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Pubblicazioni recenti - cardiac fibroblast
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MicroRNA-494 Inhibits the LRG1 Expression to Induce Proliferation and Migration of VECs in Rats following Myocardial Infarction.
Mol Ther Nucleic Acids2019 Aug;18():110-122. doi: S2162-2531(19)30219-7.
Su Qiang, Lv Xiang-Wei, Sun Yu-Han, Ye Zi-Liang, Kong Bing-Hui, Qin Zhen-Bai,
Abstract
Myocardial infarction (MI) is a life-threatening cardiac event that results in extreme damage to the heart muscle. The Wnt signaling pathway has been implicated in the development of heart diseases. Hence, the current study aimed to investigate the role of microRNA (miRNA) in association with the Wnt signaling pathway to identify potential candidates for MI therapy. Differentially expressed miRNAs associated with MI occurrence were screened, and miR-494 was selected for subsequent experiments. Sprague-Dawley rats were included to establish a MI model via intraperitoneal injection of 0.1 mg/kg atropine sulfate and 40 mg/kg pentobarbital sodium. Then, the interaction between miR-494 and LRG1 was identified. The effect of miR-494 on expression of the Wnt signaling pathway-related genes, proliferation, migration, and invasion ability of fibroblasts and vascular endothelial cells (VECs) was subsequently evaluated through a series of gain- and loss-of-function experiments. The results revealed that miR-494 was poorly expressed and LRG1 was highly expressed in MI rats. miR-494 targets and downregulates LRG1, which resulted in the inactivation of the Wnt signaling pathway and promoted proliferation, migration, and invasion ability of fibroblasts and VECs. In conclusion, this study provided evidence suggesting that overexpressed miR-494 could potentially promote the proliferation, migration, and invasion of fibroblasts and VECs in MI through the inactivation of the Wnt signaling pathway by binding to LRG1.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.
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Key Regulators of Cardiovascular Differentiation and Regeneration: Harnessing the Potential of Direct Reprogramming to Treat Heart Failure.
J Card Fail2019 Sep;():. doi: S1071-9164(19)30366-5.
Ieda Masaki,
Abstract
Cardiovascular diseases remain a leading cause of death worldwide, with the number of patients with heart failure increasing rapidly in aging societies. As adult cardiomyocytes are terminally differentiated cells and opportunities for heart transplantation are very limited, regenerative medicine may become a game changer in heart failure treatment. To develop strategies for generating cardiomyocytes, vascular cells, and other supporting cells, it is necessary to understand the mechanism of cardiovascular differentiation during development and from pluripotent stem cells. Master regulators for cardiovascular differentiation can generate new cardiomyocytes and vascular cells directly from other differentiated cells such as fibroblasts. Fibroblasts can be directly reprogrammed into cardiomyocytes by overexpressing a combination of three cardiac-specific transcription factors (Gata4, Mef2c, Tbx5) both in vitro and in vivo, which restores cardiac function after myocardial infarction in mice. Moreover, a direct reprogramming-based approach can be used to identify new key regulators of the cardiovascular mesoderm, which can differentiate into all three types of cardiovascular cells including cardiomyocytes, endothelial cells, and smooth muscle cells. This review provides a perspective on how key regulators for cardiovascular differentiation and regeneration can be identified and used to develop new treatments for heart failure.
Copyright © 2019. Published by Elsevier Inc.
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FGF23-Mediated Activation of Local RAAS Promotes Cardiac Hypertrophy and Fibrosis.
Int J Mol Sci2019 Sep;20(18):. doi: E4634.
Böckmann Ineke, Lischka Jonas, Richter Beatrice, Deppe Jennifer, Rahn Anja, Fischer Dagmar-Christiane, Heineke Jörg, Haffner Dieter, Leifheit-Nestler Maren,
Abstract
Patients with chronic kidney disease (CKD) are prone to developing cardiac hypertrophy and fibrosis, which is associated with increased fibroblast growth factor 23 (FGF23) serum levels. Elevated circulating FGF23 was shown to induce left ventricular hypertrophy (LVH) via the calcineurin/NFAT pathway and contributed to cardiac fibrosis by stimulation of profibrotic factors. We hypothesized that FGF23 may also stimulate the local renin-angiotensin-aldosterone system (RAAS) in the heart, thereby further promoting the progression of FGF23-mediated cardiac pathologies. We evaluated LVH and fibrosis in association with cardiac FGF23 and activation of RAAS in heart tissue of 5/6 nephrectomized (5/6Nx) rats compared to sham-operated animals followed by in vitro studies with isolated neonatal rat ventricular myocytes and fibroblast (NRVM, NRCF), respectively. Uremic rats showed enhanced cardiomyocyte size and cardiac fibrosis compared with sham. The cardiac expression of and RAAS genes were increased in 5/6Nx rats and correlated with the degree of cardiac fibrosis. In NRVM and NRCF, FGF23 stimulated the expression of RAAS genes and induced indicating mineralocorticoid receptor activation. The FGF23-mediated hypertrophic growth of NRVM and induction of NFAT target genes were attenuated by cyclosporine A, losartan and spironolactone. In NRCF, FGF23 induced and , which were suppressed by losartan and spironolactone, only. Our data suggest that FGF23-mediated activation of local RAAS in the heart promotes cardiac hypertrophy and fibrosis.
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Knocking down PFL can improve myocardial ischemia/reperfusion injury in rats by up-regulating heat shock protein-20.
Eur Rev Med Pharmacol Sci2019 Sep;23(17):7619-7627. doi: 18885.
Yin R-L, You H, Wu Y-M, Ye F-L, Gu W-X, Shen J,
Abstract
OBJECTIVE:
To investigate the effect and mechanism of long non-coding ribonucleic acid (lncRNA) PFL on myocardial ischemia/reperfusion (I/R) injury in rats, and to provide a reference for the prevention and treatment of myocardial infarction (MI) in clinic.
MATERIALS AND METHODS:
According to the random number table, 60 male Sprague-Dawley (SD) rats were randomly divided into 3 groups: Control group (n=20), I/R group (n=20), and I/R + PFL small interfering ribonucleic acid (siRNA) group (n=20). The I/R model was established by ligating the left anterior descending coronary artery (LAD) and then recanalizing it. PFL siRNAs were injected intravenously into the tail vein of rats in I/R + PFL siRNA group to construct a PFL knockout model. Triphenyl tetrazolium chloride (TTC) test was used to detect the infarction area of each group. Echocardiography was adopted to measure the ejection fraction [EF (%)] and fraction shortening [FS (%)] of rats in each group. Hematoxylin and eosin (H&E) staining was applied to detect the morphological changes in myocardial cells in each group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted to detect the apoptosis levels of myocardial cells and fibroblasts in heart tissues in each group. Meanwhile, the protein expression levels of apoptosis-related genes, Bcl-2-associated X protein (BAX), and Bcl-2, were measured via Western blotting. Also, the expression level of heat shock protein 20 (HSP-20) in the heart of three groups of rats was examined using immunohistochemical staining. Finally, the effects of PFL siRNAs on the expression level of HSP-20 were detected via Western blotting.
RESULTS:
PFL siRNAs could significantly improve I/R-induced cardiac insufficiency in rats, thus increasing EF (%) and FS (%) (p<0.05). Besides, PFL siRNAs could remarkably inhibit cardiac infarction caused by I/R injury and reduce the infarction area from (59.54±3.45)% to (24.85±1.30)% (p<0.05). H&E staining results manifested that, compared with those in I/R group, the cardiac myofilament was better in alignment, degradation and necrosis were milder, and cell edema was notably reduced in I/R + PFL siRNA group. Immunohistochemistry and Western blotting results showed that PFL siRNAs could remarkably reverse the decrease in the HSP-20 expression caused by I/R (p<0.05).
CONCLUSIONS:
We found that PFL knockdown can significantly improve the myocardial injury caused by I/R and improve the cardiac function in rats. The mechanism may be related to the activation of HSP-20 by PFL siRNAs. Therefore, PFL is expected to become a new target for the treatment of MI.
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MicroRNA-150 alleviates acute myocardial infarction through regulating cardiac fibroblasts in ventricular remodeling.
Eur Rev Med Pharmacol Sci2019 Sep;23(17):7611-7618. doi: 18884.
Tian H-B, Li S-H, Hu K-Q, Zan Y-S, Zhang X-L, Su G-H,
Abstract
OBJECTIVE:
The aim of this study was to investigate the effect of microRNA-150 on the regulation of myocardial fibrosis and ventricular remodeling in rats with acute myocardial infarction (AMI).
MATERIALS AND METHODS:
The AMI rats model was established by the ligation of the left anterior descending coronary artery (LAD) in vivo. After AMI procedures, the rats were injected with microRNA-150 lentivirus overexpression or negative control, respectively. Cardiac function of rats was evaluated by echocardiography. Hematoxylin and eosin (HE) staining and Masson trichrome were performed to evaluate myocardial fibrosis in each rat. Meanwhile, cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. The expression levels of microRNA-150, col1?1, col1?2, col3 and ? smooth muscle actin (?-SMA) in the border zone of rat infarct myocardium were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively.
RESULTS:
MicroRNA-150 expression in the border zone of infarct myocardium decreased significantly at day 28 after AMI (p<0.05). Overexpressing microRNA-150 significantly improved cardiac function, decreased collagen volume fraction (CVF) and attenuated cardiomyocyte apoptosis in rats. Furthermore, the expression levels of col1?1, col1?2, col3 and ?-SMA in the border zone of infarct myocardium were remarkably down-regulated in rats overexpressing microRNA-150 compared with those of controls (p<0.001).
CONCLUSIONS:
MicroRNA-150 expression in the border zone of rat infarct myocardium decreased at day 28 after AMI. In addition, the upregulation of microRNA-150 in myocardial tissue could inhibit myocardial fibrosis and improve ventricular remodeling at post-AMI.
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Long noncoding RNA GAS5 attenuates cardiac fibroblast proliferation in atrial fibrillation via repressing ALK5.
Eur Rev Med Pharmacol Sci2019 Sep;23(17):7605-7610. doi: 18883.
Lu J, Xu F-Q, Guo J-J, Lin P-L, Meng Z, Hu L-G, Li J, Li D, Lu X-H, An Y,
Abstract
OBJECTIVE:
Recently, long noncoding RNAs (lncRNAs) have caught more attention for their role in the progression of many diseases. Among them, lncRNA GAS5 (Growth Inhibition Specificity 5) was studied in this research to identify how it affects the progression of atrial fibrillation (AF).
PATIENTS AND METHODS:
In 40 patients with AF and 30 patients with sinus rhythm (SR), the GAS5 expression of the right atrial appendage (RAA) tissues was detected by the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Moreover, the cell proliferation assay was conducted in AC16 cells transfected with GAS5 inhibitor and mimics, respectively. Furthermore, the qRT-PCR was performed to uncover the mechanism.
RESULTS:
In the research, the expression of GAS5 in RAA tissues was decreased significantly in AF patients than that in SR ones. Moreover, overexpression of GAS5 inhibited cell growth in AC16 cells, while knockdown of GAS5 promoted cell growth in AC16 cells. In addition, further experiments revealed that ALK5 was a target of GAS5 and its expression in AF tissues negatively correlated to GAS5 expression.
CONCLUSIONS:
These results indicate that GAS5 could inhibit cell proliferation of AF via suppressing ALK5, which may offer a new vision for interpreting the mechanism of AF development.
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Cadherin-11 blockade reduces inflammation-driven fibrotic remodeling and improves outcomes after myocardial infarction.
JCI Insight2019 Sep;4(18):. doi: 10.1172/jci.insight.131545.
Schroer Alison K, Bersi Matthew R, Clark Cynthia R, Zhang Qinkun, Sanders Lehanna H, Hatzopoulos Antonis K, Force Thomas L, Majka Susan M, Lal Hind, Merryman W David,
Abstract
Over one million Americans experience myocardial infarction (MI) annually, and the resulting scar and subsequent cardiac fibrosis gives rise to heart failure. A specialized cell-cell adhesion protein, cadherin-11 (CDH11), contributes to inflammation and fibrosis in rheumatoid arthritis, pulmonary fibrosis, and aortic valve calcification but has not been studied in myocardium after MI. MI was induced by ligation of the left anterior descending artery in mice with either heterozygous or homozygous knockout of CDH11, wild-type mice receiving bone marrow transplants from Cdh11-deficient animals, and wild-type mice treated with a functional blocking antibody against CDH11 (SYN0012). Flow cytometry revealed significant CDH11 expression in noncardiomyocyte cells after MI. Animals given SYN0012 had improved cardiac function, as measured by echocardiogram, reduced tissue remodeling, and altered transcription of inflammatory and proangiogenic genes. Targeting CDH11 reduced bone marrow-derived myeloid cells and increased proangiogenic cells in the heart 3 days after MI. Cardiac fibroblast and macrophage interactions increased IL-6 secretion in vitro. Our findings suggest that CDH11-expressing cells contribute to inflammation-driven fibrotic remodeling after MI and that targeting CDH11 with a blocking antibody improves outcomes by altering recruitment of bone marrow-derived cells, limiting the macrophage-induced expression of IL-6 by fibroblasts and promoting vascularization.
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Participation of PKG and PKA-related pathways in the IFN-? induced modulation of the BK channel activity in human cardiac fibroblasts.
J Pharmacol Sci2019 Aug;():. doi: S1347-8613(19)35702-0.
Mitrokhin M V, Kalsin V, Kamkina O, Babkina I, Zotov A, Troitskiy V A, Mladenov M I, Kamkin G A,
Abstract
This study was devoted to elucidating the interferon (IFN)-?-induced signaling pathway and the interaction between protein kinase G (PKG) and protein kinase A (PKA) through large-conductance Ca(2+)-activated K(+) channels in human cardiac fibroblasts. The I currents were recorded using a whole-cell patch clamp method. A large depolarization (+50 mV) and a high Ca concentration (pCa 6.0) were used in the internal pipette solution to activate only the K channels. Iberiotoxin (Ibtx), which selectively inhibits BK channels at a concentration of 100 nmol/l, caused a significant reduction of basal I. Adding IFN-? in the presence of Ibtx had no effect on I. Application of the IFN-? caused a significant reduction in total K current amplitude, recorded with a 500 ms depolarizing pulse duration, to +50 mV from a holding potential of -80 mV. We tested the involvement of the sGC/cGMP/PKG signaling pathway by using specific PKG inhibitor KT 5823, potent sGC inhibitor NS 2028, and specific sGC agonist BAY 41-8543. The obtained data confirmed that only sGC participated in the IFN-?-mediated BK channel modulation, which was mediated further by PKA. This study represents first evidence about the participation of the IFN-? in the mechanisms responsible for BK modulation in HCFs. We also believe that this process occurs via negative crosstalk between the PKG- and PKA-associated pathways.
Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.
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Force Sensing by Piezo Channels in Cardiovascular Health and Disease.
Arterioscler Thromb Vasc Biol2019 Sep;():ATVBAHA119313348. doi: 10.1161/ATVBAHA.119.313348.
Beech David J, Kalli Antreas C,
Abstract
Mechanical forces are fundamental in cardiovascular biology, and deciphering the mechanisms by which they act remains a testing frontier in cardiovascular research. Here, we raise awareness of 2 recently discovered proteins, Piezo1 and Piezo2, which assemble as transmembrane triskelions to combine exquisite force sensing with regulated calcium influx. There is emerging evidence for their importance in endothelial shear stress sensing and secretion, NO generation, vascular tone, angiogenesis, atherosclerosis, vascular permeability and remodeling, blood pressure regulation, insulin sensitivity, exercise performance, and baroreceptor reflex, and there are early suggestions of relevance to cardiac fibroblasts and myocytes. Human genetic analysis points to significance in lymphatic disease, anemia, varicose veins, and potentially heart failure, hypertension, aneurysms, and stroke. These channels appear to be versatile force sensors, used creatively to inform various force-sensing situations. We discuss emergent concepts and controversies and suggest that the potential for new important understanding is substantial.
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Function analysis of differentially expressed microRNAs in TGF-?1-induced cardiac fibroblasts differentiation.
Biosci Rep2019 Sep;():. doi: BSR20182048.
Liu Suxuan, Ke Wen, Liu Yang, Zhao Zhenzhen, An Lina, You Xiaohua, Yang Fan, Yang Xiangqun, Wang Guokun, Zhao Xianxian,
Abstract
BACKGROUND:
Cardiac fibroblasts differentiation plays a critical role in cardiac remodeling and failure, but the underlying molecular mechanisms are still poorly understood. MicroRNAs (miRNAs) had been identified as important regulators during cell differentiation. The aim of this study was to screen the miRNAs involved in regulation of cardiac fibroblasts differentiation.
METHODS:
The differentiation of rat cardiac fibroblasts into myofibroblasts was induced by transforming growth factor-?1 (TGF-?1). Small RNA sequencing was then applied to detect the differentially expressed miRNAs.
RESULTS:
A total of 450 known miRNAs were detected, and 127 putative novel miRNAs were predicted by miRDeep2 analysis. DEGseq analysis and real-time PCR confirmed that 24 known miRNAs were differentially expressed in TGF-?1 induced cardiac fibroblasts, including 3 upregulated miRNAs and 21 downregulated miRNAs. After miRNAs target genes prediction by miRanda algorithm, pathway analysis showed that these potential target genes were involved in Calcium signaling pathway, Type II diabetes mellitus, and Glutamatergic synapse pathway, etc.. Meanwhile, 7 putative miRNAs were also detected differentially expressed during TGF-?1 induced cardiac fibroblasts differentiation.
CONCLUSIONS:
These differentially expressed miRNAs might play critical roles in cardiac fibroblasts differentiation. Altered expression of miRNAs may yield new insights into the underlying mechanisms of cardiac fibrosis and provide novel mechanism-based therapeutic strategies for cardiac fibrosis.
©2019 The Author(s).
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Generation of two Duchenne muscular dystrophy patient-specific induced pluripotent stem cell lines DMD02 and DMD03 (MUNIi001-A and MUNIi003-A).
Stem Cell Res2019 Sep;40():101562. doi: S1873-5061(19)30192-8.
Jelinkova Sarka, Markova Lenka, Pesl Martin, Valá?kova Iveta, Makaturová Eva, Jurikova Lenka, Vondracek Petr, Lacampagne Alain, Dvorak Petr, Meli Albano C, Rotrekl Vladimir,
Abstract
Duchenne muscular dystrophy (DMD) affects 1:3500-5000 newborn boys and manifests with progressive skeletal muscle wasting, respiratory failure and eventual heart failure. Symptoms show different onset from patients' childhood to the second decade of age. We reprogrammed fibroblasts from two independent DMD patients with a complete loss of dystrophin expression, carrying deletions of exons 45-50 and 48-50. The resulting hiPSCs show expression of pluripotency markers (NANOG, OCT4, SSEA4), differentiation capacity into all three germ layers, normal karyotype, genetic identity to the originating parental fibroblasts and the patient-specific dystrophin mutation.
Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.
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Molecular mechanisms of circular RNAs, transforming growth factor-?, and long noncoding RNAs in hepatocellular carcinoma.
Cancer Med2019 Sep;():. doi: 10.1002/cam4.2553.
Shang Wenkang, Adzika Gabriel Komla, Li Yujie, Huang Qike, Ding Ningding, Chinembiri Bianca, Rashid Mohammad Sajjad Ibn, Machuki Jeremiah Ong'achwa,
Abstract
At the heart of hepatocellular carcinoma (HCC) lies disruption of signaling pathways at the level of molecules, genes, and cells. Non-coding RNAs (ncRNAs) have been implicated in the disease progression of HCC. For instance, dysregulated expression of circular RNAs (circRNAs) has been observed in patients with HCC. As such, these RNAs are potential therapeutic targets and diagnostic markers for HCC. Long non-coding RNAs (lncRNAs), a type of ncRNA, have also been recognized to participate in the initiation and progression of HCC. Transforming growth factor-beta (TGF-?) is another element which is now recognized to play crucial roles in HCC. It has been implicated in many biological processes such as survival, immune surveillance, and cell proliferation. In HCC, TGF-? promotes disease progression by two mechanisms: an intrinsic signaling pathway and the extrinsic pathway. Through these pathways, it modulates various microenvironment factors such as inflammatory mediators and fibroblasts. An interesting yet-to-be resolved concept is whether the HCC-promoting role of TGF-? pathways is limited to a subset of HCC patients or it is involved in the whole process of HCC development. This review summarizes recent advancements to highlight the roles of circRNAs, lncRNAs, and TGF-? in HCC.
© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
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Spatiotemporal delivery of basic fibroblast growth factor to directly and simultaneously attenuate cardiac fibrosis and promote cardiac tissue vascularization following myocardial infarction.
J Control Release2019 Sep;():. doi: S0168-3659(19)30538-3.
Fan Zhaobo, Xu Zhaobin, Niu Hong, Sui Yang, Li Haichang, Ma Jianjie, Guan Jianjun,
Abstract
Following myocardial infarction (MI), the destruction of vasculature in the infarcted heart muscle and progression of cardiac fibrosis lead to cardiac function deterioration. Vascularization of the damaged tissue and prevention of cardiac fibrosis represent promising strategies to improve cardiac function. Herein we have developed a bFGF release system with suitable release kinetics to simultaneously achieve the two goals. The release system was based on an injectable, thermosensitive, and fast gelation hydrogel and bFGF. The hydrogel had gelation time <7?s. It can quickly solidify upon injection into tissue so as to increase drug retention in the tissue. Hydrogel complex modulus can be tuned by hydrogel solution concentration. The complex modulus of 176.6?Pa and lower allowed cardiac fibroblast to maintain its phenotype. Bioactive bFGF was able to gradually release from the hydrogel for 4?weeks. The released bFGF promoted cardiac fibroblast survival under ischemic conditions mimicking those of the infarcted hearts. It also attenuated cardiac fibroblasts from differentiating into myofibroblasts in the presence of TGF? when tested in 3D collagen model mimicking the scenario when the bFGF release system was injected into hearts. Furthermore, the released bFGF stimulated human umbilical endothelial cells to form endothelial lumen. After 4?weeks of implantation into infarcted hearts, the bFGF release system significantly increased blood vessel density, decreased myofibroblast density and collagen content, augmented cardiac cell survival/proliferation, and reduced macrophage density. In addition, the bFGF release system significantly increased cardiac function. These results demonstrate that delivery of bFGF with appropriate release kinetics alone may represent an efficient approach to control cardiac remodeling after MI.
Copyright © 2018. Published by Elsevier B.V.
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The axis of local cardiac endogenous Klotho-TGF-?1-Wnt signaling mediates cardiac fibrosis in human.
J Mol Cell Cardiol2019 Sep;():. doi: S0022-2828(19)30187-7.
Liu Qinghua, Zhu Lang-Jing, Waaga-Gasser Ana Maria, Ding Yan, Cao Minghua, Jadhav Shreyas J, Kirollos Sandra, Shekar Prem S, Padera Robert F, Chang Yu-Chun, Xu Xingbo, Zeisberg Elisabeth M, Charytan David M, Hsiao Li-Li,
Abstract
BACKGROUND:
Cardiovascular fibrosis is a major contributor to cardiovascular disease, the primary cause of death in patients with chronic kidney disease (CKD). We previously reported expression of endogenous Klotho in human arteries, and that CKD is a state of Klotho deficiency, resulting in vascular calcification, but myocardial expression of Klotho is poorly understood. This study aimed to further clarify endogenous Klotho's functional roles in cardiac fibrosis in patients with underlying CKD.
METHODS AND RESULTS:
Human atrial appendage specimens were collected during cardiac surgery from individuals with or without CKD. Cardiac fibrosis was quantified using trichrome staining. For endogenous Klotho functional studies, primary human cardiomyocytes (HCMs) were treated with uremic serum from CKD patients or recombinant human TGF-?1. The effects of endogenous Klotho in HCMs were studied using Klotho-siRNA and Klotho-plasmid transfection. Both gene and protein expression of endogenous Klotho are found in human heart, but decreased Klotho expression is clearly associated with the degree of cardiac fibrosis in CKD patients. Moreover, we show that endogenous Klotho is expressed by HCMs and cardiac fibroblasts (HCFs) but that HCM expression is suppressed by uremic serum or TGF-?1. Klotho knockdown or overexpression aggravates or mitigates TGF-?1-induced fibrosis and canonical Wnt signaling in HCMs, respectively. Furthermore, co-culture of HCMs with HCFs increases TGF-?1-induced fibrogenic proteins in HCFs, but overexpression of endogenous Klotho in HCMs mitigates this effect, suggesting functional crosstalk between HCMs and HCFs.
CONCLUSIONS:
Our data from analysis of human hearts as well as functional in vitro studies strongly suggests that the loss of cardiac endogenous Klotho in CKD patients, specifically in cardiomyocytes, facilitates intensified TGF-?1 signaling which enables more vigorous cardiac fibrosis through upregulated Wnt signaling. Upregulation of endogenous Klotho inhibits pathogenic Wnt/?-catenin signaling and may offer a novel strategy for prevention and treatment of cardiac fibrosis in CKD patients.
Copyright © 2018. Published by Elsevier Ltd.
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Impact of statins on cellular respiration and de-differentiation of myofibroblasts in human failing hearts.
ESC Heart Fail2019 Sep;():. doi: 10.1002/ehf2.12509.
Emelyanova Larisa, Sra Amar, Schmuck Eric G, Raval Amish N, Downey Francis X, Jahangir Arshad, Rizvi Farhan, Ross Gracious R,
Abstract
AIMS:
Fibroblast to myofibroblast trans-differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de-differentiation could mitigate CF-related pathologies. We determined whether 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors-statins, commonly prescribed to patients at risk of heart failure (HF)-can de-differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients.
METHODS AND RESULTS:
Either in vitro statin treatment of differentiated myofibroblasts (n = 3-6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4-12). In vitro, hVFs were differentiated by transforming growth factor-?1 (TGF-?1) for 72 h (TGF-72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by ?-smooth muscle actin (?-SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P < 0.05, considered significant. In vitro, statins concentration-dependently de-differentiated the myofibroblasts. The respective half-maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 ?mol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 ?mol/L), the reduced product of HMG-CoA, prevented the statin-induced de-differentiation (?-SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 ?mol/L), a cholesterol synthesis-independent HMG-CoA reductase pathway intermediate, completely prevented the statin-induced de-differentiation (?-SMA/GAPDH ratios: 0.89 ± 0.05 [TGF-72 h + 72 h], 0.63 ± 0.02 [TGF-72 h + simvastatin], and 1.2 ± 0.08 [TGF-72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co-incubation of simvastatin (200 nmol/L) with glibenclamide (10 ?mol/L), a K channel inhibitor, attenuated the simvastatin-induced de-differentiation (0.84 ± 0.05). Direct inhibition of mitochondrial respiration by oligomycin (1 ng/mL) also produced a de-differentiation effect (0.33 ± 0.02). OCR (pmol O /min/?g protein) was significantly decreased in the simvastatin-treated hVFs, including basal (P = 0.002), ATP-linked (P = 0.01), proton leak-linked (P = 0.01), and maximal (P < 0.001). The OCR inhibition was prevented by GGPP (basal OCR [P = 0.02], spare capacity OCR [P = 0.008], and maximal OCR [P = 0.003]). Congruently, hVFs from HF showed an increased population of myofibroblasts while HF + statin group showed significantly reduced cellular respiration (basal OCR [P = 0.021], ATP-linked OCR [P = 0.047], maximal OCR [P = 0.02], and spare capacity OCR [P = 0.025]) and myofibroblast differentiation (?-SMA/GAPDH: 1 ± 0.19 vs. 0.23 ± 0.06, P = 0.01).
CONCLUSIONS:
This study demonstrates the de-differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of K channels, and their impact on the differentiation magnitude of hVFs in HF patients. This novel pleiotropic effect of statins may be exploited to reduce excessive CF in patients at risk of HF.
© 2019 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.
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Rapid, proteomic urine assay for monitoring progressive organ disease in Fabry disease.
J Med Genet2019 Sep;():. doi: jmedgenet-2019-106030.
Doykov Ivan D, Heywood Wendy E, Nikolaenko Valeria, ?piewak Justyna, Hällqvist Jenny, Clayton Peter Theodore, Mills Philippa, Warnock David G, Nowak Albina, Mills Kevin,
Abstract
BACKGROUND:
Fabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker for monitoring treatment is plasma globotriaosylsphingosine (lyso-Gb3), but it is not informative about the underlying and developing disease pathology.
METHODS:
We have created a urine proteomic assay containing a panel of biomarkers designed to measure disease-related pathology which include the inflammatory system, lysosome, heart, kidney, endothelium and cardiovascular system. Using a targeted proteomic-based approach, a series of 40 proteins for organ systems affected in Fabry disease were multiplexed into a single 10?min multiple reaction monitoring Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) assay and using only 1?mL of urine.
RESULTS:
Six urinary proteins were elevated in the early-stage/asymptomatic Fabry group compared with controls including albumin, uromodulin, ?1-antitrypsin, glycogen phosphorylase brain form, endothelial protein receptor C and intracellular adhesion molecule 1. Albumin demonstrated an increase in urine and could indicate presymptomatic disease. The only protein elevated in the early-stage/asymptomatic patients that continued to increase with progressive multiorgan involvement was glycogen phosphorylase brain form. Podocalyxin, fibroblast growth factor 23, cubulin and Alpha-1-Microglobulin/Bikunin Precursor (AMBP) were elevated only in disease groups involving kidney disease. Nephrin, a podocyte-specific protein, was elevated in all symptomatic groups. Prosaposin was increased in all symptomatic groups and showed greater specificity (p<0.025-0.0002) according to disease severity.
CONCLUSION:
This work indicates that protein biomarkers could be helpful and used in conjunction with plasma lyso-Gb3 for monitoring of therapy or disease progression in patients with Fabry disease.
© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.
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Effects of nitric oxide on apoptosis and voltage-gated calcium channels in human cardiac myofibroblasts.
Clin Exp Pharmacol Physiol2019 Sep;():. doi: 10.1111/1440-1681.13178.
Bae Hyemi, Taeho Kim, Lim Inja,
Abstract
We characterised the voltage-gated Ca channels (VGCCs) in human cardiac fibroblasts (HCFs) and myofibroblasts (HCMFs) and investigated the effects of nitric oxide (NO) on apoptosis and on these channels. Western blotting and immunofluorescence analyses show that ?-smooth muscle actin (a myofibroblast marker) was markedly expressed in passage (P) 12-15 but not in P4 HCF cells, whereas calponin (a fibroblast marker) was expressed only in P4 cells. Ca 1.2 (L-type) and Ca 3.3 (T-type) of VGCCs were highly expressed in P12-15 cells, but only weak Ca 2.3 (R-type) expression was identified in P4 cells using reverse transcription-polymerase chain reaction analysis. S-Nitroso-N-acetylpenicillamine (SNAP, an NO donor) decreased cell viability of HCMFs in a dose-dependent manner and induced apoptotic changes, and nifedipine (an L-type Ca channel blocker) prevented apoptosis as shown with immunofluorescence staining and flow cytometry. Whole-cell mode patch clamp recordings demonstrate the presence of L-type Ca (I ) and T-type Ca (I ) currents in HCMFs. SNAP inhibited I of HCMFs, but pre-treatment with ODQ (a guanylate cyclase inhibitor) or KT5823 (a PKG inhibitor) prevented it. Pre-treating cells with KT5720 (a PKA inhibitor) or SQ22536 (an adenylate cyclase inhibitor) blocked SNAP-induced inhibition of I . 8-Bromo-cyclic GMP or 8-bromo-cyclic AMP also inhibited I . However, pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) did not block the SNAP effect, nor did DL-dithiothreitol (a reducing agent) reverse it. These data suggest that high concentrations of NO injure HCMFs and inhibit I through the PKG and PKA signalling pathways but not through the S-nitrosylation pathway.
© 2019 John Wiley & Sons Australia, Ltd.
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Geleophysic dysplasia: novel missense variants and insights into ADAMTSL2 intracellular trafficking.
Mol Genet Metab Rep2019 Dec;21():100504. doi: 10.1016/j.ymgmr.2019.100504.
Piccolo Pasquale, Sabatino Valeria, Mithbaokar Pratibha, Polishchuck Elena, Law Simon K, Magraner-Pardo Lorena, Pons Tirso, Polishchuck Roman, Brunetti-Pierri Nicola,
Abstract
Geleophysic dysplasia (GPHYSD1, MIM231050; GPHYSD2, MIM614185; GPHYSD3, MIM617809) is an autosomal disorder characterized by short-limb dwarfism, brachydactyly, cardiac valvular disease, and laryngotracheal stenosis. Mutations in , , and genes are responsible for this condition. We found that three previously described cases of GPHYSD diagnosed clinically were homozygote or compound heterozygotes for five variants, four of which not being previously reported. By electron microscopy, skin fibroblasts available in one case homozygote for an variant showed a defective intracellular localization of mutant ADAMTSL2 protein that did not accumulate within lysosome-like intra-cytoplasmic inclusions. Moreover, this mutant ADAMTSL2 protein was less secreted in medium and resulted in increased SMAD2 phosphorylation in transfected HEK293 cells.
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?-Arrestin 2 mediates arginine vasopressin-induced IL-6 induction via the ERK-NF-?B signal pathway in murine hearts.
Acta Pharmacol Sin2019 Sep;():. doi: 10.1038/s41401-019-0292-y.
Sun Shu-Zhen, Cao Hong, Yao Na, Zhao Ling-Ling, Zhu Xiao-Fang, Ni Er-An, Zhu Qi, Zhu Wei-Zhong,
Abstract
Evidence to date suggests that ?-arrestins act beyond their role as adapter proteins. Arginine vasopressin (AVP) may be a factor in inflammation and fibrosis in the pathogenesis of heart failure. In the present study we investigated the effect of AVP on inflammatory cytokine IL-6 production in murine hearts and the impact of ?-arrestin 2-dependent signaling on AVP-induced IL-6 production. We found that administration of AVP (0.5?U/kg, iv) markedly increased the levels of IL-6 mRNA in rat hearts with the maximum level occurred at 6?h. In ?-arrestin 2 KO mouse hearts, deletion of ?-arrestin 2 decreased AVP-induced IL-6 mRNA expression. We then performed in vitro experiments in adult rat cardiac fibroblasts (ARCFs). We found that AVP (10-10?M) dose-dependently increased the expression of IL-6 mRNA and protein, activation of NF-?B signaling and ERK phosphorylation, whereas knockdown of ?-arrestin 2 blocked AVP-induced IL-6 increase, NF-?B activation and ERK phosphorylation. Pharmacological blockade of ERK using PD98059 diminished AVP-induced NF-?B activation and IL-6 production. The selective V receptor antagonist SR49059 effectively blocked AVP-induced NF-?B phosphorylation and activation as well as IL-6 expression in ARCFs. In AVP-treated mice, pre-injection of SR49059 (2?mg/kg, iv) abolished AVP-induced NF-?B activation and IL-6 production in hearts. The above results suggest that AVP induces IL-6 induction in murine hearts via the V receptor-mediated ?-arrestin2/ERK/NF-?B pathway, thus reveal a novel mechanism of myocardial inflammation in heart failure involving the V/?-arrestin 2/ERK/NF-?B signaling pathway.
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Cell cycle regulators control mesoderm specification in human pluripotent stem cells.
J Biol Chem2019 Sep;():. doi: jbc.RA119.008251.
Yiangou Loukia, Grandy Rodrigo A, Osnato Anna, Ortmann Daniel, Sinha Sanjay, Vallier Ludovic,
Abstract
The mesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys, and the cardiovascular system originate. Understanding the mechanisms that control mesoderm specification could inform many applications, including the development of regenerative medicine therapies to manage diseases affecting these tissues. Here, we used human pluripotent stem cells (hPSCs) to investigate the role of cell cycle in mesoderm formation. To this end, using small molecules or conditional gene knockdown, we inhibited proteins controlling G1 and G2/M cell cycle phases during the differentiation of hPSCs into lateral plate, cardiac, and presomitic mesoderm. These loss-of-function experiments revealed that regulators of the G1 phase such as cyclin-dependent kinases (CDKs) and phosphorylation of retinoblastoma protein (pRb) are necessary for efficient mesoderm formation in a context-dependent manner. Further investigations disclosed that inhibition of the G2/M regulator CDK1 decreases bone morphogenetic protein (BMP) signaling activity specifically during lateral plate mesoderm formation while reducing fibroblast growth factor (FGF)/extracellular signaling-regulated kinase 1/2 (ERK1/2) activity in all mesoderm subtypes. Taken together, our findings reveal that cell cycle regulators direct mesoderm formation by controlling the activity of key developmental pathways.
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
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