MetS), affecting at least 30% of adults in the Western World, is characterized by three out of five variables, from high triglycerides, to elevated waist circumference and blood pressure. MetS is not characterized by elevated cholesterolemia, but is rather the consequence of a complex interaction of factors generally leading to increased insulin resistance. Drug treatments are of difficult handling, whereas well-characterized nutraceuticals may offer an effective alternative. Among these, functional foods, e.g. plant proteins, have been shown to improve insulin resistance and reduce triglyceride secretion. Pro- and pre-biotics, that are able to modify intestinal microbiome, reduce absorption of specific nutrients and improve the metabolic handling of energy-rich foods. Finally, specific nutraceuticals have proven to be of benefit, in particular, red-yeast rice, berberine, curcumin as well as vitamin D. All these can improve lipid handling by the liver as well as ameliorate insulin resistance. While lifestyle approaches, such as with the Mediterranean diet, may prove to be too complex for the single patient, better knowledge of selected nutraceuticals and more appropriate formulations leading to improved bioavailability will certainly widen the use of these agents, already in large use for the management of these very frequent patient groups. Key messages Functional foods, e.g. plant proteins, improve insulin resistance. Pro- and pre-biotics improve the metabolic handling of energy-rich foods. Nutraceutical can offer a significant help in handling MetS patients being part of lifestyle recommendations.
  
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• Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.
  J Pharmacol Exp Ther

  2017 Aug;362(2):306-318. doi: 10.1124/jpet.117.240457.
  
  Freeman Lita A, Demosky Stephen J, Konaklieva Monika, Kuskovsky Rostislav, Aponte Angel, Ossoli Alice F, Gordon Scott M, Koby Ross F, Manthei Kelly A, Shen Min, Vaisman Boris L, Shamburek Robert D, Jadhav Ajit, Calabresi Laura, Gucek Marjan, Tesmer John J G, Levine Rodney L, Remaley Alan T
  
  Abstract
  
  Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (
  U.S. Government work not protected by U.S. copyright.
  
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• Efficacy of Lomitapide in the Treatment of Familial Homozygous Hypercholesterolemia: Results of a Real-World Clinical Experience in Italy.
  Adv Ther

  2017 May;34(5):1200-1210. doi: 10.1007/s12325-017-0531-x.
  
  D'Erasmo Laura, Cefalù Angelo Baldassare, Noto Davide, Giammanco Antonina, Averna Maurizio, Pintus Paolo, Medde Paolo, Vigna Giovanni Battista, Sirtori Cesare, Calabresi Laura, Pavanello Chiara, Bucci Marco, Sabbà Carlo, Suppressa Patrizia, Natale Francesco, Calabrò Paolo, Sampietro Tiziana, Bigazzi Federico, Sbrana Francesco, Bonomo Katia, Sileo Fulvio, Arca Marcello
  
  Abstract
  
  INTRODUCTION:
  Homozygous familial hypercholesterolaemia (HoFH) is a rare form of inherited dyslipidemia resistant to conventional cholesterol-lowering medications so that lipoprotein apheresis (LA) is usually required. Lomitapide has been approved for the treatment of HoFH. The aim of this study was to evaluate the benefits of lomitapide in HoFH patients followed with the usual clinical care.
  
  METHODS:
  Clinical and biochemical data were retrospectively collected in 15 HoFH patients (10 with mutations in the LDLR gene and 5 in the LDLRAP1 gene) treated for at least 6 months with lomitapide in addition to lipid-lowering therapies (LLT) in different Lipid Clinics across Italy.
  
  RESULTS:
  The mean follow-up period was 32.3 ± 29.7 months. During background therapies, HoFH patients showed a mean LDL-C level of 426.0 ± 204.0 mg/dl. The addition of lomitapide at the average dosage of 19 mg/day lowered LDL-C levels by 68.2 ± 24.8%. At their last visit, 60% of patients showed LDL-C 5× ULN or had to stop treatment due to side effects. A subset of patients was evaluated by liver ultrasound and fibroscan (n = 5) or nuclear magnetic resonance with spectroscopy (MRS) (n = 1) not showing clinical evidence of liver damage.
  
  CONCLUSION:
  In this real-world experience, lomitapide was confirmed to be a very powerful cholesterol-lowering agent in HoFH showing a good safety profile.
  
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• Depletion in LpA-I:A-II particles enhances HDL-mediated endothelial protection in familial LCAT deficiency.
  J Lipid Res

  2017 May;58(5):994-1001. doi: 10.1194/jlr.P072371.
  
  Gomaraschi Monica, Ossoli Alice, Castelnuovo Samuela, Simonelli Sara, Pavanello Chiara, Balzarotti Gloria, Arca Marcello, Di Costanzo Alessia, Sampietro Tiziana, Vaudo Gaetano, Baldassarre Damiano, Veglia Fabrizio, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  The aim of this study was to evaluate the vasoprotective effects of HDL isolated from carriers of LCAT deficiency, which are characterized by a selective depletion of LpA-I:A-II particles and predominance of pre? migrating HDL. HDLs were isolated from LCAT-deficient carriers and tested in vitro for their capacity to promote NO production and to inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in cultured endothelial cells. HDLs from carriers were more effective than control HDLs in promoting eNOS activation with a gene-dose-dependent effect ( = 0.048). As a consequence, NO production induced by HDL from carriers was significantly higher than that promoted by control HDL (1.63 ± 0.24-fold vs. 1.34 ± 0.07-fold, = 0.031). HDLs from carriers were also more effective than control HDLs in inhibiting the expression of VCAM-1 (homozygotes, 65.0 ± 8.6%; heterozygotes, 53.1 ± 7.2%; controls, 44.4 ± 4.1%; = 0.0003). The increased efficiency of carrier HDL was likely due to the depletion in LpA-I:A-II particles. The in vitro findings might explain why carriers of LCAT deficiency showed flow-mediated vasodilation and plasma-soluble cell adhesion molecule concentrations comparable to controls, despite low HDL-cholesterol levels. These results indicate that selective depletion of apoA-II-containing HDL, as observed in carriers of LCAT deficiency, leads to an increased capacity of HDL to stimulate endothelial NO production, suggesting that changes in HDL apolipoprotein composition may be the target of therapeutic interventions designed to improve HDL functionality.
  
  Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.
  
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• [Lecithin:Cholesterol Acyltransferase Deficiency, from genes to therapy].
  G Ital Nefrol

  ;33(S68):. doi: gin/33.S68.3.
  
  Lucca Fabio, Ossoli Alice, Boscutti Giuliano, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  LCAT synthesizes most of the plasma cholesteryl esters, and plays a major role in HDL metabolism. Mutations in the LCAT gene cause two syndromes, familial LCAT deficiency (FLD) and fish-eye disease (FED), both characterized by severe alterations in plasma lipoprotein profile. Renal disease is the major cause of morbidity and mortality in FLD cases, but an established therapy is not currently available. The present therapy of LCAT deficiency is mainly aimed at correcting the dyslipidemia associated with the disease and at delaying evolution of chronic nephropathy. LCAT deficiency represents a candidate disease for enzyme replacement therapy. In vitro and in vivo studies proved the efficacy of recombinant human LCAT (rhLCAT) in correcting dyslipidemia, and rhLCAT is presently under development.
  
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• Persistent changes in lipoprotein lipids after a single infusion of ascending doses of MDCO-216 (apoA-IMilano/POPC) in healthy volunteers and stable coronary artery disease patients.
  Atherosclerosis

  2016 Dec;255():17-24. doi: 10.1016/j.atherosclerosis.2016.10.042.
  
  Kempen Herman J, Gomaraschi Monica, Simonelli Sara, Calabresi Laura, Moerland Matthijs, Otvos James, Jeyarajah Elias, Kallend David, Wijngaard Peter L J
  
  Abstract
  
  BACKGROUND AND AIMS:
  Effects of single ascending doses of MDCO-216 on plasma lipid and lipoprotein levels were assessed in human healthy volunteers and in patients with stable coronary artery disease (CAD).
  
  METHODS:
  MDCO-216 was infused at a single dose of 5, 10, 20, 30 or 40 mg/kg over 2 h and blood was collected at 2, 4, 8, 24, 48, 168 and 720 h after start of infusion (ASOI). Lipoprotein lipids were assessed by FLPC and by 1H NMR.
  
  RESULTS:
  Plasma concentrations of free cholesterol (FC) displayed a rapid and dose-dependent rise, peaking at 8 h, but remaining above baseline until 48 h ASOI, whereas levels of esterified cholesterol (CE) increased at lower doses but not at higher doses, and even decreased below baseline at the highest dose. Plasma cholesterol esterification rate (CER) decreased with a first nadir between 4 and 8 h and a second nadir at 48 h ASOI. Taken over all subjects receiving MDCO-216, the increase in FC at 8 h correlated inversely with the drop in CER at 4 h but positively with the increase in basal and scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacities at 2 h ASOI. Upon FPLC analysis, FC was found to increase first in high density lipoproteins (HDL) and very low density lipoproteins (VLDL) and later (at 48 or 168 h ASOI) in low density lipoproteins (LDL). CE initially decreased in LDL and HDL but after 24 h started to increase in VLDL and LDL whereas HDL-CE was still below baseline at 48 h. Phospholipids (PL) showed the same pattern as FC. Triglycerides (TG) also rose rapidly, most prominently in VLDL, but also in LDL and HDL. Apolipoprotein E (Apo-E) in VLDL increased at 4-8 h but returned to baseline at 24 h ASOI. 1H NMR analysis showed a rapid and dose-dependent increase in HDL particle size, peaking at 2 h and returning to baseline at 24 h, and a small increase in HDL particle concentration. After infusion of the 40 mg/kg dose, LDL and VLDL-particles also increased in number and size.
  
  CONCLUSIONS:
  A single administration of MDCO-216 caused rapid changes in lipid levels and lipoprotein composition, some of which persisted for at least 7 days.
  
  Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
  
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• Effect of soy on metabolic syndrome and cardiovascular risk factors: a randomized controlled trial.
  Eur J Nutr

  2018 Mar;57(2):499-511. doi: 10.1007/s00394-016-1333-7.
  
  Ruscica Massimiliano, Pavanello Chiara, Gandini Sara, Gomaraschi Monica, Vitali Cecilia, Macchi Chiara, Morlotti Beatrice, Aiello Gilda, Bosisio Raffaella, Calabresi Laura, Arnoldi Anna, Sirtori Cesare R, Magni Paolo
  
  Abstract
  
  BACKGROUND:
  Cardiovascular diseases are currently the commonest cause of death worldwide. Different strategies for their primary prevention have been planned, taking into account the main known risk factors, which include an atherogenic lipid profile and visceral fat excess.
  
  METHODS:
  The study was designed as a randomized, parallel, single-center study with a nutritional intervention duration of 12 weeks. Whole soy foods corresponding to 30 g/day soy protein were given in substitution of animal foods containing the same protein amount.
  
  RESULTS:
  Soy nutritional intervention resulted in a reduction in the number of MetS features in 13/26 subjects. Moreover, in the soy group we observed a significant improvement of median percentage changes for body weight (-1.5 %) and BMI (-1.5 %), as well as for atherogenic lipid markers, namely TC (-4.85 %), LDL-C (-5.25 %), non-HDL-C (-7.14 %) and apoB (-14.8 %). Since the majority of the studied variables were strongly correlated, three factors were identified which explained the majority (52 %) of the total variance in the whole data set. Among them, factor 1, which loaded lipid and adipose variables, explained the 22 % of total variance, showing a statistically significant difference between treatment arms (p = 0.002).
  
  CONCLUSIONS:
  The inclusion of whole soy foods (corresponding to 30 g/day protein) in a lipid-lowering diet significantly improved a relevant set of biomarkers associated with cardiovascular risk.
  
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• High-Density Lipoprotein, Lecithin: Cholesterol Acyltransferase, and Atherosclerosis.
  Endocrinol Metab (Seoul)

  2016 Jun;31(2):223-9. doi: 10.3803/EnM.2016.31.2.223.
  
  Ossoli Alice, Pavanello Chiara, Calabresi Laura
  
  Abstract
  
  Epidemiological data clearly show the existence of a strong inverse correlation between plasma high-density lipoprotein cholesterol (HDL-C) concentrations and the incidence of coronary heart disease. This relation is explained by a number of atheroprotective properties of HDL, first of all the ability to promote macrophage cholesterol transport. HDL are highly heterogeneous and are continuously remodeled in plasma thanks to the action of a number of proteins and enzymes. Among them, lecithin:cholesterol acyltransferase (LCAT) plays a crucial role, being the only enzyme able to esterify cholesterol within lipoproteins. LCAT is synthetized by the liver and it has been thought to play a major role in reverse cholesterol transport and in atheroprotection. However, data from animal studies, as well as human studies, have shown contradictory results. Increased LCAT concentrations are associated with increased HDL-C levels but not necessarily with atheroprotection. On the other side, decreased LCAT concentration and activity are associated with decreased HDL-C levels but not with increased atherosclerosis. These contradictory results confirm that HDL-C levels per se do not represent the functionality of the HDL system.
  
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• An Essential Role for Liver ER? in Coupling Hepatic Metabolism to the Reproductive Cycle.
  Cell Rep

  2016 Apr;15(2):360-71. doi: 10.1016/j.celrep.2016.03.019.
  
  Della Torre Sara, Mitro Nico, Fontana Roberta, Gomaraschi Monica, Favari Elda, Recordati Camilla, Lolli Federica, Quagliarini Fabiana, Meda Clara, Ohlsson Claes, Crestani Maurizio, Uhlenhaut Nina Henriette, Calabresi Laura, Maggi Adriana
  
  Abstract
  
  Lipoprotein synthesis is controlled by estrogens, but the exact mechanisms underpinning this regulation and the role of the hepatic estrogen receptor ? (ER?) in cholesterol physiology are unclear. Utilizing a mouse model involving selective ablation of ER? in the liver, we demonstrate that hepatic ER? couples lipid metabolism to the reproductive cycle. We show that this receptor regulates the synthesis of cholesterol transport proteins, enzymes for lipoprotein remodeling, and receptors for cholesterol uptake. Additionally, ER? is indispensable during proestrus for the generation of high-density lipoproteins efficient in eliciting cholesterol efflux from macrophages. We propose that a specific interaction with liver X receptor ? (LXR?) mediates the broad effects of ER? on the hepatic lipid metabolism.
  
  Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
  
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• Lipoprotein X Causes Renal Disease in LCAT Deficiency.
  PLoS One

  2016 ;11(2):e0150083. doi: e0150083.
  
  Ossoli Alice, Neufeld Edward B, Thacker Seth G, Vaisman Boris, Pryor Milton, Freeman Lita A, Brantner Christine A, Baranova Irina, Francone Nicolás O, Demosky Stephen J, Vitali Cecilia, Locatelli Monica, Abbate Mauro, Zoja Carlamaria, Franceschini Guido, Calabresi Laura, Remaley Alan T
  
  Abstract
  
  Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
  
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• Protective Effects of HDL Against Ischemia/Reperfusion Injury.
  Front Pharmacol

  2016 ;7():2. doi: 2.
  
  Gomaraschi Monica, Calabresi Laura, Franceschini Guido
  
  Abstract
  
  Several lines of evidence suggest that, besides being a strong independent predictor of the occurrence of primary coronary events, a low plasma high density lipoprotein (HDL) cholesterol level is also associated with short- and long-term unfavorable prognosis in patients, who have recovered from a myocardial infarction, suggesting a direct detrimental effect of low HDL on post-ischemic myocardial function. Experiments performed in ex vivo and in vivo models of myocardial ischemia/reperfusion (I/R) injury have clearly shown that HDL are able to preserve cardiac function when given before ischemia or at reperfusion; the protective effects of HDL against I/R injury have been also confirmed in other tissues and organs, as brain and hind limb. HDL were shown to act on coronary endothelial cells, by limiting the increase of endothelium permeability and promoting vasodilation and neoangiogenesis, on white blood cells, by reducing their infiltration into the ischemic tissue and the release of pro-inflammatory and matrix-degrading molecules, and on cardiomyocytes, by preventing the activation of the apoptotic cascade. Synthetic HDL retains the cardioprotective activity of plasma-derived HDL and may become a useful adjunctive therapy to improve clinical outcomes in patients with acute coronary syndromes or undergoing coronary procedures.
  
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• A complex phenotype in a child with familial HDL deficiency due to a novel frameshift mutation in APOA1 gene (apoA-IGuastalla).
  J Clin Lipidol

  2015 ;9(6):837-846. doi: 10.1016/j.jacl.2015.09.001.
  
  Pisciotta Livia, Vitali Cecilia, Favari Elda, Fossa Paola, Adorni Maria Pia, Leone Daniela, Artom Nathan, Fresa Raffaele, Calabresi Laura, Calandra Sebastiano, Bertolini Stefano
  
  Abstract
  
  BACKGROUND:
  We describe a kindred with high-density lipoprotein (HDL) deficiency due to APOA1 gene mutation in which comorbidities affected the phenotypic expression of the disorder.
  
  METHODS:
  An overweight boy with hypertriglyceridemia (HTG) and HDL deficiency (HDL cholesterol 0.39 mmol/L, apoA-I 40 mg/dL) was investigated. We sequenced the candidate genes for HTG (LPL, APOC2, APOA5, GPIHBP1, LMF1) and HDL deficiency (LCAT, ABCA1 and APOA1), analyzed HDL subpopulations, measured cholesterol efflux capacity (CEC) of sera and constructed a model of the mutant apoA-I.
  
  RESULTS:
  No mutations in HTG-related genes, ABCA1 and LCAT were found. APOA1 sequence showed that the proband, his mother and maternal grandfather were heterozygous of a novel frameshift mutation (c.546_547delGC), which generated a truncated protein (p.[L159Afs*20]) containing 177 amino acids with an abnormal C-terminal tail of 19 amino acids. Trace amounts of this protein were detectable in plasma. Mutation carriers had reduced levels of LpA-I, pre?-HDL and large HDL and no detectable HDL-2 in their plasma; their sera had a reduced CEC specifically the ABCA1-mediated CEC. Metabolic syndrome in the proband explains the extremely low HDL cholesterol level (0.31 mmol/L), which was half of that found in the other carriers. The proband's mother and grandfather, both presenting low plasma low-density lipoprotein cholesterol, were carriers of the ?-thalassemic trait, a condition known to be associated with a reduced low-density lipoprotein cholesterol and a reduced prevalence of cardiovascular disease. This trait might have delayed the development of atherosclerosis related to HDL deficiency.
  
  CONCLUSIONS:
  In these heterozygotes for apoA-I truncation, the metabolic syndrome has deleterious effect on HDL system, whereas ?-thalassemia trait may delay the onset of cardiovascular disease.
  
  Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.
  
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• Role of LCAT in Atherosclerosis.
  J Atheroscler Thromb

  2016 ;23(2):119-27. doi: 10.5551/jat.32854.
  
  Ossoli Alice, Simonelli Sara, Vitali Cecilia, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Lecithin:cholesterol acyltransferase (LCAT) is the only enzyme capable of esterifying cholesterol in plasma, thus determining the maturation of high-density lipoproteins. Because it maintains an unesterified cholesterol gradient between peripheral cells and extracellular acceptors, for a long time, LCAT has been considered as a key enzyme in reverse cholesterol transport. However, despite the fact that it has been more than 50 years since the identification of LCAT, the role of this enzyme in the pathogenesis of atherosclerosis is still debated. A number of studies have been conducted in different animal models, with contradictory results. Studies in humans, in particular in the general population, in subjects at high cardiovascular risk, and in carriers of genetic LCAT deficiency in an excellent model to evaluate the correlation between the reduction of LCAT activity and atherosclerosis also gave conflicting results. This review provides a comprehensive overview of the controversial findings obtained in animals and humans, strengthening the necessity of further investigation to establish how LCAT could be regulated in a promising therapeutic strategy to reduce cardiovascular risk.
  
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• Plasma-derived and synthetic high-density lipoprotein inhibit tissue factor in endothelial cells and monocytes.
  Biochem J

  2016 Jan;473(2):211-9. doi: 10.1042/BJ20151000.
  
  Ossoli Alice, Remaley Alan T, Vaisman Boris, Calabresi Laura, Gomaraschi Monica
  
  Abstract
  
  HDL (high-density lipoproteins) exert anti-thrombotic activities by preventing platelet adhesion and activation and by stimulating the protein C pathway and fibrinolysis. The aim of the present study was to assess the effect of plasma-derived and synthetic HDL on endothelial and monocyte expression of TF (tissue factor), the primary initiator of coagulation. HDL inhibited TF expression and activity in stimulated endothelial cells and monocytes in a dose-dependent way. Synthetic HDL fully retain the ability to inhibit TF expression in a dose-dependent manner; lipid-free apoA-I (apolipoprotein A-I) was not effective and neither was sphingosine 1-phosphate involved. HDL-mediated TF inhibition was due to a modulation of cellular cholesterol content through the interaction with SR-BI (scavenger receptor BI); downstream, HDL inhibited the activation of p38 MAPK (mitogen-activated protein kinase) and the repression of the PI3K (phosphoinositide 3-kinase) pathway responsible for TF expression. In vivo, human apoA-I-transgenic mice displayed a reduced aortic TF expression compared with wild-type animals and TF plasma levels were increased in subjects with low HDL-C (HDL-cholesterol) levels compared with high HDL-C subjects. Thus the anti-thrombotic activity of HDL could also be mediated by the inhibition of TF expression and activity in endothelial cells and monocytes; synthetic HDL retain the inhibitory activity of plasma-derived HDL, supporting the hypothesis that synthetic HDL infusion may be beneficial in the setting of acute coronary syndrome.
  
  © 2016 Authors; published by Portland Press Limited.
  
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• Cholesterol trafficking-related serum lipoprotein functions in children with cholesteryl ester storage disease.
  Atherosclerosis

  2015 Oct;242(2):443-9. doi: 10.1016/j.atherosclerosis.2015.08.007.
  
  Zimetti Francesca, Favari Elda, Cagliero Paola, Adorni Maria Pia, Ronda Nicoletta, Bonardi Renato, Gomaraschi Monica, Calabresi Laura, Bernini Franco, Guardamagna Ornella
  
  Abstract
  
  OBJECTIVE:
  Serum lipoproteins influence cell cholesterol content by delivering and removing cholesterol to/from cells, functions mainly exerted by LDL and HDL, respectively. Especially in the case of HDL, structure and composition are crucial for function, beyond serum levels. Cholesteryl ester storage disease (CESD) is caused by LIPA gene mutations and reduced activity of lysosomal acid lipase (LAL), the enzyme responsible for hydrolysis of cholesteryl esters and TG. CESD patients typically present dyslipidaemia, liver damage and premature atherosclerosis. The objective of this work was to evaluate serum HDL cholesterol efflux capacity (CEC) and serum cholesterol loading capacity (CLC) in CESD pediatric patients and to study lipoprotein qualitative modifications.
  
  METHODS:
  HDL CEC was evaluated by radioisotopic techniques, serum CLC was measured by a fluorimetric assay, HDL subclasses were determined by two-dimensional electrophoresis.
  
  RESULTS:
  CESD patients (n = 3) displayed on average increased LDL cholesterol (+163%; p = 0.019), TG (+203; p = 0.012), phospholipids (+40%; p = 0.024) and lower HDL cholesterol (-57%; p = 0.012) compared to controls (n = 9). CESD HDL CEC was impaired both as a whole (average reduction of 26%; p
  CONCLUSION:
  These new data demonstrate that the pro-atherogenic modifications of serum include disturbances in lipoprotein functions involved in cell cholesterol homeostasis occurring from very early age in CESD patients.
  
  Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
  
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• Vascular alterations in apolipoprotein A-I amyloidosis (Leu75Pro). A case-control study.
  Amyloid

  2015 ;22(3):187-93. doi: 10.3109/13506129.2015.1066771.
  
  Muiesan Maria Lorenza, Salvetti Massimo, Paini Anna, Agabiti Rosei Claudia, Rubagotti Giulia, Negrinelli Alessandro, Gregorini Gina, Cancarini Giovanni, Calabresi Laura, Franceschini Guido, Obici Laura, Perlini Stefano, Merlini Giampaolo, Agabiti Rosei Enrico
  
  Abstract
  
  BACKGROUND:
  Among hereditary amyloidoses, apolipoprotein A-I (APO A-I) amyloidosis (Leu75Pro) is a rare, autosomal dominant condition in which renal, hepatic, and testicular involvement has been demonstrated.
  
  OBJECTIVE:
  To investigate vascular structural as well as functional alterations.
  
  METHODS:
  In 131 carriers of the amyloidogenic Leu75Pro APO A-I mutation (mean age 52?+?16 years, 56 women) and in 131 subjects matched for age, sex, body mass index and clinic blood pressure (BP), arterial stiffness (pulse wave velocity, PWV) and carotid intima-media thickness (IMT) were measured.
  
  RESULTS:
  By definition no differences for age, sex, body mass index, and BP were observed. Meanmax IMT (Mmax-IMT) in the common (CC), bifurcation (BIF) and internal (ICA) carotid artery were comparable in the two groups. After adjustment for high-density lipoprotein cholesterol and renal function differences between the two groups, a lower meanmax-IMT was observed in APO A-I Leu75Pro mutation carriers than in controls (CC Mmax-IMT 0.87?±?0.21 versus 0.93?±?0.2?mm, p?=?0.07; BIF Mmax-IMT 1.19?±?0.48 versus 1.36?±?0.46?mm, p?=?0.025; ICA Mmax-IMT 0.9?±?0.37 versus 1.02?±?0.35?mm, p?=?0.028). On the other hand, aortic stiffness was significantly greater in patients with APO A-I amyloidosis than controls (PWV 11.5?±?2.9 and 10.7?±?2.3?m/s, p?0.05), even after adjusting for confounders.
  
  CONCLUSIONS:
  In carriers of the amyloidogenic Leu75Pro APO A-I mutation, a significant increase in arterial stiffness is observed; on the contrary, carotid artery IMT is comparable to that of control subjects. These results may add significant information to the clinical features of this rare genetic disorder.
  
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• Fenofibrate and extended-release niacin improve the endothelial protective effects of HDL in patients with metabolic syndrome.
  Vascul Pharmacol

  2015 Nov;74():80-86. doi: 10.1016/j.vph.2015.06.014.
  
  Gomaraschi Monica, Ossoli Alice, Adorni Maria Pia, Damonte Elisabetta, Niesor Eric, Veglia Fabrizio, Franceschini Guido, Benghozi Renee, Calabresi Laura
  
  Abstract
  
  BACKGROUND:
  Fibrates and niacin are at present the most effective therapies to increase plasma levels of high density lipoprotein-cholesterol (HDL-C); to date, limited data are available on their effects on HDL protective functions.
  
  METHODS AND RESULTS:
  Within a multicenter, randomized, open-label, cross-over study, 37 patients with metabolic syndrome received 6weeks' treatment with fenofibrate or extended-release niacin (ER niacin), with a 4weeks' wash-out period. HDL ability to preserve endothelial cell homeostasis was assessed by incubating cultured endothelial cells with HDL isolated from patients at baseline and after each treatment. HDL isolated from patients at baseline were as effective as control HDL in inhibiting vascular cell adhesion molecule-1 (VCAM-1) expression, but less efficient in promoting endothelial cell nitric oxide (NO) release. Both fenofibrate and ER niacin increased HDL ability to inhibit TNF?-induced VCAM-1 expression (+7% and +11%, respectively). Fenofibrate and ER niacin also improved the impaired HDL ability to induce the expression of endothelial nitric oxide synthase and NO production (+10% and +8%, respectively). Interestingly, HDL isolated after treatment showed an ability to promote endothelial NO release similar to HDL isolated from controls. No differences were observed between the two drugs. With both drugs, HDL function was improved irrespective of baseline HDL-C levels.
  
  CONCLUSION:
  Treatment with fenofibrate or ER niacin in patients with metabolic syndrome not only increased HDL-C levels but also improved the endothelial protective effects of HDL.
  
  Copyright © 2015 Elsevier Inc. All rights reserved.
  
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• Gender-related lipid and/or lipoprotein responses to statins in subjects in primary and secondary prevention.
  J Clin Lipidol

  2015 ;9(2):226-33. doi: 10.1016/j.jacl.2014.12.003.
  
  Mombelli Giuliana, Bosisio Raffaella, Calabresi Laura, Magni Paolo, Pavanello Chiara, Pazzucconi Franco, Sirtori Cesare R
  
  Abstract
  
  BACKGROUND:
  Cardiovascular risk in men rises around the fourth decade of life, whereas women appear to be protected by sex hormones until menopause. This, in turn, tends to negatively affect the lipid profile. Since the 1980s, the incidence of cardiovascular diseases has been reported to progressively decline in men, but it has persisted almost unchanged in women. Major clinical trials on statins have been mostly conducted in men and have fostered the introduction of these agents into clinical practice worldwide. However, only few reports have examined a possible differential activity of statins in the 2 genders, providing in some cases opposite findings.
  
  OBJECTIVE:
  To evaluate gender-related differences in statin responses.
  
  METHODS:
  Variations of lipid profile after 1-year of treatment with different statins in 337 dyslipidemic patients (171 men and 166 women).
  
  RESULTS:
  In this series of patients, a significantly attenuated reduction of total cholesterol and low-density lipoprotein cholesterol in women vs men on drug treatment was noted after adjustment for dose and statin power (low-density lipoprotein cholesterol: -28.5 ± 11.8% in men vs -22.7 ± 11.8% in women; P 
  CONCLUSIONS:
  The present study indicates that statin treatment has a reduced effectiveness in improving the plasma lipid profile in dyslipidemic women vs men. Whether such gender-related differences may have an impact on clinical outcomes remains to be elucidated.
  
  Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.
  
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• Effects of established hypolipidemic drugs on HDL concentration, subclass distribution, and function.
  Handb Exp Pharmacol

  2015 ;224():593-615. doi: 10.1007/978-3-319-09665-0_19.
  
  Gomaraschi Monica, Adorni Maria Pia, Banach Maciej, Bernini Franco, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  The knowledge of an inverse relationship between plasma high-density lipoprotein cholesterol (HDL-C) concentrations and rates of cardiovascular disease has led to the concept that increasing plasma HDL-C levels would be protective against cardiovascular events. Therapeutic interventions presently available to correct the plasma lipid profile have not been designed to specifically act on HDL, but have modest to moderate effects on plasma HDL-C concentrations. Statins, the first-line lipid-lowering drug therapy in primary and secondary cardiovascular prevention, have quite modest effects on plasma HDL-C concentrations (2-10%). Fibrates, primarily used to reduce plasma triglyceride levels, also moderately increase HDL-C levels (5-15%). Niacin is the most potent available drug in increasing HDL-C levels (up to 30%), but its use is limited by side effects, especially flushing.The present chapter reviews the effects of established hypolipidemic drugs (statins, fibrates, and niacin) on plasma HDL-C levels and HDL subclass distribution, and on HDL functions, including cholesterol efflux capacity, endothelial protection, and antioxidant properties.
  
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• Structure of HDL: particle subclasses and molecular components.
  Handb Exp Pharmacol

  2015 ;224():3-51. doi: 10.1007/978-3-319-09665-0_1.
  
  Kontush Anatol, Lindahl Mats, Lhomme Marie, Calabresi Laura, Chapman M John, Davidson W Sean
  
  Abstract
  
  A molecular understanding of high-density lipoprotein (HDL) will allow a more complete grasp of its interactions with key plasma remodelling factors and with cell-surface proteins that mediate HDL assembly and clearance. However, these particles are notoriously heterogeneous in terms of almost every physical, chemical and biological property. Furthermore, HDL particles have not lent themselves to high-resolution structural study through mainstream techniques like nuclear magnetic resonance and X-ray crystallography; investigators have therefore had to use a series of lower resolution methods to derive a general structural understanding of these enigmatic particles. This chapter reviews current knowledge of the composition, structure and heterogeneity of human plasma HDL. The multifaceted composition of the HDL proteome, the multiple major protein isoforms involving translational and posttranslational modifications, the rapidly expanding knowledge of the HDL lipidome, the highly complex world of HDL subclasses and putative models of HDL particle structure are extensively discussed. A brief history of structural studies of both plasma-derived and recombinant forms of HDL is presented with a focus on detailed structural models that have been derived from a range of techniques spanning mass spectrometry to molecular dynamics.
  
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• HDL and atherosclerosis: Insights from inherited HDL disorders.
  Biochim Biophys Acta

  2015 Jan;1851(1):13-8. doi: 10.1016/j.bbalip.2014.07.015.
  
  Calabresi Laura, Gomaraschi Monica, Simonelli Sara, Bernini Franco, Franceschini Guido
  
  Abstract
  
  Plasma high density lipoproteins (HDL) comprise a highly heterogeneous family of lipoprotein particles, differing in density, size, surface charge, and lipid and protein composition. Epidemiological studies have shown that plasma HDL level inversely correlates with atherosclerotic cardiovascular disease. The most relevant atheroprotective function of HDL is to promote the removal of cholesterol from macrophages within the arterial wall and deliver it to the liver for excretion in a process called reverse cholesterol transport. In addition, HDLs can contribute to the maintenance of endothelial cell homeostasis and have potent antioxidant properties. It has been long suggested that individual HDL subclasses may differ in terms of their functional properties, but which one is the good particle remains to be defined. Inherited HDL disorders are rare monogenic diseases due to mutations in genes encoding proteins involved in HDL metabolism. These disorders are not only characterized by extremely low or high plasma HDL levels but also by an abnormal HDL subclass distribution, and thus represent a unique tool to understand the relationship between plasma HDL concentration, HDL function, and HDL-mediated atheroprotection. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.
  
  Copyright © 2014 Elsevier B.V. All rights reserved.
  
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• HDL and cholesterol handling in the brain.
  Cardiovasc Res

  2014 Aug;103(3):405-13. doi: 10.1093/cvr/cvu148.
  
  Vitali Cecilia, Wellington Cheryl L, Calabresi Laura
  
  Abstract
  
  Cholesterol is an essential component of both the peripheral nervous system and central nervous system (CNS) of mammals. Brain cholesterol is synthesized in situ by astrocytes and oligodendrocytes and is almost completely isolated from other pools of cholesterol in the body, but a small fraction can be taken up from the circulation as 27-hydroxycholesterol, or via the scavenger receptor class B type I. Glial cells synthesize native high-density lipoprotein (HDL)-like particles, which are remodelled by enzymes and lipid transfer proteins, presumably as it occurs in plasma. The major apolipoprotein constituent of HDL in the CNS is apolipoprotein E, which is produced by astrocytes and microglia. Apolipoprotein A-I, the major protein component of plasma HDL, is not synthesized in the CNS, but can enter and become a component of CNS lipoproteins. Low HDL-C levels have been shown to be associated with cognitive impairment and various neurodegenerative diseases. On the contrary, no clear association with brain disorders has been shown in genetic HDL defects, with the exception of Tangier disease. Mutations in a wide variety of lipid handling genes can result in human diseases, often with a neuronal phenotype caused by dysfunctional intracellular lipid trafficking.
  
  Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.
  
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• eNOS activation by HDL is impaired in genetic CETP deficiency.
  PLoS One

  2014 ;9(5):e95925. doi: e95925.
  
  Gomaraschi Monica, Ossoli Alice, Pozzi Silvia, Nilsson Peter, Cefalù Angelo B, Averna Maurizio, Kuivenhoven Jan Albert, Hovingh G Kees, Veglia Fabrizio, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Mutations in the CETP gene resulting in defective CETP activity have been shown to cause remarkable elevations of plasma HDL-C levels, with the accumulation in plasma of large, buoyant HDL particles enriched in apolipoprotein E. Genetic CETP deficiency thus represents a unique tool to evaluate how structural alterations of HDL impact on HDL atheroprotective functions. Aim of the present study was to assess the ability of HDL obtained from CETP-deficient subjects to protect endothelial cells from the development of endothelial dysfunction. HDL isolated from one homozygous and seven heterozygous carriers of CETP null mutations were evaluated for their ability to down-regulate cytokine-induced cell adhesion molecule expression and to promote NO production in cultured endothelial cells. When compared at the same protein concentration, HDL and HDL3 from carriers proved to be as effective as control HDL and HDL3 in down-regulating cytokine-induced VCAM-1, while carrier HDL2 were more effective than control HDL2 in inhibiting VCAM-1 expression. On the other hand, HDL and HDL fractions from carriers of CETP deficiency were significantly less effective than control HDL and HDL fractions in stimulating NO production, due to a reduced eNOS activating capacity, likely because of a reduced S1P content. In conclusion, the present findings support the notion that genetic CETP deficiency, by affecting HDL particle structure, impacts on HDL vasculoprotective functions. Understanding of these effects might be important for predicting the outcomes of pharmacological CETP inhibition.
  
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• Distant homology modeling of LCAT and its validation through in silico targeting and in vitro and in vivo assays.
  PLoS One

  2014 ;9(4):e95044. doi: e95044.
  
  Sensi Cristina, Simonelli Sara, Zanotti Ilaria, Tedeschi Gabriella, Lusardi Giulia, Franceschini Guido, Calabresi Laura, Eberini Ivano
  
  Abstract
  
  LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late '90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the ?/? hydrolase superfamily, and its topology is characterized by a combination of ?-helices covering a central 7-strand ?-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.
  
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• Hepatic ACAT2 knock down increases ABCA1 and modifies HDL metabolism in mice.
  PLoS One

  2014 ;9(4):e93552. doi: e93552.
  
  Pedrelli Matteo, Davoodpour Padideh, Degirolamo Chiara, Gomaraschi Monica, Graham Mark, Ossoli Alice, Larsson Lilian, Calabresi Laura, Gustafsson Jan-Åke, Steffensen Knut R, Eriksson Mats, Parini Paolo
  
  Abstract
  
  OBJECTIVES:
  ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism.
  
  DESIGN:
  WT and LXR?/? double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet.
  
  RESULTS:
  ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE.
  
  CONCLUSIONS:
  The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1.
  
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• Nutraceutical approach to moderate cardiometabolic risk: results of a randomized, double-blind and crossover study with Armolipid Plus.
  J Clin Lipidol

  2014 ;8(1):61-8. doi: 10.1016/j.jacl.2013.11.003.
  
  Ruscica Massimiliano, Gomaraschi Monica, Mombelli Giuliana, Macchi Chiara, Bosisio Raffaella, Pazzucconi Franco, Pavanello Chiara, Calabresi Laura, Arnoldi Anna, Sirtori Cesare R, Magni Paolo
  
  Abstract
  
  BACKGROUND:
  Primary cardiovascular prevention may be achieved by lifestyle/nutrition improvements and specific drugs, although a relevant role is now emerging for specific functional foods and nutraceuticals.
  
  OBJECTIVES:
  The aim of this study was to evaluate the usefulness of a nutraceutical multitarget approach in subjects with moderate cardiovascular risk and to compare it with pravastatin treatment.
  
  SUBJECTS:
  Thirty patients with moderate dyslipidemia and metabolic syndrome (according to the Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults) were included in an 8-week randomized, double-blind crossover study and took either placebo or a nutraceutical combination that contained red yeast rice extract, berberine, policosanol, astaxanthin, coenzyme Q10, and folic acid (Armolipid Plus). Subsequently, they were subjected to another 8-week treatment with pravastatin 10 mg/d. This dosage was selected on the basis of its expected -20% efficacy in reducing low-density lipoprotein-cholesterol.
  
  RESULTS:
  Treatment with Armolipid Plus led to a significant reduction of total cholesterol (-12.8%) and low-density lipoprotein-cholesterol (-21.1%), similar to pravastatin (-16% and -22.6%, respectively), and an increase of high-density lipoprotein-cholesterol (4.8%). Armolipid Plus improved the leptin-to-adiponectin ratio, whereas adiponectin levels were unchanged.
  
  CONCLUSIONS:
  These results indicate that this nutraceutical approach shows a lipid-lowering activity comparable to pravastatin treatment. Hence, it may be a safe and useful option, especially in conditions of moderate cardiovascular risk, in which a pharmacologic intervention may not be appropriate.
  
  Copyright © 2014 National Lipid Association. Published by Elsevier Inc. All rights reserved.
  
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• Lipoprotein glomerulopathy associated with a mutation in apolipoprotein e.
  Clin Med Insights Case Rep

  2013 ;6():189-96. doi: 10.4137/CCRep.S12209.
  
  Magistroni Riccardo, Bertolotti Marco, Furci Luciana, Fano Rita Adriana, Leonelli Marco, Pisciotta Livia, Pellegrini Elisa, Calabresi Laura, Bertolini Stefano, Calandra Sebastiano
  
  Abstract
  
  Lipoprotein glomerulopathy is a pathological condition characterized by lipid accumulation in the glomerular capillaries that has been associated with the presence of rare mutants of apolipoprotein E (ApoE). We describe a 51-year-old Italian patient presenting Type III hyperlipidemia and proteinuria in whom renal biopsy showed capillary ectasia and intraluminal lipid deposits, suggesting the diagnosis of lipoprotein glomerulopathy. The patient, who had elevated plasma ApoE level, was found to be heterozygous for a mutation in ApoE (Arg150Cys), designated apoEMODENA. This mutation induces the formation of ApoE dimers that are detectable under non-reducing conditions. Treatment with hypolipidemic drugs did not result in a complete remission of the proteinuria and was accompanied by a slow but progressive worsening of renal function with the persistence of intracapillary lipid thrombi. The introduction of low-density lipoprotein aphaeresis combined with a more aggressive lipid lowering and antihypertensive therapy resulted in the remission of proteinuria and a substantial improvement of renal function. Switching from low-density lipoprotein aphaeresis to plasma filtration did not result in an equivalent control of renal damage. The patient died of intracranial hemorrhage during an acute episode of malignant hypertension.
  
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• Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency.
  Biologicals

  2013 Nov;41(6):446-9. doi: 10.1016/j.biologicals.2013.09.007.
  
  Simonelli Sara, Tinti Cristina, Salvini Laura, Tinti Laura, Ossoli Alice, Vitali Cecilia, Sousa Vitor, Orsini Gaetano, Nolli Maria Luisa, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (-30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small pre?-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL.
  
  Copyright © 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
  
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• Differential effects of fenofibrate and extended-release niacin on high-density lipoprotein particle size distribution and cholesterol efflux capacity in dyslipidemic patients.
  J Clin Lipidol

  2013 ;7(5):414-22. doi: 10.1016/j.jacl.2013.06.007.
  
  Franceschini Guido, Favari Elda, Calabresi Laura, Simonelli Sara, Bondioli Alighiero, Adorni Maria Pia, Zimetti Francesca, Gomaraschi Monica, Coutant Karine, Rossomanno Simona, Niesor Eric J, Bernini Franco, Benghozi Renee
  
  Abstract
  
  BACKGROUND:
  The effectiveness of therapies that raise high-density lipoprotein cholesterol (HDL-C) to lower cardiovascular disease risk is currently under debate, and further research into the relationship between HDL-C and function is required.
  
  OBJECTIVE:
  o investigate whether 2 established HDL-C-raising therapies had differential effects on parameters of high-density lipoprotein (HDL) quality and function, such as HDL particle profile and cholesterol efflux capacity (CEC), in patients with dyslipidemia.
  
  METHODS AND RESULTS:
  Sixty-six patients with dyslipidemia, 24 with low HDL-C levels (
  CONCLUSIONS:
  Fenofibrate and ER niacin increased plasma HDL-C level similarly, but modulated HDL particle size distribution differently; however, these changes did not result in differential effects on serum CECs.
  
  Copyright © 2013 National Lipid Association. Published by Elsevier Inc. All rights reserved.
  
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• Effect of repeated apoA-IMilano/POPC infusion on lipids, (apo)lipoproteins, and serum cholesterol efflux capacity in cynomolgus monkeys.
  J Lipid Res

  2013 Sep;54(9):2341-53. doi: 10.1194/jlr.M033779.
  
  Kempen Herman J, Gomaraschi Monica, Bellibas S Eralp, Plassmann Stephanie, Zerler Brad, Collins Heidi L, Adelman Steven J, Calabresi Laura, Wijngaard Peter L J
  
  Abstract
  
  MDCO-216, a complex of dimeric recombinant apoA-IMilano (apoA-IM) and palmitoyl-oleoyl-phosphatidylcholine (POPC), was administered to cynomolgus monkeys at 30, 100, and 300 mg/kg every other day for a total of 21 infusions, and effects on lipids, (apo)lipoproteins, and ex-vivo cholesterol efflux capacity were monitored. After 7 or 20 infusions, free cholesterol (FC) and phospholipids (PL) were strongly increased, and HDL-cholesterol (HDL-C), apoA-I, and apoA-II were strongly decreased. We then measured short-term effects on apoA-IM, lipids, and (apo)lipoproteins after the first or the last infusion. After the first infusion, PL and FC went up in the HDL region and also in the LDL and VLDL regions. ApoE shifted from HDL to LDL and VLDL regions, while ApoA-IM remained located in the HDL region. On day 41, ApoE levels were 8-fold higher than on day 1, and FC, PL, and apoE resided mostly in LDL and VLDL regions. Drug infusion quickly decreased the endogenous cholesterol esterification rate. ABCA1-mediated cholesterol efflux on day 41 was markedly increased, whereas scavenger receptor type B1 (SRB1) and ABCG1-mediated effluxes were only weakly increased. Strong increase of FC is due to sustained stimulation of ABCA1-mediated efflux, and drop in HDL and formation of large apoE-rich particles are due to lack of LCAT activation.
  
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• Inflammation impairs eNOS activation by HDL in patients with acute coronary syndrome.
  Cardiovasc Res

  2013 Oct;100(1):36-43. doi: 10.1093/cvr/cvt169.
  
  Gomaraschi Monica, Ossoli Alice, Favari Elda, Adorni Maria Pia, Sinagra Gianfranco, Cattin Luigi, Veglia Fabrizio, Bernini Franco, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  AIMS:
  The aim of the present study was to evaluate the high-density lipoprotein (HDL) structure and endothelial NO synthase (eNOS) activation capacity in ST-elevation myocardial infarction (STEMI) patients with different acute-phase inflammatory response (APR).
  
  METHODS AND RESULTS:
  Forty-five STEMI patients were stratified in quartiles according to the delta CRP level, calculated by subtracting the CRP value at admission from the CRP peak value (APR peak). The HDL structure and HDL capacity to stimulate NO production were evaluated at admission and at APR peak. STEMI patients with a low APR had a completely preserved HDL structure and HDL ability to activate eNOS and promote NO production, which did not change during STEMI. On the contrary, HDL from STEMI patients developing a significant APR had compromised ability to stimulate eNOS and promote NO production, and underwent a significant particle remodelling during STEMI. The defective capacity to stimulate NO production of HDL isolated from STEMI patients with high APR was explained, at least in part, by the reduced PON-1 and S1P content. The HDL ability to promote cell cholesterol efflux through different pathways was preserved in ACS patients independently of the inflammatory response.
  
  CONCLUSION:
  The present results extend previous studies reporting an impaired eNOS-activating capacity of HDL from ACS patients, showing that only a subset of patients undergoing STEMI, and in particular those developing an important inflammatory response, have circulating HDL defective in stimulating endothelial eNOS and NO production.
  
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• A proteomic portrait of atherosclerosis.
  J Proteomics

  2013 Apr;82():92-112. doi: 10.1016/j.jprot.2013.02.007.
  
  Eberini Ivano, Wait Robin, Calabresi Laura, Sensi Cristina, Miller Ingrid, Gianazza Elisabetta
  
  Abstract
  
  We have arranged in this review the main evidence about proteome alterations in different cell and body fluid compartments along the progression of atherosclerosis. With time the description of the molecular phenomena is becoming more and more detailed yet the complex interrelationships among different factors are still elusive and previously neglected aspects (such as size for lipoprotein particles) emerge as not less relevant than the absolute abundance of individual proteins. Physiological limits to the kinetics of protein distribution through the biological fluids seem to hinder the early diagnosis of acute conditions through plasma analysis but suggest urine analysis as a workable alternative for the assessment of chronic conditions. The survey of literature data is complemented with a few unpublished results from our laboratories, featuring 2DE maps of the proteins extracted from human thrombi.
  
  Copyright © 2013. Published by Elsevier B.V.
  
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• Off-target effects of thrombolytic drugs: apolipoprotein A-I proteolysis by alteplase and tenecteplase.
  Biochem Pharmacol

  2013 Feb;85(4):525-30. doi: 10.1016/j.bcp.2012.11.023.
  
  Gomaraschi Monica, Ossoli Alice, Vitali Cecilia, Pozzi Silvia, Vitali Serdoz Laura, Pitzorno Cristina, Sinagra Gianfranco, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  The administration of thrombolytic drugs is of proven benefit in a variety of clinical conditions requiring acute revascularization, including acute myocardial infarction (AMI), ischemic stroke, pulmonary embolism, and venous thrombosis. Generated plasmin can degrade non-target proteins, including apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoproteins (HDL). Aim of the present study was to compare the extent of apoA-I proteolytic degradation in AMI patients treated with two thrombolytic drugs, alteplase and the genetically engineered t-PA variant tenecteplase. ApoA-I degradation was evaluated in sera from 38 AMI patients treated with alteplase or tenecteplase. In vitro, apoA-I degradation was tested by incubating control sera or purified HDL with alteplase or tenecteplase at different concentrations (5-100 ?g/ml). Treatment with alteplase and tenecteplase results in apoA-I proteolysis; the extent of apoA-I degradation was more pronounced in alteplase-treated patients than in tenecteplase-treated patients. In vitro, the extent of apoA-I proteolysis was higher in alteplase-treated sera than in tenecteplase-treated sera, in the whole drug concentration range. No direct effect of the two thrombolytic agents on apoA-I degradation was observed. In addition to apoA-I, apoA-IV was also degraded by the two thrombolytic agents and again proteolytic degradation was higher with alteplase than tenecteplase. In conclusion, this study indicates that both alteplase and tenecteplase cause plasmin-mediated proteolysis of apoA-I, with alteplase resulting in a greater apoA-I degradation than tenecteplase, potentially causing a transient impairment of HDL atheroprotective functions.
  
  Copyright © 2012 Elsevier Inc. All rights reserved.
  
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• Novel missense variants in LCAT and APOB genes in an Italian kindred with familial lecithin:cholesterol acyltransferase deficiency and hypobetalipoproteinemia.
  J Clin Lipidol

  2012 ;6(3):244-50. doi: 10.1016/j.jacl.2012.01.006.
  
  Conca Paola, Pileggi Silvana, Simonelli Sara, Boer Emanuela, Boscutti Giuliano, Magnolo Lucia, Tarugi Patrizia, Penco Silvana, Franceschini Guido, Calabresi Laura, Gomaraschi Monica
  
  Abstract
  
  BACKGROUND:
  Lecithin:cholesterol acyltransferase (LCAT) is responsible for cholesterol esterification in plasma. Mutations of LCAT gene cause familial LCAT deficiency, a metabolic disorder characterized by hypoalphalipoproteinemia. Apolipoprotein B (apoB) is the main protein component of very-low-density lipoproteins and low-density lipoprotein (LDL). Mutations of APOB gene cause familial hypobetalipoproteinemia, a codominant disorder characterized by low plasma levels of LDL cholesterol and apoB.
  
  OBJECTIVE:
  This was a genetic and biochemical analysis of an Italian kindred with hypobetalipoproteinemia whose proband presented with hypoalphalipoproteinemia and severe chronic kidney disease.
  
  METHODS:
  Plasma lipids and apolipoproteins, cholesterol esterification, and high-density lipoprotein (HDL) subclass distribution were analyzed. LCAT and APOB genes were sequenced.
  
  RESULTS:
  The proband had severe impairment of plasma cholesterol esterification and high pre?-HDL content. He was heterozygote for the novel LCAT P406L variant, as were two other family members. The proband's wife and children presented with familial hypobetalipoproteinemia and were heterozygotes for the novel apoB H1401R variant. Cholesterol esterification rate of apoB H1401R carriers was reduced, likely attributable to the low amount of circulating LDL. After renal transplantation, proband's lipid profile, HDL subclass distribution, and plasma cholesterol esterification were almost at normal levels, suggesting a mild contribution of the LCAT P406L variant to his pretransplantation severe hypoalphalipoproteinemia and impairment of plasma cholesterol esterification.
  
  CONCLUSION:
  LCAT P406L variant had a mild effect on lipid profile, HDL subclass distribution, and plasma cholesterol esterification. ApoB H1401R variant was identified as possible cause of familial hypobetalipoproteinemia and resulted in a reduction of cholesterol esterification rate.
  
  Copyright © 2012 National Lipid Association. Published by Elsevier Inc. All rights reserved.
  
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• Lipid and apoprotein composition of HDL in partial or complete CETP deficiency.
  Curr Vasc Pharmacol

  2012 Jul;10(4):422-31.
  
  Niesor Eric J, von der Mark Elisabeth, Calabresi Laura, Averna Maurizio, Cefalù Angelo B, Tarugi Patrizia, Nilsson Peter, Dernick Gregor
  
  Abstract
  
  Hyperalphalipoproteinemia, as observed in patients who are either homozygous or heterozygous for cholesteryl ester transfer protein (CETP) deficiency, has been shown to be associated with striking changes in apolipoprotein size distribution, namely, of high-density lipoprotein (HDL) and HDL-like particles. We compared the effect of varying degrees of CETP activity on the HDL apolipoprotein profile in Caucasian CETP-deficient subjects and following pharmacological decrease in CETP activity, using Size Exclusion Chromatography followed by Reverse Phase Protein Array (SEC RPA). The main HDL-associated apolipoproteins (Apo), i.e. ApoA-I, ApoA-II, ApoC-I, and ApoC-III, co-eluted with the HDL peak. The presence of a HDL-like peak migrating between the ApoB-LDL and ApoA-I-HDL was identified in a Caucasian patient with homozygosity for a point mutation in exon 2 of the CETP gene (c.109 C > T) resulting in a premature termination codon (R37X) and complete CETP deficiency. This HDL-like peak was not observed either in healthy volunteers treated with the CETP modulator dalcetrapib, patients heterozygous for the same mutation, or in patients heterozygous with G165X mutations. SEC RPA offers the possibility to investigate the distribution of a large number of apolipoproteins simultaneously under non-denaturing separation in normal and dyslipidemic subjects. This is only limited by the availability of antibodies against specific apolipoproteins to be investigated.
  
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• Severe high-density lipoprotein deficiency associated with autoantibodies against lecithin:cholesterol acyltransferase in non-Hodgkin lymphoma.
  Arch Intern Med

  2012 Jan;172(2):179-81. doi: 10.1001/archinternmed.2011.661.
  
  Simonelli Sara, Gianazza Elisabetta, Mombelli Giuliana, Bondioli Alighiero, Ferraro Giovanni, Penco Silvana, Sirtori Cesare R, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  An antibody against the lecithin:cholesterol acyltransferase (LCAT) enzyme, which negates cholesterol esterification in plasma, causing severe high-density lipoprotein deficiency (HD), was identified in a woman with a large-cell non-Hodgkin lymphoma. Successful treatment of the lymphoma resulted in clearance of the antibody and complete correction of the defective cholesterol esterification and HD. To our knowledge, an acquired LCAT deficiency leading to severe HD has not been reported previously in association with a malignant disease, and this patient represents the first such documented case.
  
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• Genetic lecithin:cholesterol acyltransferase deficiency and cardiovascular disease.
  Atherosclerosis

  2012 Jun;222(2):299-306. doi: 10.1016/j.atherosclerosis.2011.11.034.
  
  Calabresi Laura, Simonelli Sara, Gomaraschi Monica, Franceschini Guido
  
  Abstract
  
  The lecithin:cholesterol acyltransferase (LCAT) enzyme is responsible for the synthesis of cholesteryl esters in human plasma and plays a critical role in high density lipoprotein (HDL) metabolism. Genetic LCAT deficiency is a rare metabolic disorder characterized by low HDL cholesterol levels. This paper reviews the genetic and biochemical features of LCAT deficiency, highlighting the absence of enhanced preclinical atherosclerosis in carriers, despite the remarkably low HDL cholesterol.
  
  Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
  
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• Characterization of three kindreds with familial combined hypolipidemia caused by loss-of-function mutations of ANGPTL3.
  Circ Cardiovasc Genet

  2012 Feb;5(1):42-50. doi: 10.1161/CIRCGENETICS.111.960674.
  
  Pisciotta Livia, Favari Elda, Magnolo Lucia, Simonelli Sara, Adorni Maria Pia, Sallo Raffaella, Fancello Tatiana, Zavaroni Ivana, Ardigò Diego, Bernini Franco, Calabresi Laura, Franceschini Guido, Tarugi Patrizia, Calandra Sebastiano, Bertolini Stefano
  
  Abstract
  
  BACKGROUND:
  Angiopoietin-like protein 3 (ANGPTL3) affects lipid metabolism by inhibiting the activity of lipoprotein and endothelial lipases. Angptl3 knockout mice have marked hypolipidemia, and heterozygous carriers of ANGPLT3, loss-of-function mutations were found among individuals in the lowest quartile of plasma triglycerides in population studies. Recently, 4 related individuals with primary hypolipidemia were found to be compound heterozygotes for ANGPTL3 loss-of-function mutations.
  
  METHODS AND RESULTS:
  We resequenced ANGPTL3 in 4 members of 3 kindreds originally identified for very low levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (0.97±0.16 and 0.56±0.20 mmol/L, respectively) in whom no mutations of known candidate genes for monogenic hypobetalipoproteinemia and hypoalphalipoproteinemia had been detected. These subjects were found to be homozygous or compound heterozygous for ANGPTL3 loss-of-function mutations (p.G400VfsX5, p.I19LfsX22/p.N147X) associated with the absence of ANGPTL3 in plasma. They had reduced plasma levels of triglyceride-containing lipoproteins and of HDL particles that contained only apolipoprotein A-I and pre-?-high-density lipoprotein. In addition, their apolipoprotein B-depleted sera had a reduced capacity to promote cell cholesterol efflux through the various pathways (ABCA1-, SR-BI-, and ABCG1-mediated efflux); however, these subjects had no clinical evidence of accelerated atherosclerosis. Heterozygous carriers of the ANGPTL3 mutations had low plasma ANGPTL3 and moderately reduced low-density lipoprotein cholesterol (2.52±0.38 mmol/L) but normal plasma high-density lipoprotein cholesterol.
  
  CONCLUSIONS:
  Complete ANGPTL3 deficiency caused by loss-of-function mutations of ANGPTL3 is associated with a recessive hypolipidemia characterized by a reduction of apolipoprotein B and apolipoprotein A-I-containing lipoproteins, changes in subclasses of high-density lipoprotein, and reduced cholesterol efflux potential of serum. Partial ANGPTL3 deficiency is associated only with a moderate reduction of low-density lipoprotein.
  
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• A woman with low HDL cholesterol and corneal opacity.
  Intern Emerg Med

  2012 Dec;7(6):533-7. doi: 10.1007/s11739-011-0712-6.
  
  Lucchi Tiziano, Calabresi Laura, Pinto Angela, Benetti Elisa, Arosio Beatrice, Simonelli Sara, Ratiglia Roberto, Vergani Carlo
  
  
  
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• Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals.
  Br J Nutr

  2012 Apr;107(8):1176-83. doi: 10.1017/S0007114511004120.
  
  Sirtori Cesare R, Triolo Michela, Bosisio Raffaella, Bondioli Alighiero, Calabresi Laura, De Vergori Viviana, Gomaraschi Monica, Mombelli Giuliana, Pazzucconi Franco, Zacherl Christian, Arnoldi Anna
  
  Abstract
  
  The present study was aimed to evaluate the effect of plant proteins (lupin protein or pea protein) and their combinations with soluble fibres (oat fibre or apple pectin) on plasma total and LDL-cholesterol levels. A randomised, double-blind, parallel group design was followed: after a 4-week run-in period, participants were randomised into seven treatment groups, each consisting of twenty-five participants. Each group consumed two bars containing specific protein/fibre combinations: the reference group consumed casein+cellulose; the second and third groups consumed bars containing lupin or pea proteins+cellulose; the fourth and fifth groups consumed bars containing casein and oat fibre or apple pectin; the sixth group and seventh group received bars containing combinations of pea protein and oat fibre or apple pectin, respectively. Bars containing lupin protein+cellulose ( - 116 mg/l, - 4·2%), casein+apple pectin ( - 152 mg/l, - 5·3%), pea protein+oat fibre ( - 135 mg/l, - 4·7%) or pea protein+apple pectin ( - 168 mg/l, - 6·4%) resulted in significant reductions of total cholesterol levels (P
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• [LCAT deficiency: a nephrological diagnosis].
  G Ital Nefrol

  2011 ;28(4):369-82.
  
  Boscutti Giuliano, Calabresi Laura, Pizzolitto Stefano, Boer Emanuela, Bosco Manuela, Mattei Piero Luigi, Martone Massimiliano, Milutinovic Neva, Berbecar Dorina, Beltram Elisabetta, Franceschini Guido
  
  Abstract
  
  A genetic mendelian autosomal recessive condition of deficiency of lecithin- cholesterol acyltransferase (LCAT) can produce two different diseases: one highly interesting nephrologic picture of complete enzymatic deficiency (lecithin:cholesterol acyltransferase deficiency; OMIM ID #245900; FLD), characterized by the association of dyslipidemia, corneal opacities, anemia and progressive nephropathy; and a partial form (fish eye disease; OMIM ID #136120; FED) with dyslipidemia and progressive corneal opacities only. The diagnosis of FLD falls first of all under the competence of nephrologists, because end-stage renal disease appears to be its most severe outcome. The diagnostic suspicion is based on clinical signs (corneal opacities, more severe anemia than expected for the degree of chronic renal failure, progressive proteinuric nephropathy) combined with histology obtained by kidney biopsy (glomerulopathy evolving toward sclerosis with distinctive lipid deposition). However, the final diagnosis, starting with a finding of extremely low levels of HDL-cholesterol, requires collaboration with lipidology Centers that can perform sophisticated investigations unavailable in common laboratories. To be heterozygous for a mutation of the LCAT gene is one of the monogenic conditions underlying primary hypoalphalipoproteinemia (OMIM ID #604091). This disease, which is characterized by levels of HDL-cholesterol below the 5th percentile of those of the examined population (
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• Plasma lecithin:cholesterol acyltransferase and carotid intima-media thickness in European individuals at high cardiovascular risk.
  J Lipid Res

  2011 Aug;52(8):1569-74. doi: 10.1194/jlr.P014977.
  
  Calabresi Laura, Baldassarre Damiano, Simonelli Sara, Gomaraschi Monica, Amato Mauro, Castelnuovo Samuela, Frigerio Beatrice, Ravani Alessio, Sansaro Daniela, Kauhanen Jussi, Rauramaa Rainer, de Faire Ulf, Hamsten Anders, Smit Andries J, Mannarino Elmo, Humphries Steve E, Giral Philippe, Veglia Fabrizio, Sirtori Cesare R, Franceschini Guido, Tremoli Elena
  
  Abstract
  
  Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. LCAT is a major factor in HDL remodeling and metabolism, and it has long been believed to play a critical role in macrophage reverse cholesterol transport (RCT). The effect of LCAT on human atherogenesis is still controversial. In the present study, the plasma LCAT concentration was measured in all subjects (n = 540) not on drug treatment at the time of enrollment in the multicenter, longitudinal, observational IMPROVE study. Mean and maximum intima-media thickness (IMT) of the whole carotid tree was measured by B-mode ultrasonography in all subjects. In the entire cohort, LCAT quartiles were not associated with carotid mean and maximum IMT (P for trend 0.95 and 0.18, respectively), also after adjustment for age, gender, HDL-cholesterol (HDL-C), and triglycerides. No association between carotid IMT and LCAT quartiles was observed in men (P=0.30 and P=0.99 for mean and maximum IMT, respectively), whereas carotid IMT increased with LCAT quartiles in women (P for trend 0.14 and 0.019 for mean and maximum IMT, respectively). The present findings support the concept that LCAT is not required for an efficient reverse cholesterol transport and that a low plasma LCAT concentration and activity is not associated with increased atherosclerosis.
  
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• Effect of the amyloidogenic L75P apolipoprotein A-I variant on HDL subpopulations.
  Clin Chim Acta

  2011 Jun;412(13-14):1262-5. doi: 10.1016/j.cca.2011.03.027.
  
  Gomaraschi Monica, Obici Laura, Simonelli Sara, Gregorini Gina, Negrinelli Alessandro, Merlini Giampaolo, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  BACKGROUND:
  Hereditary amyloidosis due to mutations of apolipoprotein A-I (apoA-I) is a rare disease characterized by the deposition of amyloid fibrils constituted by the N-terminal fragment of apoA-I in several organs. L75P is a variant of apoA-I associated with systemic amyloidosis predominantly involving the liver, kidneys, and testis, identified in a large number of unrelated subjects. Objective of the present paper was to evaluate the impact of the L75P apoA-I variant on HDL subpopulations and cholesterol esterification in carriers.
  
  METHODS AND RESULTS:
  Plasma samples were collected from 30 carriers of the amyloidogenic L75P apoA-I (Carriers) and from 15 non affected relatives (Controls). Carriers displayed significantly reduced plasma levels of HDL-cholesterol, apoA-I, and apoA-II compared to Controls. Plasma levels of LpA-I, but not LpA-I:A-II, were significantly reduced in Carriers. HDL subclass distribution was not affected by the presence of the variant. The unesterified to total cholesterol ratio was higher, and cholesterol esterification rate and LCAT activity were lower in Carriers than in Controls.
  
  CONCLUSIONS:
  The L75P apoA-I variant is associated with hypoalphalipoproteinemia, a selective reduction of LpA-I particles, and a partial defect in cholesterol esterification.
  
  Copyright © 2011 Elsevier B.V. All rights reserved.
  
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• Macrophage, but not systemic, apolipoprotein E is necessary for macrophage reverse cholesterol transport in vivo.
  Arterioscler Thromb Vasc Biol

  2011 Jan;31(1):74-80. doi: 10.1161/ATVBAHA.110.213892.
  
  Zanotti Ilaria, Pedrelli Matteo, Potì Francesco, Stomeo Grazia, Gomaraschi Monica, Calabresi Laura, Bernini Franco
  
  Abstract
  
  OBJECTIVE:
  To assess the role of apolipoprotein (apo) E in macrophage reverse cholesterol transport (RCT) in vivo.
  
  METHODS AND RESULTS:
  ApoE exerts an antiatherosclerotic activity by regulating lipoprotein metabolism and promoting cell cholesterol efflux. We discriminated between macrophage and systemic apoE contribution using an assay of macrophage RCT in mice. The complete absence of apoE lead to an overall impairment of the process and, similarly, the absence of apoE exclusively in macrophages resulted in the reduction of cholesterol mobilization from macrophages to plasma, liver, and feces. Conversely, expression of apoE in macrophages is sufficient to promote normal RCT even in apoE-deficient mice. The mechanisms accounting for these results were investigated by evaluating the first step of RCT (ie, cholesterol efflux from cells). Macrophages isolated from apoE-deficient mice showed a reduced ability to release cholesterol into the culture medium, whereas the apoB-depleted plasma from apoE-deficient and healthy mice possessed a similar capacity to promote cellular lipid release from cultured macrophages.
  
  CONCLUSIONS:
  Our data demonstrate, for the first time to our knowledge, that apoE significantly contributes to macrophage RCT in vivo and that this role is fully attributable to apoE expressed in macrophages.
  
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• Structural and dynamic features of apolipoprotein A-I cysteine mutants, Milano and Paris, in synthetic HDL.
  J Mol Graph Model

  2010 Nov;29(3):406-14. doi: 10.1016/j.jmgm.2010.08.002.
  
  Rocco Alessandro Guerini, Sensi Cristina, Gianazza Elisabetta, Calabresi Laura, Franceschini Guido, Sirtori Cesare R, Eberini Ivano
  
  Abstract
  
  Pursuing an established research interest in our group, we built two models for synthetic HDL containing the natural cysteine mutants of apolipoprotein A-I, apolipoprotein A-I Milano (apoA-IM) and apolipoprotein A-I Paris (apoA-IP), both in their homodimeric form. Data on the structural and dynamic properties of such s-HDL are an essential preliminary step for the understanding of the biological activity of the two mutants. Furthermore, comparison between apoA-IM and apoA-IP allows evaluating the effects of the same mutation in a different position in the primary structure and to directly compare our findings with previously published models. We computed for 50 ns in explicit solvent the molecular dynamics of the two complexes and analyzed different properties as a function of time. The proposed s-HDL structures differ significantly from one another and from wild type apolipoprotein A-I. All features of the apoA-IM model are consistent with experimental data. The higher RMSF of apoA-IM has a counterpart in the finding that trypsin, matrix metalloproteases, and chymase degrade apoA-IM much faster than wild type apoA-I; the primary cutting site is correctly identified by molecular dynamics data on our model of apoA-IM-containing s-HDL. The few experimental data for apoA-IP prevent direct comparison with our findings.
  
  Copyright © 2010 Elsevier Inc. All rights reserved.
  
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• Intestinal specific LXR activation stimulates reverse cholesterol transport and protects from atherosclerosis.
  Cell Metab

  2010 Aug;12(2):187-93. doi: 10.1016/j.cmet.2010.07.002.
  
  Lo Sasso Giuseppe, Murzilli Stefania, Salvatore Lorena, D'Errico Ilenia, Petruzzelli Michele, Conca Paola, Jiang Zhao-Yan, Calabresi Laura, Parini Paolo, Moschetta Antonio
  
  Abstract
  
  Several steps of the HDL-mediated reverse cholesterol transport (RCT) are transcriptionally regulated by the nuclear receptors LXRs in the macrophages, liver, and intestine. Systemic LXR activation via synthetic ligands induces RCT but also causes increased hepatic fatty acid synthesis and steatosis, limiting the potential therapeutic use of LXR agonists. During the last few years, the participation of the intestine in the control of RCT has appeared more evident. Here we show that while hepatic-specific LXR activation does not contribute to RCT, intestinal-specific LXR activation leads to decreased intestinal cholesterol absorption, improved lipoprotein profile, and increased RCT in vivo in the absence of hepatic steatosis. These events protect against atherosclerosis in the background of the LDLR-deficient mice. Our study fully characterizes the molecular and metabolic scenario that elects the intestine as a key player in the LXR-driven protective environment against cardiovascular disease.
  
  Copyright 2010 Elsevier Inc. All rights reserved.
  
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• Lecithin:cholesterol acyltransferase, high-density lipoproteins, and atheroprotection in humans.
  Trends Cardiovasc Med

  2010 Feb;20(2):50-3. doi: 10.1016/j.tcm.2010.03.007.
  
  Calabresi Laura, Franceschini Guido
  
  Abstract
  
  The lecithin:cholesterol acyltransferase (LCAT) enzyme is responsible for the synthesis of cholesteryl esters in human plasma. Lecithin:cholesterol acyltransferase is a critical enzyme in high-density lipoprotein (HDL) metabolism, and deficiency of LCAT-mediated cholesterol esterification leads to defective HDL maturation with accumulation of nascent pre-beta HDL. In addition to its function in HDL metabolism, LCAT has also long been believed to play a critical role in macrophage reverse cholesterol transport (RCT). However, recent findings have shown that human LCAT overexpression in mice does not enhance macrophage RCT in vivo, and conversely, LCAT-deficient mice display a preserved macrophage RCT despite the severe plasma HDL reduction. In agreement with this observation, defective LCAT activity does not result in enhanced atherosclerosis, despite the reduced HDL cholesterol levels. These findings challenge the notion that LCAT is required for effective atheroprotection and suggest that elevating LCAT expression and/or activity is not a promising therapeutic strategy to reduce cardiovascular risk.
  
  Copyright (c) 2010 Elsevier Inc. All rights reserved.
  
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• Conformation of dimeric apolipoprotein A-I milano on recombinant lipoprotein particles.
  Biochemistry

  2010 Jun;49(25):5213-24. doi: 10.1021/bi1003734.
  
  Bhat Shaila, Sorci-Thomas Mary G, Calabresi Laura, Samuel Michael P, Thomas Michael J
  
  Abstract
  
  Apolipoprotein A-I Milano (apoA-I(Milano)) is a naturally occurring human mutation of wild-type apolipoprotein A-I (apoA-I(WT)) having cystine substituted for arginine(173). Two molecules of apo-I(WT) form disks with phospholipid having a defined relationship between the apoA-I(WT) molecules. ApoA-I(Milano) forms cystine homodimers that would not allow the protein to adopt the conformation reported for apoA-I(WT). The conformational constraints for dimeric apoA-I(Milano) recombinant high-density lipoprotein (rHDL) disks made with phospholipid were deduced from a combination of chemical cross-linking and mass spectrometry. Lysine-selective homobifunctional cross-linkers were reacted with homogeneous rHDL having diameters of 78 and 125 A. After reduction, cross-linked apoA-I(Milano) was separated from monomeric apoprotein by gel electrophoresis and then subjected to in-gel trypsin digest. Cross-linked peptides were confirmed by MS/MS sequencing. The cross-links provided distance constraints that were used to refine models of lipid-bound dimeric apoA-I(Milano). These studies suggest that a single dimeric apoA-I(Milano) on 78 A diameter rHDL girdles the edge of a phospholipid disk assuming a "belt" conformation similar to the "belt" region of apoA-I(WT) on rHDL. However, the C-terminal end of dimeric apoA-I(Milano) wraps around the periphery of the particle to shield the fatty acid chains from water rather than folding back onto the "belt" as does apoA-I(WT). The two apoA-I(Milano) dimers on a 125 A diameter rHDL do not encircle the periphery of a phospholipid disk but appear to reside on the surface of a laminar micelle.
  
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• Paradoxical decrease in high-density lipoprotein cholesterol with fenofibrate: a quite rare phenomenon indeed.
  Cardiovasc Ther

  2010 Jun;28(3):153-60. doi: 10.1111/j.1755-5922.2009.00121.x.
  
  Mombelli Giuliana, Pazzucconi Franco, Bondioli Alighiero, Zanaboni AnnaMaria, Gaito Sabrina, Calabresi Laura, Sirtori Cesare R
  
  Abstract
  
  Some recent clinical reports have suggested that paradoxical decreases in high-density lipoprotein cholesterol (HDL-C) levels after fenofibrate treatment may be quite common. These appear to occur mainly in patients with combined fibrate/statin therapy and possibly in those with low baseline HDL-C. Reports on HDL-C reductions after fenofibrate are possibly supported by the disappointing results in terms of HDL-C responses from the recent FIELD study. A survey on 581 patients treated for 1 year or longer was carried out in our Clinical Center. This indicated that paradoxical HDL-C reductions are a relatively uncommon phenomenon. Not more than 15.3% of the present series showed an HDL-C reduction, mostly of a modest degree. Further, reductions of HDL-C appear to occur mainly in individuals with significant HDL-C elevations (>50 mg/dL), almost never in patients with low HDL-C. Otherwise, there seems to be no impact of a previous diagnosis of diabetes or hypertension on the HDL-C changes. From a very recent pharmacogenomic study on the apo A1/C3/A4/A5 gene cluster, genetic influences appear only to reduce the positive impact of fenofibrate on HDL-C, but do not indicate any risk of occurrence of HDL-C reductions. Also based on our very long experience with this drug, it appears that fenofibrate raises HDL-C levels in the vast majority of treated patients, with a particularly dramatic effect in individuals with low HDL-C and hypertriglyceridemia.
  
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• High-density lipoprotein quantity or quality for cardiovascular prevention?
  Curr Pharm Des

  2010 May;16(13):1494-503.
  
  Calabresi Laura, Gomaraschi Monica, Franceschini Guido
  
  Abstract
  
  Plasma concentrations of HDL cholesterol (HDL-C) are strongly and inversely associated with cardiovascular risk, leading to the concept that therapies to enhance plasma HDL-C levels would be anti-atherogenic and protective against cardiovascular events. However, HDL are highly heterogeneous, with subclasses that can be separated and identified according to density, size, charge, and protein composition. There is evidence that these subclasses may differ in their functional anti-atherogenic properties. As a snapshot of the steady-state cholesterol carried by all HDL subclasses together, the individual HDL-C measurement is insufficient to capture the structural and functional variation in HDL particles. This review addresses the current knowledge on the structural and functional heterogeneity of HDL particles, and their relationship to cardiovascular disease, in the attempt of answering the question on whether certain subclasses of HDL may be better predictors of cardiovascular risk than HDL-C, and may be better targets than HDL-C for further improving cardiovascular disease reduction in the statin era.
  
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• Structure and function of the apoA-IV T347S and Q360H common variants.
  Biochem Biophys Res Commun

  2010 Feb;393(1):126-30. doi: 10.1016/j.bbrc.2010.01.099.
  
  Gomaraschi Monica, Putt Wendy E, Pozzi Silvia, Iametti Stefania, Barbiroli Alberto, Bonomi Francesco, Favari Elda, Bernini Franco, Franceschini Guido, Talmud Philippa J, Calabresi Laura
  
  Abstract
  
  Human apolipoprotein A-IV (apoA-IV) is involved in chylomicron assembly and secretion, and in reverse cholesterol transport. Several apoA-IV isoforms exist, the most common in Caucasian populations being apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). The objective of the present study was to investigate the impact of these common aminoacid substitutions on the ability of apoA-IV to bind lipids, to promote cell cholesterol efflux via ABCA1, and to maintain endothelial homeostasis. Recombinant forms of wild-type apoA-IV, apoA-IV Q360H, and apoA-IV T347S were produced in Escherichia coli. ApoA-IV Q360H and apoA-IV T347S showed a slightly higher alpha-helical content compared to wild-type apoA-IV, and associated with phospholipids faster than wild-type apoA-IV. The capacity to promote ABCA1-mediated cholesterol efflux was significantly greater for the apoA-IV T347S than the other apoA-IV isoforms. No differences were observed in the ability of apoA-IV isoforms to inhibit the production of VCAM-1 and IL-6 in TNFalpha-stimulated endothelial cells. In conclusion, the apoA-IV T347S common variant has increased lipid binding properties and cholesterol efflux capacity, while the apoA-IV Q360H variant has only slightly increased lipid binding properties. The two common aminoacid substitutions have no effect on the ability of apoA-IV to maintain endothelial homeostasis.
  
  Copyright 2010 Elsevier Inc. All rights reserved.
  
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• Native LDL-induced oxidative stress in human proximal tubular cells: multiple players involved.
  J Cell Mol Med

  2011 Feb;15(2):375-95. doi: 10.1111/j.1582-4934.2009.00946.x.
  
  Piccoli Claudia, Quarato Giovanni, D'Aprile Annamaria, Montemurno Eustacchio, Scrima Rosella, Ripoli Maria, Gomaraschi Monica, Cirillo Pietro, Boffoli Domenico, Calabresi Laura, Gesualdo Loreto, Capitanio Nazzareno
  
  Abstract
  
  Dyslipidemia is a well-established condition proved to accelerate the progression of chronic kidney disease leading to tubulo-interstitial injury. However, the molecular aspects of the dyslipidemia-induced renal damage have not been fully clarified and in particular the role played by low-density lipoproteins (LDLs). This study aimed to examine the effects of native non-oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS-induced ROS release mechanism. The LDL-dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mt??, increased Ca(2+) uptake and loss of cytochrome c. All the above LDL-induced effects were completely abrogated by chelating extracellular Ca(2+) as well as by inhibition of the Ca(2+) -activated cytoplasmic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL-induced intracellular redox unbalance is triggered by a Ca(2+) inward flux-dependent commencement of cPLA2 followed by activation of a lipid- and ROS-based cross-talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox- and Ca(2+) -dependent dysfunctions leading to cell-harming conditions. These findings may help to clarify the mechanism of dyslipidemia-induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.
  
  © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
  
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• Small discoidal pre-beta1 HDL particles are efficient acceptors of cell cholesterol via ABCA1 and ABCG1.
  Biochemistry

  2009 Nov;48(46):11067-74. doi: 10.1021/bi901564g.
  
  Favari Elda, Calabresi Laura, Adorni Maria Pia, Jessup Wendy, Simonelli Sara, Franceschini Guido, Bernini Franco
  
  Abstract
  
  The aim of this study was to correlate the lipid content and size of discoidal reconstituted HDL particles with their ability to promote cellular cholesterol efflux. Homogeneous discoidal rHDL particles containing apoA-I and POPC, with diameters of 7.8, 9.6, 10.8, 12.5, and 17.0 nm, were prepared by the cholate dialysis technique. Cholesterol efflux to rHDL was evaluated in pathway-specific cell models for ABCA1-, ABCG1-, and SR-BI-mediated efflux. ABCA1-mediated efflux was efficiently promoted by the 7.8 nm rHDL containing 82 POPC molecules per particle. This rHDL also promoted ABCG1, but not SR-BI, cholesterol efflux. All large and lipid-rich rHDLs, with a diameter of >or=9.6 nm and a phospholipid content of >/=202 molecules per particle, promoted both SR-BI- and ABCG1-mediated efflux. Our results indicated that the ABCA1-mediated cell cholesterol efflux can be efficiently driven not only by monomolecular lipid free/poor apoA-I but also by a small discoidal phospholipid-containing particle resembling plasma pre-beta1 HDL. This same particle also promotes ABCG1- but not SR-BI-mediated efflux. These results help to clarify the role of plasma pre-beta1 HDL in reverse cholesterol transport.
  
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• Structural features and dynamics properties of human apolipoprotein A-I in a model of synthetic HDL.
  J Mol Graph Model

  2009 Nov;28(4):305-12. doi: 10.1016/j.jmgm.2009.08.008.
  
  Rocco Alessandro Guerini, Gianazza Elisabetta, Calabresi Laura, Sensi Cristina, Franceschini Guido, Sirtori Cesare R, Eberini Ivano
  
  Abstract
  
  High-density lipoproteins (HDL) play a major role in the reverse transport of cholesterol and have antiatherogenic activities. Their major protein component is apolipoprotein (apo) A-I. While apoA-I amphipathic alpha-helix based secondary structure has been extensively investigated, for its lipid-bound tertiary structure only theoretical models have been proposed. In the past years, experimental approaches aimed at a direct visualization of HDL structure have been exploited, but data obtained through different microscopy techniques are conflicting and do not settle the issue. Here we present a 50 ns molecular dynamics simulation of a synthetic HDL containing two molecules of apoA-I and 101 of l-alpha-palmitoyl-oleoyl-phosphatidylcholine. Essential dynamics and structural property investigations suggest that the stabilization of the system is obtained through specific motions, whose driving forces are protein-phospholipid interactions. The most important are: the relative sliding of the two apoA-I molecules along their major axes, the relative rotation of the protein chains, and the out-of-plane deformation around proline hinges. The sliding and the out-of-plane deformation allow apoA-I to optimize its interactions with phospholipids, while the rotation is useful to maximize protein-protein salt bridges. The correspondence between computed parameters and their experimental counterparts contributes to validate our model and its dynamic behaviors. Our findings help in defining a molecular model for apoA-I contained in HDL and suggest a possible mechanism through which apoA-I can vary its diameter and accommodate different numbers of phospholipids during the metabolism of HDL.
  
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• Functional lecithin: cholesterol acyltransferase is not required for efficient atheroprotection in humans.
  Circulation

  2009 Aug;120(7):628-35. doi: 10.1161/CIRCULATIONAHA.108.818143.
  
  Calabresi Laura, Baldassarre Damiano, Castelnuovo Samuela, Conca Paola, Bocchi Letizia, Candini Chiara, Frigerio Beatrice, Amato Mauro, Sirtori Cesare R, Alessandrini Paola, Arca Marcello, Boscutti Giuliano, Cattin Luigi, Gesualdo Loreto, Sampietro Tiziana, Vaudo Gaetano, Veglia Fabrizio, Calandra Sebastiano, Franceschini Guido
  
  Abstract
  
  BACKGROUND:
  Mutations in the LCAT gene cause lecithin:cholesterol acyltransferase (LCAT) deficiency, a very rare metabolic disorder with 2 hypoalphalipoproteinemia syndromes: classic familial LCAT deficiency (Online Mendelian Inheritance in Man No. 245900), characterized by complete lack of enzyme activity, and fish-eye disease (Online Mendelian Inheritance in Man No. 136120), with a partially defective enzyme. Theoretically, hypoalphalipoproteinemia cases with LCAT deficiency should be at increased cardiovascular risk because of high-density lipoprotein deficiency and defective reverse cholesterol transport.
  
  METHODS AND RESULTS:
  The extent of preclinical atherosclerosis was assessed in 40 carriers of LCAT gene mutations from 13 Italian families and 80 healthy controls by measuring carotid intima-media thickness (IMT). The average and maximum IMT values in the carriers were 0.07 and 0.21 mm smaller than in controls (P=0.0003 and P=0.0027), respectively. Moreover, the inheritance of a mutated LCAT genotype had a remarkable gene-dose-dependent effect in reducing carotid IMT (P=0.0003 for average IMT; P=0.001 for maximum IMT). Finally, no significant difference in carotid IMT was found between carriers of LCAT gene mutations that cause total or partial LCAT deficiency (ie, familial LCAT deficiency or fish-eye disease).
  
  CONCLUSIONS:
  Genetically determined low LCAT activity in Italian families is not associated with enhanced preclinical atherosclerosis despite low high-density lipoprotein cholesterol levels. This finding challenges the notion that LCAT is required for effective atheroprotection and suggests that elevating LCAT expression or activity is not a promising therapeutic strategy to reduce cardiovascular risk.
  
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• A novel homozygous mutation in CETP gene as a cause of CETP deficiency in a Caucasian kindred.
  Atherosclerosis

  2009 Aug;205(2):506-11. doi: 10.1016/j.atherosclerosis.2009.01.006.
  
  Calabresi Laura, Nilsson Peter, Pinotti Elisa, Gomaraschi Monica, Favari Elda, Adorni Maria Pia, Bernini Franco, Sirtori Cesare R, Calandra Sebastiano, Franceschini Guido, Tarugi Patrizia
  
  Abstract
  
  OBJECTIVE:
  To analyze the cholesteryl ester transfer protein (CETP) gene and the plasma HDL phenotype in a Caucasian subject with extremely elevated plasma high density lipoprotein-cholesterol (HDL-C).
  
  METHODS AND RESULTS:
  The proband, a 63-year-old male of Swedish ancestry with elevated HDL-C (208mg/dl) and apoA-I (and 272mg/dl), was found to be homozygous for a point mutation in exon 2 of CETP gene (c.109 C>T) resulting in a premature termination codon (R37X). Plasma CETP mass and activity were undetectable. Plasma HDL were characterized by predominance of large HDL with enhanced prebeta-HDL content. The proband's sons, heterozygotes for the mutation, had reduced plasma CETP activity and moderately elevated HDL-C. Serum of CETP deficient subjects showed a normal or enhanced cholesterol efflux capacity via ABCG1/SR-BI; cholesterol efflux via ABCA1 and macrophage cholesterol removal were lower than normal. The proband was healthy and had no atherosclerotic plaques in carotid or femoral arteries.
  
  CONCLUSION:
  Complete CETP deficiency caused by mutations in CETP gene is exceedingly rare in Caucasians; the description of this single case indicates that CETP deficiency does not predispose to atherosclerosis in the absence of major cardiovascular risk factors.
  
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• Novel mutations of CETP gene in Italian subjects with hyperalphalipoproteinemia.
  Atherosclerosis

  2009 May;204(1):202-7. doi: 10.1016/j.atherosclerosis.2008.08.031.
  
  Cefalù Angelo B, Noto Davide, Magnolo Lucia, Pinotti Elisa, Gomaraschi Monica, Martini Scipione, Vigna Giovanni B, Calabresi Laura, Tarugi Patrizia, Averna Maurizio R
  
  Abstract
  
  Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that catalyses the transfer of cholesteryl esters from HDL to the other plasma lipoproteins. Genetic deficiency of CETP is one of the known causes of elevation of plasma HDL-C (primary hyperalphalipoproteinemia, HALP). We sequenced CETP gene in a group of 24 Italian subjects with primary HALP (HDL-C>80 mg/dl) suspected to have CETP deficiency. Two unrelated subjects both coming from the same geographical district, were found to be heterozygous for a nucleotide substitution in exon 6 (c.544C>T) and another subject was found to be heterozygous for a C>T transition in exon 9 (c.802C>T). Both mutations introduce a premature stop codon and are predicted to cause the production of truncated proteins (Q165X and R268X, respectively) devoid of function. The fourth proband was found to carry a T>C substitution in intron 15 (c.1407+2T>C) predicted to abolish the function of the donor splice site. To define the effect of this mutation on CETP pre-mRNA splicing we analysed CETP mRNA in COS-1 cells expressing a CETP minigene harbouring the mutation. The analysis of minigene transcript in COS-1 cells showed that IVS15+2T>C mutation caused the formation of an abnormal mRNA in which exon 14 joins directly to exon 16, predicted to encode a truncated peptide of 435 amino acids. In mutation carriers plasma CETP activity was found to be reduced by 38-60%. These are the first mutations in the CETP gene found in Italian subjects with HALP.
  
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• Functional LCAT is not required for macrophage cholesterol efflux to human serum.
  Atherosclerosis

  2009 May;204(1):141-6. doi: 10.1016/j.atherosclerosis.2008.08.038.
  
  Calabresi Laura, Favari Elda, Moleri Elsa, Adorni Maria Pia, Pedrelli Matteo, Costa Sara, Jessup Wendy, Gelissen Ingrid C, Kovanen Petri T, Bernini Franco, Franceschini Guido
  
  Abstract
  
  OBJECTIVES:
  To evaluate the capacity of serum from carriers of LCAT gene mutations to promote cell cholesterol efflux through the ABCA1, ABCG1, and SR-BI pathways.
  
  METHODS:
  Serum was obtained from 41 carriers of mutant LCAT alleles (14 carriers of two mutant LCAT alleles and 27 heterozygotes) and 10 non-carrier relatives (controls). The capacity of serum to promote cholesterol efflux was tested in pathway-specific cell models.
  
  RESULTS:
  LCAT deficient sera were significantly more efficient than control sera in promoting cell cholesterol efflux via ABCA1 (3.1+/-0.3% for carriers of two mutant LCAT alleles and 2.6+/-0.2% for heterozygotes vs. 1.5+/-0.4% for controls), and less efficient in promoting ABCG1- and SR-BI-mediated cholesterol efflux. The enhanced capacity of LCAT deficient serum for ABCA1 efflux is explained by the increased content of prebeta-HDL, as indicated by the significant positive correlation between ABCA1 efflux and serum prebeta-HDL content (R=0.468, P
  CONCLUSIONS:
  Serum from carriers of LCAT gene mutations has the same capacity of control serum to decrease the cholesterol content of cholesterol-loaded macrophages due to a greater cholesterol efflux capacity via ABCA1.
  
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• The plasma concentration of Lpa-I:A-II particles as a predictor of the inflammatory response in patients with ST-elevation myocardial infarction.
  Atherosclerosis

  2009 Jan;202(1):304-11. doi: 10.1016/j.atherosclerosis.2008.04.004.
  
  Gomaraschi Monica, Sinagra Gianfranco, Serdoz Laura Vitali, Pitzorno Cristina, Fonda Maurizio, Cattin Luigi, Calabresi Laura, Franceschini Guido
  
  Abstract
  
  OBJECTIVE:
  To investigate the relationship between plasma HDL at admission and the extent of the inflammatory response during an ST-elevation myocardial infarction (STEMI), and to analyse structural HDL changes during STEMI as related to the extent of inflammation.
  
  METHODS AND RESULTS:
  CRP and IL-6 were monitored for 96h in 45 patients with STEMI. Plasma apoA-II and LpA-I:A-II levels at admission, but not HDL cholesterol or other HDL-related biomarkers, were associated with the extent of the inflammatory response during STEMI, as indicated by the positive correlations with CRP AUC (apoA-II: F=7.44, p=0.009; LpA-I:A-II: F=14.29, p
  CONCLUSION:
  An elevated plasma concentration of LpA-I:A-II particles was an independent predictor of a more severe inflammatory response in patients with STEMI.
  
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• The LXR agonist T0901317 promotes the reverse cholesterol transport from macrophages by increasing plasma efflux potential.
  J Lipid Res

  2008 May;49(5):954-60. doi: 10.1194/jlr.M700254-JLR200.
  
  Zanotti Ilaria, Potì Francesco, Pedrelli Matteo, Favari Elda, Moleri Elsa, Franceschini Guido, Calabresi Laura, Bernini Franco
  
  Abstract
  
  The liver X receptors (LXRs) have been shown to affect lipoprotein plasma profile, lipid metabolism, and reverse cholesterol transport (RCT). In the present study, we investigated whether a short-term administration of the synthetic LXR agonist T0901317 (T0) to mice may affect RCT by modulating the capacity of plasma to promote cellular lipid efflux. Consistent with previous data, the pharmacological treatment of mice caused a significant increase of macrophage-derived [3H]cholesterol content in plasma, liver, and feces and resulted in improved capacity of plasma to promote cellular cholesterol release through passive diffusion and scavenger receptor class B type I (SR-BI)-mediated mechanisms. Differently, plasma from treated mice possessed similar or reduced capacity to drive lipid efflux via ABCA1. Consistent with these data, the analysis of plasma HDL fractions revealed that T0 caused the formation of larger, lipid-enriched particles. These results suggest that T0 promotes in vivo RCT from macrophages at least in part by inducing an enrichment of those HDL subclasses that increase plasma capacity to promote cholesterol efflux by passive diffusion and SR-BI-mediated mechanisms.
  
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• Anti-inflammatory and cardioprotective activities of synthetic high-density lipoprotein containing apolipoprotein A-I mimetic peptides.
  J Pharmacol Exp Ther

  2008 Feb;324(2):776-83.
  
  Gomaraschi Monica, Calabresi Laura, Rossoni Giuseppe, Iametti Stefania, Franceschini Guido, Stonik John A, Remaley Alan T
  
  Abstract
  
  Apolipoprotein A-I (apoA-I) mimetic peptides may represent an alternative to apoA-I for large-scale production of synthetic high-density lipoproteins (sHDL) as a therapeutic agent. In this study, the cardioprotective activity of sHDL made with either L37pA peptide or its d-stereoisomer, D37pA, was compared to sHDL made with apoA-I. The peptides were reconstituted with palmitoyl-oleoyl-phosphatidylcholine, which yielded sHDL particles comparable to apoA-I sHDL in diameter, molecular weight, and alpha-helical content. Pretreatment of endothelial cells with either peptide sHDL reduced tumor necrosis factor alpha-stimulated vascular cell adhesion molecule-1 expression to the same extent as apoA-I sHDL. In an isolated rat heart model of ischemia/reperfusion (I/R) injury, L37pA and D37pA sHDL significantly reduced postischemic cardiac contractile dysfunction compared to the saline control, as indicated by a 49.7 +/- 6.4% (L37pA; P
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• Normal vascular function despite low levels of high-density lipoprotein cholesterol in carriers of the apolipoprotein A-I(Milano) mutant.
  Circulation

  2007 Nov;116(19):2165-72.
  
  Gomaraschi Monica, Baldassarre Damiano, Amato Mauro, Eligini Sonia, Conca Paola, Sirtori Cesare R, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  BACKGROUND:
  Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) mutant have very low plasma high-density lipoprotein cholesterol (HDL-C) levels but do not show any history of premature cardiovascular disease or any evidence of preclinical vascular disease. HDL is believed to prevent the development of vascular dysfunction, which may well contribute to HDL-mediated atheroprotection. Whether the low HDL level of apoA-I(M) carriers is associated with impaired vascular function is presently unknown.
  
  METHODS AND RESULTS:
  The vascular response to reactive hyperemia, assessed by measuring postischemic increase in forearm arterial compliance, and the plasma concentration of soluble cell adhesion molecules were evaluated in 21 adult apoA-I(M) carriers, 21 age- and gender-matched nonaffected relatives (control subjects), and 21 healthy subjects with low HDL-C (low-HDL subjects). The average plasma HDL-C and apoA-I levels of apoA-I(M) carriers were remarkably lower than those of control subjects and significantly lower than those of low-HDL subjects. The postischemic increase in forearm arterial compliance in the apoA-I(M) carriers was 2-fold greater than in low-HDL subjects and remarkably similar to that of control subjects. Plasma soluble cell adhesion molecule levels were similar in apoA-I(M) carriers and control subjects but were greater in low-HDL subjects. When incubated with endothelial cells, HDL isolated from apoA-I(M) carriers was more effective than HDL from control and low-HDL subjects in stimulating endothelial nitric oxide synthase expression and activation and in downregulating tumor necrosis factor-alpha-induced expression of vascular cell adhesion molecule-1.
  
  CONCLUSIONS:
  Despite their very low HDL levels, apoA-I(M) carriers do not display typical features of impaired vascular function because of an improved activity of apoA-I(M) HDL in maintaining endothelial cell homeostasis.
  
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• A novel mutation of the apolipoprotein A-I gene in a family with familial combined hyperlipidemia.
  Atherosclerosis

  2008 May;198(1):145-51.
  
  Pisciotta Livia, Fasano Tommaso, Calabresi Laura, Bellocchio Antonella, Fresa Raffaele, Borrini Claudia, Calandra Sebastiano, Bertolini Stefano
  
  Abstract
  
  We report a large family in which four members showed a plasma lipid profile consistent with the clinical diagnosis of familial combined hyperlipidemia (FCHL). One of these patients was found to have markedly reduced HDL cholesterol (HDL-C) (0.72 mmol/l) and Apo A-I (72 mg/dl) levels, a condition suggestive of the presence of a mutation in one of the HDL-related genes. The analysis of APOA1 gene revealed that this patient was heterozygous for a cytosine insertion in exon 3 (c.49-50 ins C), resulting in a frame-shift and premature stop codon at position 26 of pro-Apo A-I (Q17PFsX10). This novel mutation, which prevents the synthesis of Apo A-I, was also found in four family members, including three siblings and the daughter of the proband. Carriers of Apo A-I mutation had significantly lower HDL-C and Apo A-I than non-carriers family members (0.77+/-0.15 mmol/l vs. 1.15+/-0.20 mmol/l, P
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• European Lipoprotein Club: Report of the 29th ELC Annual Conference, Tutzing, 4-7 September 2006.
  Atherosclerosis

  2007 Aug;193(2):461-8.
  
  Hofker Marten, Calabresi Laura, von Eckardstein Arnold, Heeren Joerg, Karpe Fredrik, Kalopissis Athina, Kronenberg Florian, Kuipers Folkert, Lindstedt Ken, Parini Paolo, Schuster Gertrud, van Dijk Ko Willems
  
  
  
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• Apolipoprotein A-I breakdown is induced by thrombolysis in coronary patients.
  Ann Med

  2007 ;39(4):306-11.
  
  Eberini Ivano, Gianazza Elisabetta, Breghi Loranni, Klugmann Silvio, Calabresi Laura, Gomaraschi Monica, Mombelli Giuliana, Brusoni Bruno, Wait Robin, Sirtori Cesare R
  
  Abstract
  
  BACKGROUND:
  The outcome of percutaneous coronary intervention (PCI) is apparently worse in patients receiving a prior thrombolytic therapy ('facilitated PCI'). Recombinant tissue-type plasminogen activator (rt-PA) can degrade circulating high-density lipoproteins (HDL) bound apolipoprotein A-I (apoA-I), thus possibly reducing the vascular protective activity. There have never been reports of the detection of apolipoprotein breakdown products in the circulation.
  
  AIM:
  We studied the potential interactions between the protein components of HDL and tenecteplase, infused as thrombolytic therapy.
  
  METHODS:
  Sera from a total of 40 patients with acute myocardial infarction (AMI), unstable angina (UA), and dilative cardiomyopathy (controls) were investigated. AMI patients underwent either immediate PCI or were treated with tenecteplase thrombolysis.
  
  RESULTS:
  Products of extensive proteolysis of apoA-I were found in many acute coronary patients treated with tenecteplase, and in some AMI patients before starting the treatment (time 0). These were not detected in controls, UA patients as well as AMI patients undergoing immediate PCI. Small pre-beta-HDLs were selectively degraded.
  
  CONCLUSION:
  Significant apoA-I degradation occurs in AMI patients after thrombolytic treatment. This finding may provide a potential mechanism for the apparent reduction of benefit of facilitated versus nonfacilitated PCI.
  
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• Molecular characterization of two patients with severe LCAT deficiency.
  Nephrol Dial Transplant

  2007 Aug;22(8):2379-82.
  
  Charlton-Menys Valentine, Pisciotta Livia, Durrington Paul N, Neary Richard, Short Colin D, Calabresi Laura, Calandra Sebastiano, Bertolini Stefano
  
  
  
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• Tolerability of statins is not linked to CYP450 polymorphisms, but reduced CYP2D6 metabolism improves cholesteraemic response to simvastatin and fluvastatin.
  Pharmacol Res

  2007 Apr;55(4):310-7.
  
  Zuccaro Piergiorgio, Mombelli Giuliana, Calabresi Laura, Baldassarre Damiano, Palmi Ilaria, Sirtori Cesare R
  
  Abstract
  
  Statin therapy, although generally well tolerated, leads not infrequently to significant subjective and at times objective adverse effects (AEs), mainly of a muscular nature. The genetic background of these AEs is not clear and possibly side effects and lipid lowering efficacy may be linked. Aim of the study was a detailed evaluation of CYP450 and apolipoprotein E gene polymorphisms in two large series of age-sex matched patients with and without muscular side effects to statins. In a Clinical Institution specialised in lipid-lipoprotein disorders, 50 statin treated patients were selected, with subjective or objective statin-associated myopathy, evaluated using standardized forms. These were sex and age matched with 50 statin-treated patients from the same Clinic, without any subjective or objective complaints. DNA samples for the evaluation of CYP450 genetic polymorphisms and apo E genotypes were collected in order to assess correlations with both genetic polymorphisms and AEs, as well as with therapeutic efficacy. None of the assessed CYP450 polymorphisms appeared to be related to an increased incidence of AEs. The CYP2D6 *1/*4 and *4/4* poor metabolizer (PM) status was associated to a higher efficacy of statins metabolized by this system and, in addition, the apo E2 genotype was, in this series, linked to increased HDL-C levels after therapy. Patients with statin associated myopathy are not characterized by significantly different genotypes for the CYP450s responsible for statin metabolism. On the other hand, CYP2D6 PM status is associated to an increased efficacy of statins metabolized by this system.
  
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• Role of LCAT in HDL remodeling: investigation of LCAT deficiency states.
  J Lipid Res

  2007 Mar;48(3):592-9.
  
  Asztalos Bela F, Schaefer Ernst J, Horvath Katalin V, Yamashita Shizuya, Miller Michael, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.
  
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• A unique protease-sensitive high density lipoprotein particle containing the apolipoprotein A-I(Milano) dimer effectively promotes ATP-binding Cassette A1-mediated cell cholesterol efflux.
  J Biol Chem

  2007 Feb;282(8):5125-32.
  
  Favari Elda, Gomaraschi Monica, Zanotti Ilaria, Bernini Franco, Lee-Rueckert Miriam, Kovanen Petri T, Sirtori Cesare R, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Carriers of the apolipoprotein A-I(Milano) (A-I(M)) variant present with severe reductions of plasma HDL levels, not associated with premature coronary heart disease (CHD). Sera from 14 A-I(M) carriers and matched controls were compared for their ability to promote ABCA1-driven cholesterol efflux from J774 macrophages and human fibroblasts. When both cell types are stimulated to express ABCA1, the efflux of cholesterol through this pathway is greater with A-I(M) than control sera (3.4 +/- 1.0% versus 2.3 +/- 1.0% in macrophages; 5.2 +/- 2.4% versus 1.9 +/- 0.1% in fibroblasts). A-I(M) and control sera are instead equally effective in removing cholesterol from unstimulated cells and from fibroblasts not expressing ABCA1. The A-I(M) sera contain normal amounts of apoA-I-containing prebeta-HDL and varying concentrations of a unique small HDL particle containing a single molecule of the A-I(M) dimer; chymase treatment of serum degrades both particles and abolishes ABCA1-mediated cholesterol efflux. The serum content of chymase-sensitive HDL correlates strongly and significantly with ABCA1-mediated cholesterol efflux (r = 0.542, p = 0.004). The enhanced capacity of A-I(M) serum for ABCA1 cholesterol efflux is thus explained by the combined occurrence in serum of normal amounts of apoA-I-containing prebeta-HDL, together with a unique protease-sensitive, small HDL particle containing the A-I(M) dimer, both effective in removing cell cholesterol via ABCA1.
  
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• Effects of fenofibrate and simvastatin on HDL-related biomarkers in low-HDL patients.
  Atherosclerosis

  2007 Dec;195(2):385-91.
  
  Franceschini Guido, Calabresi Laura, Colombo Cinzia, Favari Elda, Bernini Franco, Sirtori Cesare R
  
  Abstract
  
  The objective of the present study was to compare the effects of fenofibrate versus simvastatin on various HDL-related biomarkers in dyslipidemic patients with low HDL-C, in whom it is as yet unclear whether a statin or a fibrate is the most appropriate treatment. Fifty-two patients received either fenofibrate (160 mg/day) or simvastatin (40 mg/day) for 8 weeks in a randomized, double-blind, parallel group trial. Simvastatin effectively lowered plasma LDL-C and apoB levels, but did not change plasma HDL levels and HDL-related biomarkers, except for a small, significant increase in the capacity of plasma to promote SR-BI mediated cholesterol efflux. Fenofibrate did not affect plasma LDL-C levels but lowered triglycerides, and exerted a remarkable HDL-C raising activity (+22%), with patients in the lowest range of HDL-C getting the maximal benefit. The HDL-C raise was associated with a shift of HDL from large to small particles, and from LpA-I to LpA-I:A-II, which might explain the observed increase in the plasma capacity to promote ABCA1 mediated efflux with no changes in SR-BI efflux. The distinct and complementary effects of fenofibrate and simvastatin on lipid parameters and HDL-related biomarkers suggest that a combination therapy with the two drugs in dyslipidemic patients with low HDL would be fully justified.
  
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• Structure, function and amyloidogenic propensity of apolipoprotein A-I.
  Amyloid

  2006 Dec;13(4):191-205.
  
  Obici Laura, Franceschini Guido, Calabresi Laura, Giorgetti Sofia, Stoppini Monica, Merlini Giampaolo, Bellotti Vittorio
  
  Abstract
  
  Apolipoprotein A-I, the major structural apolipoprotein of high-density lipoproteins, efficiently protects humans from cholesterol accumulation in tissues; however, it can cause systemic amyloidosis in the presence of peculiar amino acid replacements. The wild-type molecule also has an intrinsic tendency to generate amyloid fibrils that localise within the atherosclerotic plaques. The structure, folding and metabolism of normal apolipoprotein A-I are extremely complex and as yet not completely clarified, but their understanding appears essential for the elucidation of the amyloid transition. We reviewed present knowledge on the structure, function and amyloidogenic propensity of apolipoprotein A-I with the aim of highlighting the possible molecular mechanisms that might contribute to the pathogenesis of this disease. Important clues on apolipoprotein A-I amyloidogenesis may be obtained from classical comparative studies of the properties of the wild-type versus the amyloidogenic counterpart. Additionally, in the case of apoA-I, further insights on the molecular mechanisms underlying its amyloidogenic propensity may derive from comparative studies between amyloidogenic variants and other mutations associated with hypoalphalipoproteinemia without amyloidosis.
  
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• Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection.
  FEBS Lett

  2006 Oct;580(25):5974-8.
  
  Bellosta Stefano, Gomaraschi Monica, Canavesi Monica, Rossoni Giuseppe, Monetti Mara, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.
  
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• Japan: are statins still good for everybody?
  Lancet

  2006 Sep;368(9542):1135-6.
  
  Sirtori Cesare R, Calabresi Laura
  
  
  
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• Synthetic high density lipoproteins for the treatment of myocardial ischemia/reperfusion injury.
  Pharmacol Ther

  2006 Sep;111(3):836-54.
  
  Calabresi Laura, Gomaraschi Monica, Rossoni Giuseppe, Franceschini Guido
  
  Abstract
  
  Synthetic high density lipoproteins (sHDL) are discoidal lipoprotein particles made of an apolipoprotein and a phospholipid, which mimic most, if not all, of the atheroprotective properties of plasma HDL, including stimulation of reverse cholesterol transport (RCT), prevention of endothelial dysfunction, and inhibition of lipid oxidation. sHDL are currently under development as a novel treatment for atherosclerotic cardiovascular disease. A number of preclinical studies have demonstrated the ability of single or multiple injections of sHDL to induce the regression of atherosclerotic plaques and prevent arterial restenosis. In the first phase II trial in patients with acute coronary syndromes, a short-term treatment with sHDL containing the disulfide-linked dimer of the apolipoprotein A-IMilano variant (A-IM/A-IM) caused a remarkable reduction of atheroma volume. sHDL also display a direct cardioprotective activity in ex vivo and in vivo models of myocardial ischemia/reperfusion (I/R) injury, and may become a useful adjunctive therapy to improve clinical outcomes in patients with acute coronary syndromes or undergoing coronary procedures.
  
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• A model structure for the heterodimer apoA-IMilano-apoA-II supports its peculiar susceptibility to proteolysis.
  Biophys J

  2006 Oct;91(8):3043-9.
  
  Rocco Alessandro Guerini, Mollica Luca, Gianazza Elisabetta, Calabresi Laura, Franceschini Guido, Sirtori Cesare R, Eberini Ivano
  
  Abstract
  
  In this study, we propose a structure for the heterodimer between apolipoprotein A-I(Milano) and apolipoprotein A-II (apoA-I(M)-apoA-II) in a synthetic high-density lipoprotein (HDL) containing L-alpha-palmitoyloleoyl phosphatidylcholine. We applied bioinformatics/computational tools and procedures, such as molecular docking, molecular and essential dynamics, starting from published crystal structures for apolipoprotein A-I and apolipoprotein A-II. Structural and energetic analyses onto the simulated system showed that the molecular dynamics produced a stabilized synthetic HDL. The essential dynamic analysis showed a deviation from the starting belt structure. Our structural results were validated by limited proteolysis experiments on HDL from apoA-I(M) carriers in comparison with control HDL. The high sensitivity of apoA-I(M)-apoA-II to proteases was in agreement with the high root mean-square fluctuation values and the reduction in secondary structure content from molecular dynamics data. Circular dichroism on synthetic HDL containing apoA-I(M)-apoA-II was consistent with the alpha-helix content computed on the proposed model.
  
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• LCAT deficiency: molecular and phenotypic characterization of an Italian family.
  J Nephrol

  2006 ;19(3):375-81.
  
  Gigante Maddalena, Ranieri Elena, Cerullo Giuseppina, Calabresi Laura, Iolascon Achille, Assmann Gerd, Morrone Luigi, Pisciotta Livia, Schena Francesco Paolo, Gesualdo Loreto
  
  Abstract
  
  Familial lecithin:cholesterol acyltransferase (LCAT) deficiency is an autosomal recessive disorder of lipoprotein metabolism, resulting from loss of function of lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43), a key enzyme in extracellular cholesterol metabolism and reverse cholesterol transport (RCT). The human LCAT gene has been mapped to chromosome band 16q22, and consists of 6 exons encoding for a mature protein of 416 amino acids. In the present study, we describe the molecular phenotype of a patient with classical LCAT deficiency and progressive renal failure. Sequence analysis of the LCAT gene showed 2 homozygous missense mutations: the common variant p.S208T, described as a homozygous change for the first time, and a missense mutation characterized by the substitution of Leu372 to Arg. Clinical, biochemical and renal histological studies were also performed to elucidate the functional effects of these variations. In the proband and his brother, LCAT activity and plasma cholesterol esterification rate (CER) were absent, while plasma LCAT concentrations were slightly reduced. By light microscopy, silver-stained renal biopsy specimens of the proband showed focal segmental glomerulosclerosis, while electron microscopy detected lipid deposits with both vacuolar lucent appearance and electron-dense lamellar structures within the mesangial matrix and glomerular basement membrane. This study describes for the first time the occurrence of two homozygous missense mutations as the common variant p.S208T and the mutation p.L372R in familial LCAT deficiency.
  
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• High-density lipoproteins: a therapeutic target for atherosclerotic cardiovascular disease.
  Expert Opin Ther Targets

  2006 Aug;10(4):561-72.
  
  Gomaraschi Monica, Calabresi Laura, Franceschini Guido
  
  Abstract
  
  Despite great progress being made during the last two decades in cardiovascular disease prevention, especially by lowering low-density lipoprotein-cholesterol with statins, cardiovascular events continue to occur. Plasma high-density lipoprotein (HDL) exerts multiple protective effects on the arterial wall, through promotion of reverse cholesterol transport, prevention of endothelial dysfunction and inhibition of lipid oxidation. Therapeutic interventions raising plasma HDL levels or directly mimicking its beneficial effects represent the next frontier in the prevention and treatment of cardiovascular disease.
  
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• CETP levels rather than polymorphisms as markers of coronary risk: healthy athlete with high HDL-C and coronary disease--effectiveness of probucol.
  Atherosclerosis

  2006 May;186(1):225-7.
  
  Sirtori Cesare R, Calabresi Laura, Baldassarre Damiano, Franceschini Guido, Cefalù Angelo B, Averna Maurizio
  
  
  
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• Recombinant apolipoprotein A-IMilano for the treatment of cardiovascular diseases.
  Curr Atheroscler Rep

  2006 Mar;8(2):163-7.
  
  Calabresi Laura, Sirtori Cesare R, Paoletti Rodolfo, Franceschini Guido
  
  Abstract
  
  Apolipoprotein A-I(Milano) (apoA-I(M)) is a natural variant of apoA-I characterized by a cysteine for arginine substitution at position 173 of the primary sequence. ApoA-I(M) carriers have much less atherosclerosis than expected from their very low plasma high-density lipoprotein (HDL) cholesterol levels, suggesting that the variant might be protective. Synthetic HDL (sHDL) made with a recombinant form of the dimeric A-I(M) (A-I(M)/A-I(M)) and phospholipids given in single or multiple injections is effective in inducing the regression of atherosclerotic plaques, preventing arterial restenosis, and limiting cardiac dysfunction after ischemia/reperfusion injury. In a phase II trial in patients with acute coronary syndromes, a short-term treatment with A-I(M)/A-I(M) sHDL caused a remarkable reduction of atheroma burden. Although at early stages of drug development, A-I(M)/A-I(M) sHDL holds vast promise for the treatment of a variety of cardiovascular diseases in humans.
  
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• High-density lipoproteins attenuate interleukin-6 production in endothelial cells exposed to pro-inflammatory stimuli.
  Biochim Biophys Acta

  2005 Sep;1736(2):136-43.
  
  Gomaraschi Monica, Basilico Nicoletta, Sisto Francesca, Taramelli Donatella, Eligini Sonia, Colli Susanna, Sirtori Cesare R, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  The purpose of the present study was to investigate the ability of high-density lipoproteins (HDL) to attenuate endothelial dysfunction, by assessing down-regulation of cytokine-induced interleukin-6 (IL-6) production in cultured endothelial cells, and measuring plasma IL-6 levels in three groups of healthy individuals with low, average, or high plasma HDL-cholesterol. Human plasma HDL caused a concentration-dependent inhibition of TNFalpha-induced IL-6 production in human endothelial cells (by 58.5+/-1.5% at 2 mg of HDL-protein/ml). Reconstituted HDL made with apolipoprotein A-I (apoA-I) and phosphatidylcholine were as effective as plasma HDL, while lipid-free apoA-I or phosphatidylcholine liposomes had no effect. HDL attenuated IL-6 mRNA levels, an effect which occurs through inhibition of p38 MAP kinase. The median plasma IL-6 concentration was significantly higher in subjects with low HDL-cholesterol (2.54 pg/ml) compared with those with average or high HDL-cholesterol (1.31 pg/ml and 1.47 pg/ml, respectively). When all subjects were considered together, a lower HDL-cholesterol was the strongest independent predictor of higher IL-6 (F=25.38, P
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• Combined monogenic hypercholesterolemia and hypoalphalipoproteinemia caused by mutations in LDL-R and LCAT genes.
  Atherosclerosis

  2005 Sep;182(1):153-9.
  
  Pisciotta Livia, Calabresi Laura, Lupattelli Graziana, Siepi Donatella, Mannarino Massimo Raffaele, Moleri Elsa, Bellocchio Antonella, Cantafora Alfredo, Tarugi Patrizia, Calandra Sebastiano, Bertolini Stefano
  
  Abstract
  
  We studied a three generation family with co-dominant monogenic hypercholesterolemia and hypoalphalipoproteinemia. The proband, a 48 year-old male, was found to be heterozygous for a previously reported mutation in LDL receptor (LDL-R) gene (IVS15-3 c>a) and a novel mutation in exon 6 of lecithin cholesterol acyltransferase (LCAT) gene (c.803 G>A) causing a non-synonymous amino acid substitution (p.R244H). These mutations segregated independently in the family. The LDL-R mutation was associated with high levels of LDL-C (6.20-9.85 mmol/L) and apo B (170-255 mg/dL), comparable to those previously reported in carriers of the same mutation. The LCAT mutation was associated with low levels of HDL-C (0.67-0.80 mmol/L) and apo A-I (96-110 mg/dL). The proband had reduced LCAT function, as measured by cholesterol esterification rate (29 nmol/(mL/h) versus 30-60 nmol/(mL/h)), LCAT activity (10 nmol/(mL/h) versus 20-55 nmol/(mL/h)) and LCAT mass (2.87 microg/mL versus 3.1-6.7 microg/mL). Carriers of LCAT mutation had lower LCAT activity and a tendency to reduced cholesterol esterification rate (CER) and LCAT mass as compared to non-carrier family members. The LCAT mutation was not found in 80 control subjects and 60 patients with primary hypoalphalipoproteinemia. Despite the unfavourable lipoprotein profile, the proband had only mild clinical signs of atherosclerosis. This unexpected finding is probably due to the intensive lipid lowering treatment the patient has been on over the last decade.
  
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• The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families.
  Arterioscler Thromb Vasc Biol

  2005 Sep;25(9):1972-8.
  
  Calabresi Laura, Pisciotta Livia, Costantin Anna, Frigerio Ilaria, Eberini Ivano, Alessandrini Paola, Arca Marcello, Bon Gabriele Bittolo, Boscutti Giuliano, Busnach Ghil, Frascà Giovanni, Gesualdo Loreto, Gigante Maddalena, Lupattelli Graziana, Montali Anna, Pizzolitto Stefano, Rabbone Ivana, Rolleri Marina, Ruotolo Giacomo, Sampietro Tiziana, Sessa Adalberto, Vaudo Gaetano, Cantafora Alfredo, Veglia Fabrizio, Calandra Sebastiano, Bertolini Stefano, Franceschini Guido
  
  Abstract
  
  OBJECTIVE:
  To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene.
  
  METHODS AND RESULTS:
  Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-beta high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene-dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former.
  
  CONCLUSIONS:
  In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene-dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.
  
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• Effect of statins on LDL particle size in patients with familial combined hyperlipidemia: a comparison between atorvastatin and pravastatin.
  Nutr Metab Cardiovasc Dis

  2005 Feb;15(1):47-55.
  
  Sirtori Cesare R, Calabresi Laura, Pisciotta Livia, Cattin Luigi, Pauciullo Paolo, Montagnani Mario, Manzato Enzo, Bittolo Bon Gabriele, Fellin Renato
  
  Abstract
  
  BACKGROUND AND AIM:
  Elevation of plasma cholesterol and/or triglycerides, and the prevalence of small dense low density lipoproteins (LDL) particles remarkably increase the risk in patients with familial combined hyperlipidemia (FCHL). There are, at present, inconsistent data on the effects of different treatments on size and density of LDL particles in FCHL patients.
  
  METHODS AND RESULTS:
  A multicenter, randomized, double-blind, double-dummy, parallel group study was designed to evaluate the effect of 3 months' treatment with atorvastatin (10mg/day) or pravastatin (20mg/day) on the lipid/lipoprotein profile and LDL size in a total of 86 FCHL patients. Both statins significantly lowered plasma total and LDL cholesterol, with a significantly higher hypocholesterolemic effect observed with atorvastatin (-26.8+/-11.1% and -35.9+/-11.1%, respectively) compared to pravastatin (-17.6+/-11.1% and -24.5+/-10.2%). The percent decrease in plasma triglycerides was highly variable, but more pronounced with atorvastatin (-19.8+/-29.2%) than with pravastatin (-5.3+/-48.6%). Opposite changes in LDL size were seen with the 2 treatments, with increased mean LDL particle diameter with atorvastatin, and decreased diameter with pravastatin, and significant between treatment difference in terms of percent modification vs baseline (+0.5+/-1.6% with atorvastatin vs -0.3+/-1.8% with pravastatin).
  
  CONCLUSIONS:
  The present results support the evidence indicative of a greater hypocholesterolemic effect of atorvastatin compared to pravastatin, and in addition show a raising effect of atorvastatin on the size of LDL particles in FCHL patients.
  
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• A 33-year-old man with nephrotic syndrome and lecithin-cholesterol acyltransferase (LCAT) deficiency. Description of two new mutations in the LCAT gene.
  Nephrol Dial Transplant

  2004 Jun;19(6):1622-4.
  
  Frascà Giovanni M, Soverini Letizia, Tampieri Elena, Franceschini Guido, Calabresi Laura, Pisciotta Livia, Preda Paola, Vangelista Alba, Stefoni Sergio, Bertolini Stefano
  
  
  
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• Liver biopsy discloses a new apolipoprotein A-I hereditary amyloidosis in several unrelated Italian families.
  Gastroenterology

  2004 May;126(5):1416-22.
  
  Obici Laura, Palladini Giovanni, Giorgetti Sofia, Bellotti Vittorio, Gregorini Gina, Arbustini Eloisa, Verga Laura, Marciano Sabrina, Donadei Simona, Perfetti Vittorio, Calabresi Laura, Bergonzi Cesare, Scolari Francesco, Merlini Giampaolo
  
  Abstract
  
  BACKGROUND & AIMS:
  Hereditary systemic amyloidoses are autosomal dominant, late-onset disorders caused by mutations in the genes for a group of plasma proteins including transthyretin, lysozyme, fibrinogen Aalpha chain, gelsolin, apolipoprotein A-I, and apolipoprotein A-II. We investigated both phenotypic and genotypic aspects of apolipoprotein A-I amyloidosis unexpectedly disclosed by liver biopsy in 13 unrelated individuals with asymptomatic, persistent elevation of alkaline phosphatase and gamma-glutamyltransferase levels.
  
  METHODS:
  Immunoelectron microscopy was used for in situ characterization of amyloid deposits on liver biopsy specimens. Mutation analysis was performed by sequencing of the apolipoprotein A-I gene in all patients. Wild-type/variant apolipoprotein A-I ratio in plasma high-density lipoproteins was assessed by a peptide mass fingerprinting approach after purification of total apolipoprotein A-I of 2 patients.
  
  RESULTS:
  Family history was informative in 5 cases. Renal failure developed in 9 cases. Hypogonadism due to testicular involvement was observed. Amyloid fibrils specifically stained with anti-apolipoprotein A-I antibody. A novel (Leu75Pro) heterozygous mutation in the apolipoprotein A-I gene was present in affected individuals but not in controls. Variant apolipoprotein A-I was about 10% of the total protein in high-density lipoproteins.
  
  CONCLUSIONS:
  The high number of individuals with apparently sporadic disease might reflect widespread occurrence of this mutation in the population and a milder phenotype of this variant compared with other apolipoprotein A-I amyloidogenic mutants. These findings suggest that specific staining for amyloid should be performed on liver biopsy of individuals with asymptomatic chronic elevation of alkaline phosphatase and gamma-glutamyltransferase levels.
  
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• Pharmacokinetic interactions between omeprazole/pantoprazole and clarithromycin in health volunteers.
  Pharmacol Res

  2004 May;49(5):493-9.
  
  Calabresi Laura, Pazzucconi Franco, Ferrara Stefano, Di Paolo Antonello, Tacca Mario Del, Sirtori Cesare
  
  Abstract
  
  The association omeprazole/clarithromycin is of current wide use in the treatment of Helicobacter pylori associated gastroduodenal ulcer. This combination may result in increased levels of omeprazole with potential interactions with commonly associated drugs. Kinetic/metabolic changes occurring after omeprazole/clarithromycin were compared to those occurring after pantoprazole/clarithromycin in healthy volunteers. Eight healthy volunteers, all males, age 25-34 years, all EM for CYP2C19, participated in a randomized, double blind crossover study in two periods of 7 days, separated by a 14-day washout. In each treatment period, subjects took either omeprazole 20mg b.i.d. together with clarithromycin 500 mg b.i.d., or pantoprazole 40 mg b.i.d. with the same dose of the antibiotic. The pharmacokinetic parameters of omeprazole and pantoprazole were compared to those after intake of both agents alone. Kinetics of unchanged clarithromycin was evaluated at the end of the two periods. The mean value of the area under the plasma concentration versus time curve (AUC) of unchanged omeprazole increased almost two-fold after concomitant administration of clarithromycin; the average 5-OH-omeprazole AUC was instead significantly reduced by 42%. Omeprazole clearance and volume of distribution were reduced significantly by 75 and 56%, respectively, after administration of the drug with clarithromicyn. No significant changes of the kinetic of pantoprazole and metabolites were observed. Kinetics of clarithromycin did not differ after the two associated treatments. The administration of clarithromycin with two different proton pump inhibitors indicates that the antibiotic can markedly increase omeprazole, not pantoprazole, levels. This observation may result in a better therapeutic response to omeprazole, but it may also potentially affect either the metabolism of CYP3A4 substrates or interfere with the absorption of drugs requiring an intact gastric digestion system.
  
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• An omega-3 polyunsaturated fatty acid concentrate increases plasma high-density lipoprotein 2 cholesterol and paraoxonase levels in patients with familial combined hyperlipidemia.
  Metabolism

  2004 Feb;53(2):153-8.
  
  Calabresi Laura, Villa Barbara, Canavesi Monica, Sirtori Cesare R, James Richard W, Bernini Franco, Franceschini Guido
  
  Abstract
  
  A remarkable reduction of plasma concentrations of high-density lipoproteins (HDL), especially of the HDL(2) subfraction, is one of the typical lipoprotein alterations found in patients with familial combined hyperlipidemia (FCHL). Fourteen FCHL patients received 4 capsules daily of Omacor (an omega-3 polyunsaturated fatty acid [omega3 FA] concentrate providing 1.88 g of eicosapentaenoic acid [EPA] and 1.48 g of docosahexaenoic acid [DHA] per day; Pronova Biocare, Oslo, Norway) or placebo for 8 weeks in a randomized, double-blind, crossover study. Plasma triglycerides were 44% lower, and LDL cholesterol and apoliporpotein (apo)B were 25% and 7% higher after Omacor than placebo. HDL cholesterol was higher (+8%) after Omacor than placebo, but this difference did not achieve statistical significance. Omacor caused a selective increase of the more buoyant HDL(2) subfraction; plasma HDL(2) cholesterol and total mass increased by 40% and 26%, respectively, whereas HDL(3) cholesterol and total mass decreased by 4% and 6%. Both HDL(2) and HDL(3) were enriched in cholesteryl esters and depleted of triglycerides after Omacor. No changes were observed in the plasma concentration of major HDL apolipoproteins, LpA-I and LpA-I:A-II particles, lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP). The plasma concentration of the HDL-bound antioxidant enzyme paraoxonase increased by 10% after Omacor. Omacor may be helpful in correcting multiple lipoprotein abnormalities and reducing cardiovascular risk in FCHL patients.
  
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• Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein.
  J Biol Chem

  2004 Mar;279(11):9930-6.
  
  Favari Elda, Lee Miriam, Calabresi Laura, Franceschini Guido, Zimetti Francesca, Bernini Franco, Kovanen Petri T
  
  Abstract
  
  The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular unesterified cholesterol and phospholipid to lipid-poor apolipoprotein A-I. Chymase, a protease secreted by mast cells, selectively cleaves pre-beta-migrating particles from high density lipoprotein (HDL)(3) and reduces the efflux of cholesterol from macrophages. To evaluate whether this effect is the result of reduction of ABCA1-dependent or -independent pathways of cholesterol efflux, in this study we examined the efflux of cholesterol to preparations of chymase-treated HDL(3) in two types of cell: 1) in J774 murine macrophages endogenously expressing low levels of scavenger receptor class B, type I (SR-BI), and high levels of ABCA1 upon treatment with cAMP; and 2) in Fu5AH rat hepatoma cells endogenously expressing high levels of the SR-BI and low levels of ABCA1. Treatment of HDL(3) with the human chymase resulted in rapid depletion of pre-beta-HDL and a concomitant decrease in the efflux of cholesterol and phospholipid (2-fold and 3-fold, respectively) from the ABCA1-expressing J774 cells. In contrast, efflux of free cholesterol from Fu5AH to chymase-treated and to untreated HDL(3) was similar. Incubation of HDL(3) with phospholipid transfer protein led to an increase in pre-beta-HDL contents as well as in ABCA1-mediated cholesterol efflux. A decreased cholesterol efflux to untreated HDL(3) but not to chymase-treated HDL(3) was observed in ABCA1-expressing J774 with probucol, an inhibitor of cholesterol efflux to lipid-poor apoA-I. Similar results were obtained using brefeldin and gliburide, two inhibitors of ABCA1-mediated efflux. These results indicate that chymase treatment of HDL(3) specifically impairs the ABCA1-dependent pathway without influencing either aqueous or SR-BI-facilitated diffusion and that this effect is caused by depletion of lipid-poor pre-beta-migrating particles in HDL(3). Our results are compatible with the view that HDL(3) promotes ABCA1-mediated lipid efflux entirely through its lipid-poor fraction with pre-beta mobility.
  
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• Synthetic high-density lipoproteins exert cardioprotective effects in myocardial ischemia/reperfusion injury.
  J Pharmacol Exp Ther

  2004 Jan;308(1):79-84.
  
  Rossoni Giuseppe, Gomaraschi Monica, Berti Ferruccio, Sirtori Cesare R, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Human high-density lipoproteins (HDLs) protect the heart against ischemia/reperfusion injury. In the present study, the cardioprotective effects of synthetic high-density lipoproteins (sHDLs) made of phosphatidylcholine and apolipoprotein A-I were investigated in isolated rat hearts, which underwent a 20-min low-flow ischemia followed by a 30-min reperfusion. The administration of sHDL during the 10 min immediately before ischemia caused a rapid, dose-dependent improvement of postischemic cardiac function: at the maximum dose (2.0 mg of sHDL protein/ml), left ventricular developed pressure (LVDP) recovered to 71.0 +/- 3.2 versus 40.5 +/- 3.8 mm Hg in saline-treated hearts, and coronary perfusion pressure (CPP) increased to 100.3 +/- 6.2 versus 132.0 +/- 9.0 mm Hg. The preservation of postischemic cardiac function was associated with a dose-dependent reduction of creatine kinase release into the coronary effluent. sHDLs administered in the first 10 min postischemia also exerted a significant, dose-dependent improvement of postischemic LVDP, CPP, and creatine kinase release, but the cardioprotective effect was less than when sHDLs were given preischemia. The preservation of postischemic cardiac function by sHDL was mediated through a reduction of cardiac tumor necrosis factor-alpha content and an enhanced cardiac production of prostaglandin E2 and I2. The present experimental data indicate that sHDLs may provide a novel therapeutic approach to clinical conditions in which myocardial ischemia/reperfusion occurs, such as acute coronary syndromes, cardiac surgery, or revascularization procedures.
  
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• Endothelial protection by high-density lipoproteins: from bench to bedside.
  Arterioscler Thromb Vasc Biol

  2003 Oct;23(10):1724-31.
  
  Calabresi Laura, Gomaraschi Monica, Franceschini Guido
  
  Abstract
  
  There are several potential mechanisms by which HDLs protect against the development of vascular disease. One relates to the unique ability of these lipoproteins to remove cholesterol from the arterial wall. Another is the ability of HDL to prevent and eventually correct endothelial dysfunction, a key variable in the pathogenesis of atherosclerosis and its complications. HDLs help maintain endothelial integrity, facilitate vascular relaxation, inhibit blood cell adhesion to vascular endothelium, reduce platelet aggregability and coagulation, and may favor fibrinolysis. These functions of HDLs complement their activity in arterial cholesterol removal by providing an excellent rationale for favorably influencing pathological processes underlying a variety of clinical conditions, such as accelerated atherosclerosis, acute coronary syndromes, and restenosis after coronary angioplasty, through a chronic or acute elevation of plasma HDL concentration.
  
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• Recurrent mutations of the apolipoprotein A-I gene in three kindreds with severe HDL deficiency.
  Atherosclerosis

  2003 Apr;167(2):335-45.
  
  Pisciotta Livia, Miccoli Roberto, Cantafora Alfredo, Calabresi Laura, Tarugi Patrizia, Alessandrini Paola, Bittolo Bon Gabriele, Franceschini Guido, Cortese Claudio, Calandra Sebastiano, Bertolini Stefano
  
  Abstract
  
  Two siblings with high density lipoprotein (HDL) deficiency and no plasma apolipoprotein A-I (Apo A-I) were found to be homozygous for a cytosine deletion in exon 3 of Apo A-I gene (c.85 del C, Q5FsX11). This mutation causes a frameshift leading to a premature stop codon and abolishes the synthesis of Apo A-I. Although both siblings had corneal opacifications and planar xanthomas, only one of them had premature coronary artery disease, probably as the result of mildly elevated LDL levels. In two other unrelated subjects HDL deficiency was due to heterozygosity for a nucleotide substitution in exon 4 of Apo A-I gene (c.494 T>G, L141R). Both Apo A-I mutations were reported previously in an Italian kindred which included compound heterozygotes and simple heterozygotes. We investigated all carriers of these mutations in the three kindreds and in the one previously reported. Plasma Apo A-I and HDL-C levels were lower in the mutation carriers than in non-carrier family members. These levels, however, were lower in L141R carriers than in carriers of c.85 del C. Haplotype analysis performed using several polymorphisms suggested that both the c.85 del C and L141R are likely to be recurrent mutations.
  
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• High-density lipoproteins protect isolated rat hearts from ischemia-reperfusion injury by reducing cardiac tumor necrosis factor-alpha content and enhancing prostaglandin release.
  Circ Res

  2003 Feb;92(3):330-7.
  
  Calabresi Laura, Rossoni Giuseppe, Gomaraschi Monica, Sisto Francesca, Berti Ferruccio, Franceschini Guido
  
  Abstract
  
  The incidence and severity of primary cardiac events are inversely related to the plasma concentration of high-density lipoproteins (HDLs). We investigated whether HDLs may exert a direct cardioprotection in buffer-perfused isolated rat hearts, which underwent a 20-minute low-flow ischemia followed by a 30-minute reperfusion. The administration of HDLs at physiological concentrations (0.5 and 1.0 mg/mL) during the 10 minutes immediately before ischemia rapidly and remarkably improved postischemic functional recovery and decreased creatine kinase release in the coronary effluent. Reconstituted HDLs containing apolipoprotein A-I (apoA-I) and phosphatidylcholine, but not lipid-free apoA-I or phosphatidylcholine liposomes, were also effective in protecting the heart from ischemia-reperfusion injury. HDLs at reperfusion were less effective than when given before ischemia. HDLs caused a dose-dependent reduction of ischemia-induced cardiac tumor necrosis factor-alpha (TNF-alpha) expression and content, which correlated with the improved functional recovery. A parallel increase of TNF-alpha release in the coronary effluent was observed, due to a direct binding of cardiac TNF-alpha to HDLs. Taken together, these findings argue for a cause-effect relationship between the HDL-mediated removal of TNF-alpha from the ischemic myocardium and the HDL-induced cardioprotection. Indeed, etanercept, a recombinant TNF-alpha-blocking protein, caused a dose-dependent improvement of postischemic functional recovery. HDLs also enhanced ischemia-induced prostaglandin release, which may contribute to the cardioprotective effect. A low plasma HDL level may expose the heart to excessive ischemia-reperfusion damage, and HDL-targeted therapies may be helpful to induce immediate or delayed myocardial protection from ischemia-reperfusion injury.
  
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• Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene.
  J Lipid Res

  2003 Feb;44(2):254-64.
  
  Altilia Serena, Pisciotta Livia, Garuti Rita, Tarugi Patrizia, Cantafora Alfredo, Calabresi Laura, Tagliabue Jacopo, Maccari Sergio, Bernini Franco, Zanotti Ilaria, Vergani Carlo, Bertolini Stefano, Calandra Sebastiano
  
  Abstract
  
  Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.
  
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• Apolipoprotein composition and particle size affect HDL degradation by chymase: effect on cellular cholesterol efflux.
  J Lipid Res

  2003 Mar;44(3):539-46.
  
  Lee Miriam, Kovanen Petri T, Tedeschi Gabriella, Oungre Emanuela, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Mast cell chymase, a chymotrypsin-like neutral protease, can proteolyze HDL3. Here we studied the ability of rat and human chymase to proteolyze discoidal pre beta-migrating reconstituted HDL particles (rHDLs) containing either apolipoprotein A-I (apoA-I) or apoA-II. Both chymases cleaved apoA-I in rHDL at identical sites, either at the N-terminus (Tyr18 or Phe33) or at the C-terminus (Phe225), so generating three major truncated polypeptides that remained bound to the rHDL. The cleavage sites were independent of the size of the rHDL particles, but small particles were more susceptible to degradation than bigger ones. Chymase-induced truncation of apoA-I yielded functionally compromised rHDL with reduced ability to promote cellular cholesterol efflux. In sharp contrast to apoA-I, apoA-II was resistant to degradation. However, when apoA-II was present in rHDL that also contained apoA-I, it was degraded by chymase. We conclude that chymase reduces the ability of apoA-I in discoidal rHDL particles to induce cholesterol efflux by cleaving off either its amino- or carboxy-terminal portion. This observation supports the concept that limited extracellular proteolysis of apoA-I is one pathophysiologic mechanism leading to the generation and maintenance of foam cells in atherosclerotic lesions.
  
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• The C-terminal domain of apolipoprotein A-I is involved in ABCA1-driven phospholipid and cholesterol efflux.
  Biochem Biophys Res Commun

  2002 Dec;299(5):801-5.
  
  Favari Elda, Bernini Franco, Tarugi Patrizia, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  ABCA1, a member of the ATP-binding cassette family, mediates the efflux of cellular lipids to free apolipoproteins, mainly apoA-I. The role of the C-terminal domain of apoA-I in this process has been evaluated by measuring the efflux capacity of a truncated form (apoA-I-(1-192)) versus intact apoA-I in different cellular models. In stimulated J774 macrophages, cholesterol efflux to apoA-I-(1-192) was remarkably lower than that to the intact apoA-I. The truncated apoA-I, lacking an important lipid-binding domain, was also significantly less efficient in removing phospholipids from stimulated macrophages. No difference was detected with stimulated Tangier fibroblasts that do not express functional ABCA1. The C-terminal domain of apoA-I is clearly involved in ABCA1-driven lipid efflux. Independent of the interaction with the cell surface, it may be the decreased ability of the truncated apoA-I to recruit membrane phospholipids that impairs its capacity to promote cell cholesterol efflux.
  
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• Mast cell chymase degrades apoE and apoA-II in apoA-I-knockout mouse plasma and reduces its ability to promote cellular cholesterol efflux.
  Arterioscler Thromb Vasc Biol

  2002 Sep;22(9):1475-81.
  
  Lee Miriam, Calabresi Laura, Chiesa Giulia, Franceschini Guido, Kovanen Petri T
  
  Abstract
  
  OBJECTIVE:
  Mast cell chymase is a chymotryptic heparin proteoglycan-bound neutral protease that exerts its activity in extracellular fluids. We studied the effect of chymase on the apolipoprotein compositions and the abilities of plasmas from apolipoprotein (apo)A-I-knockout (A-I-KO) and wild-type (C57BL/6J) mice to stimulate efflux of cellular cholesterol from mouse macrophage foam cells.
  
  METHODS AND RESULTS:
  The A-I-KO apolipoproteins compared with the wild-type (apoA-I, apoA-II, apoA-IV, and apoE) showed total lack of apoA-I, unaltered apoA-II, an absence of apoA-IV, and an increase of apoE. Despite these major differences, the 2 plasmas induced similar high-affinity efflux of cholesterol from the foam cells. Quantitative analysis of chymase-treated plasmas revealed (1) in A-I-KO plasma, complete loss of apoE and apoA-II, and (2) in wild-type plasma, slight reduction of apoA-I associated with complete depletion of the minor pre-beta-high density lipoprotein fraction, strong reduction of apoA-II, and complete depletion of apoA-IV and apoE. Both proteolyzed plasmas had lost the ability to induce cellular cholesterol efflux with high affinity. Addition of discoidal pre-beta-migrating reconstituted high density lipoprotein particles containing human apoA-I or apoA-II to the chymase-treated A-I-KO plasma fully restored its cholesterol efflux-inducing ability, indicating functional replacement of the proteolyzed apoE and apoA-II. Thus, chymase degraded all the nondeleted apolipoproteins of the A-I-KO plasma involved in the high-affinity efflux of cellular cholesterol.
  
  CONCLUSIONS:
  This is the first indication that genetically engineered mice could be used as models for examining the hypothesis that extracellular proteases are involved in the development of atherosclerosis by inhibiting the apolipoprotein-mediated removal of macrophage cholesterol.
  
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• Omega-3 fatty acid ethyl esters increase heart rate variability in patients with coronary disease.
  Pharmacol Res

  2002 Jun;45(6):475.
  
  Villa Barbara, Calabresi Laura, Chiesa Giulia, Risè Patrizia, Galli Claudio, Sirtori Cesare R
  
  Abstract
  
  n-3 Fatty acids may reduce the incidence of sudden cardiac death, a property potentially related to their activity on myocardial excitability. We carried out a cross-over trial in which 10 coronary patients were treated with n-3 ethyl esters at two different dosages (3 and 6 g day (-1)) for 4 weeks. Plasma fatty acid composition, lipid profile, and heart rate variability (HRV) were analysed. n-3 Fatty acid intake significantly reduced plasma cholesterol and triglyceride levels and decreased the low to high frequency ratio. In addition, significant positive correlations were found between n-3 phospholipid content and HRV indices, thus confirming, in a prospective trial, retrospective data suggesting that an increased HRV may be achieved in coronary patients by exogenous provision of n-3 fatty acids.
  
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• Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins.
  Biochem J

  2002 Aug;366(Pt 1):245-53.
  
  Gianazza Elisabetta, Eberini Ivano, Sirtori Cesare R, Franceschini Guido, Calabresi Laura
  
  Abstract
  
  Lipid-free apolipoprotein A-I (apoA-I) and A-I(Milano) (A-I(M)) were compared for their denaturation behaviour by running across transverse gradients of a chaotrope, urea, and of a ionic detergent, SDS. For both apo A-I and monomeric apoA-I(M) in the presence of increasing concentrations of urea the transition from high to low mobility had a sigmoidal course, whereas for dimeric A-I(M)/A-I(M) a non-sigmoidal shape was observed. The co-operativity of the unfolding process was lower for dimeric A-I(M)/A-I(M) than for apoA-I or for monomeric apoA-I(M). A slightly higher susceptibility to denaturation was observed for dimeric A-I(M)/A-I(M) than for monomeric apoA-I(M). A similar behaviour of A-I(M)/A-IM versus apoA-I(M) was observed in CD experiments. Large- (12.7/12.5 nm) and small- (7.8 nm) sized reconstituted high-density lipoproteins (rHDL) containing either apoA-I or A-I(M)/A-I(M) were compared with respect to their protein-lipid dissociation behaviour by subjecting them to electrophoresis in the presence of urea, of SDS and of a non-ionic detergent, Nonidet P40. A higher susceptibility to dissociation of small-sized versus large-sized rHDL, regardless of the apolipoprotein component, was observed in all three instances. Our data demonstrate that the differential plasticity of the various classes of rHDL is a function of their size; the higher stability of 12.5/12.7 nm rHDL is likely connected to the higher number of protein-lipid and lipid-lipid interactions in larger as compared with smaller rHDL.
  
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• Elevated soluble cellular adhesion molecules in subjects with low HDL-cholesterol.
  Arterioscler Thromb Vasc Biol

  2002 Apr;22(4):656-61.
  
  Calabresi Laura, Gomaraschi Monica, Villa Barbara, Omoboni Laura, Dmitrieff Camille, Franceschini Guido
  
  Abstract
  
  The purpose of this study was to investigate whether the expression of cellular adhesion molecules (CAMs) is enhanced in individuals with low HDL cholesterol (HDL-C). Plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), and E-selectin (sE-selectin) were measured in subjects with low (below the 10th percentile for the Italian population), average, or high (above the 90th percentile) HDL-C. Average sICAM-1 and sE-selectin levels were significantly higher in two groups of 65 individuals with low HDL levels, either hyperlipidemic (320.5+/-16.0 and 61.4+/-3.5 ng/mL) or normolipidemic (309.6+/-13.0 and 60.0+/-2.7 ng/mL), than in subjects with average HDL levels, either hyperlipidemic (267.0+/-10.1 and 50.4+/-2.8 ng/mL) or normolipidemic (257.9+/-5.4 and 51.1+/-2.4 ng/mL), or with high HDL levels (254.8+/-10.2 and 52.5+/-3.2 ng/mL). No significant difference was found in the plasma sVCAM-1 concentration. HDL-C was inversely correlated with sICAM-1 and sE-selectin in the low-HDL subjects (r(2)=0.087 and 0.035, P=0.0007 and 0.033, respectively), but not in individuals with normal or elevated HDL-C (r(2)=0.012 and 0.006). A fenofibrate-induced increase of HDL-C in 20 low-HDL subjects was associated with a significant reduction of plasma sICAM-1 and sE-selectin concentrations. An increased CAMs expression may be a mechanism by which a low plasma HDL level promotes atherogenesis and causes acute atherothrombotic events.
  
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• Macrophage metalloproteinases degrade high-density-lipoprotein-associated apolipoprotein A-I at both the N- and C-termini.
  Biochem J

  2002 Mar;362(Pt 3):627-34.
  
  Eberini Ivano, Calabresi Laura, Wait Robin, Tedeschi Gabriella, Pirillo Angela, Puglisi Lina, Sirtori Cesare R, Gianazza Elisabetta
  
  Abstract
  
  Atheromatous plaques contain various cell types, including macrophages, endothelial cells and smooth-muscle cells. To investigate the possible interactions between secreted matrix metalloproteinases and high-density lipoprotein (HDL) components, we tested the above cell types by culturing them for 24 h. HDL(3) (HDL subfractions with average sizes of between 8.44 nm for HDL(3A) and 7.62 nm for HDL(3C)) were then incubated in their cell-free conditioned media. Proteolytic degradation of apolipoprotein A-I was observed with macrophages, but not with endothelial-cell- or muscle-cell-conditioned supernatant. Absence of calcium or addition of EDTA to incubation media prevented all proteolytic processes. The identified apolipoprotein A-I fragments had sizes of 26, 22, 14 and 9 kDa. Two-dimensional electrophoresis and MS resolved the 26 and the 22 kDa components and identified peptides resulting from both N- and C-terminal cleavage of apolipoprotein A-I. The higher abundance of C- than N-terminally cleaved peptides agrees with data in the literature for a fully structured alpha-helix around Tyr(18) compared with an unstructured region around Gly(185) and Gly(186). The flexibility in the latter region of apolipoprotein A-I may explain its susceptibility to proteolysis. In our experimental set-up, HDL(3C) was more extensively degraded than the other HDL(3) subclasses (HDL(3A) and HDL(3B)). Proteolytic fragments produced by metalloproteinase action were shown by gel filtration and electrophoresis to be neither associated with lipids nor self-associated.
  
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• Increased carotid artery intima-media thickness in subjects with primary hypoalphalipoproteinemia.
  Arterioscler Thromb Vasc Biol

  2002 Feb;22(2):317-22.
  
  Baldassarre Damiano, Amato Mauro, Pustina Linda, Tremoli Elena, Sirtori Cesare R, Calabresi Laura, Franceschini Guido
  
  Abstract
  
  The plasma concentration of high-density lipoprotein cholesterol (HDL-C) is inversely correlated with the incidence of atherosclerotic vascular events. In the present study, we evaluated pre-intrusive atherosclerosis in subjects with plasma HDL-C at the extremities of normal distribution. Fifty-five subjects with primary hypoalphalipoproteinemia (HypoALP) or hyperalphalipoproteinemia (HyperALP) were compared with fifty-five control subjects with average HDL-C levels, matched for sex, age, and plasma cholesterol. The average and maximal intima-media thicknesses (Avg-IMT and Max-IMT) of 48 carotid segments for each subject were approximately 40% greater in HypoALP than in control subjects (0.94 +/- 0.06 versus 0.69 +/- 0.04 mm, P=0.004, and 1.86 +/- 0.16 versus 1.35 +/- 0.10 mm, P=0.025, respectively). The IMT values in HyperALP subjects (Avg-IMT, 0.71 +/- 0.04 and Max-IMT, 1.38 +/- 0.14 mm) were the same as in controls. In a large cohort of hyperlipidemic subjects (n=559), significantly greater Avg-IMT and Max-IMT were found in subjects belonging to the first HDL-C quintile (
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• Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism.
  J Biol Chem

  2002 Feb;277(6):4301-8.
  
  Deakin Sara, Leviev Ilia, Gomaraschi Monica, Calabresi Laura, Franceschini Guido, James Richard W
  
  Abstract
  
  Paraoxonase-1 (PON1) is a high density lipoprotein (HDL)-associated serum enzyme that protects low density lipoproteins from oxidative modifications. There is a relative lack of information on mechanisms implicated in PON1 release from cells. The present study focused on a model derived from stable transfection of CHO cells, to avoid co-secretion of apolipoprotein (apo) A-I and lipids, which could lead to formation of HDL-like complexes. Our results indicate that, in the absence of an appropriate acceptor, little PON1 is released. The results designate HDL as the predominant, physiological acceptor, whose efficiency is influenced by size and composition. Neither lipid-poor apoA-I or apoA-II nor low density lipoproteins could substitute for HDL. Protein-free phospholipid complexes promoted PON1 release. However, the presence of both apolipoprotein and phospholipid were necessary to promote release and stabilize the enzyme. Immunofluorescence studies demonstrated that PON1 was inserted into the external membrane of CHO cells, where it was enzymatically active. Accumulation of PON1 in the cell membrane was not influenced by the ability of the cell to co-secrete of apoA-I. Release appeared to involve desorption by HDL; human and reconstituted HDL promoted PON1 release in a saturable, high affinity manner (apparent affinity 1.59 +/- 0.3 microg of HDL protein/ml). Studies with PON1-transfected hepatocytes (HuH-7) revealed comparable structural features with the peptide located in a punctate pattern at the external membrane and enzymatically active. We hypothesize that release of PON1 involves a docking process whereby HDL transiently associate with the cell membrane and remove the peptide from the external membrane. The secretory process may be of importance for assuring the correct lipoprotein destination of PON1 and thus its functional efficiency.
  
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