Role In Cardiac Fibrosis Research Articles

MFGE8 is down-regulated in cardiac fibrosis and attenuates endothelial-mesenchymal transition through Smad2/3-Snail signalling pathway.

Endothelial‐mesenchymal transition (EndMT) is a major source of transformed cardiac fibroblasts, which is reported to play a key role in cardiac fibrosis (CF), a pathogenesis of cardiovascular diseases such as heart failure, myocardial infarction and atrial fibrillation. Nonetheless, the specific mechanism underlying the progression of EndMT to CF is still largely unknown. In this study, we aimed to investigate the role of milk fat globule‐EGF factor 8 (MFGE8), a kind of soluble glycoprotein, in TGF‐β1‐induced EndMT. In animal experiments, the expression of MFGE8 was found down‐regulated in the left ventricle and aorta of rats after transverse aortic constriction (TAC) compared with the sham group, especially in endothelial cells (ECs). In in vitro cultured ECs, silencing MFGE8 with small interfering RNA (siRNA) was found to promote the process of TGF‐β1‐induced EndMT, whereas administration of recombinant human MFGE8 (rh‐MFGE8) attenuated the process. Moreover, activated Smad2/3 signalling pathway after TGF‐β1 treatment and EndMT‐related transcription factors, such as Snail, Twist and Slug, was potentiated by MFGE8 knock‐down but inhibited by rh‐MFGE8. In conclusion, our experiments indicate that MFGE8 might play a protective role in TGF‐β1‐induced EndMT and might be a potential therapeutic target for cardiac fibrosis.

Epigenetic Control of circHNRNPH1 in Postischemic Myocardial Fibrosis through Targeting of TGF-β Receptor Type I

Postischemic myocardial fibrosis is a factor for the development of cardiac dysfunction and malignant cardiac arrhythmias, and no effective therapy is currently available. Circular RNAs are emerging as important epigenetic players in various biological functions; however, their roles in cardiac fibrosis are unknown. With the use of a rat model of postischemic myocardial fibrosis, we identified an increase in circHNRNPH1 in the ischemic myocardium after myocardial infarction, particularly in cardiac fibroblasts. In cardiac fibroblasts, circHNRNPH1 was responsive to transforming growth factor β1 (TGF-β1), the principal profibrotic factor. The downregulation of circHNRNPH1, in contrast to its overexpression, promoted myofibroblast migration and α-smooth muscle actin and collagen I expression and inhibited myofibroblast apoptosis. The recombinant adeno-associated virus 9 (rAAV9)-mediated, cardiac-specific knockdown of circHNNRPH1 accordingly facilitated cardiac fibrosis and aggravated cardiac dysfunction. Mechanistically, circHNRNPH1 colocalized with and sponged microRNA (miR)-216-5p in the cytoplasm of cardiac fibroblasts to induce SMAD7 (protein family of signal transduction component of the canonical transforming growth factor-β signaling pathway) expression, accelerating the degradation of TGF-β receptor I. Thus, our results indicated that circHNRNPH1 negatively regulates the fibrogenesis of cardiac fibroblasts and may provide a new therapeutic strategy for postischemic myocardial fibrosis.

Paired box 6 inhibits cardiac fibroblast differentiation

Cardiac fibroblast (CF) differentiation plays a crucial role in cardiac fibrosis, which is a specific manifestation distinguishing pathological cardiac hypertrophy from physiological hypertrophy. The DNA-binding activity of paired box 6 (Pax6) has been shown to be oppositely regulated in physiological and pathological hypertrophy; however, it remains unclear whether Pax6 is involved in CF differentiation during cardiac fibrosis. We found that Pax6 is expressed in the heart of and CFs isolated from adult mice. Moreover, angiotensin II (Ang II) induced the downregulation of Pax6 mRNA and protein expression in fibrotic heart tissue and cardiac myofibroblasts. Pax6 knockdown in CFs promoted the expression of the myofibroblast marker α-smooth muscle actin (α-SMA) and the synthesis of the extracellular matrix (ECM) proteins collagen I and fibronectin. Furthermore, we validated the ability of Pax6 to bind to the promoter regions of Cxcl10 and Il1r2 and the intronic region of Tgfb1. Pax6 knockdown in CFs decreased CXC chemokine 10 (CXCL10) and interleukin-1 receptor 2 (IL-1R2) expression and increased transforming growth factor β1 (TGFβ1) expression, mimicking the effects of Ang II. In conclusion, Pax6 exerts an inhibitory effect on CF differentiation and ECM synthesis by transcriptionally activating the expression of the anti-fibrotic factors CXCL10 and IL-1R2 and repressing the expression of the pro-fibrotic factor TGFβ1. Therefore, maintaining Pax6 expression in CFs is essential for preventing CF differentiation, and provides a new therapeutic target for cardiac fibrosis.

Cardiac Fibrosis and Cardiac Fibroblast Lineage-Tracing: Recent Advances.

Cardiac fibrosis is a common pathological change associated with cardiac injuries and diseases. Even though the accumulation of collagens and other extracellular matrix (ECM) proteins may have some protective effects in certain situations, prolonged fibrosis usually negatively affects cardiac function and often leads to deleterious consequences. While the development of cardiac fibrosis involves several cell types, the major source of ECM proteins is cardiac fibroblast. The high plasticity of cardiac fibroblasts enables them to quickly change their behaviors in response to injury and transition between several differentiation states. However, the study of cardiac fibroblasts in vivo was very difficult due to the lack of specific research tools. The development of cardiac fibroblast lineage-tracing mouse lines has greatly promoted cardiac fibrosis research. In this article, we review the recent cardiac fibroblast lineage-tracing studies exploring the origin of cardiac fibroblasts and their complicated roles in cardiac fibrosis, and briefly discuss the translational potential of basic cardiac fibroblast researches.

Secreted Frizzled-Related Protein 2 and Extracellular Volume Fraction in Patients with Heart Failure.

Background Quantification of extracellular volume (ECV) fraction by cardiovascular magnetic resonance (CMR) has emerged as a noninvasive diagnostic tool to assess myocardial fibrosis. Secreted frizzled-related protein 2 (SFRP2) appears to play an important role in cardiac fibrosis. We aimed to evaluate the association between SFRP2 and myocardial fibrosis and the prognostic value of ECV fraction in patients with heart failure (HF). Methods In this prospective cohort study, 72 hospitalized adult patients (age ≥ 18 years) with severe decompensated HF were included. CMR measurements and T1 mapping were performed to calculate ECV fraction. Serum SFRP2 level was detected by an enzyme-linked immunosorbent assay kit. All patients were followed up, and the primary outcomes were composite events including all-cause mortality and HF hospitalization. Results During the median follow-up of 12 months, 27 (37.5%) patients experienced primary outcome events and had higher levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP), SFRP2, and ECV fraction compared with those without events. In Pearson correlation analysis, levels of SFRP2 (r = 0.33), high-sensitivity C-reactive protein (r = 0.31), and hemoglobin A1c (r = 0.29) were associated with ECV fraction (all P < 0.05); however, in multivariate linear regression analysis, SFRP2 was the only significant factor determined for ECV fraction (rpartial = 0.33, P = 0.02). In multivariate Cox regression analysis, age (each 10 years, hazard ratio (HR) 1.13, 95% confidence interval (CI) 1.04–1.22), ECV fraction (per doubling, HR 1.68, 95% CI 1.03–2.74), and NT-proBNP (per doubling, HR 2.46, 95% CI 1.05–5.76) were independent risk factors for primary outcomes. Conclusions Higher ECV fraction is associated with worsened prognosis in HF. SFRP2 is an independent biomarker for myocardial fibrosis. Further studies are needed to explore the potential therapeutic value of SFRP2 in myocardial fibrosis.

Cardiomyocyte β2‐Adrenergic Receptor Plays A Protective Role in Cardiac Fibrosis

BackgroundChronic stimulation of β adrenergic receptor (βAR) has been implicated in cardiac remodeling including hypertrophy and fibrosis associated with heart failure. Previous studies indicate a role of β2AR on inhibiting collagen production in cultured cardiac fibroblasts, however, our recent studies show that global deletion of β2AR prevent cardiac fibrosis in diabetic cardiomyopathy. Therefore, this study aims to determine the specific role of cardiomyocyte β2AR in cardiac function and the development of cardiac fibrosis in vivo.Methods2‐month old male β2AR flox/flox (f/f) mice (n=10) and cardiomyocyte‐specific knockout of β2AR (CKO) mice (n=10) were fed with either normal chow (NC), or high fat diet (HFD) for 6 months to induce obesity and diabetes. Echocardiography was performed using a Vevo 2100 imaging system from VisualSonics. Biochemistry and histological analyses were performed on heart tissues. Real‐time PCR was performed for mRNA expression examination.ResultsWhile cardiac function was normal at 2‐month old CKO mice, a significant decrease in cardiac contractility was observed in CKO mice compared to f/f mice at 8‐month old age (EF%: CKO 48.43 ± 1.314 vs. f/f 52.69 ± 0.8270, p&lt;0.05). Moreover, HFD feeding exacerbated cardiac dysfunction in CKO mice when compared to f/f mice (EF%: CKO 42.93 ± 0.69 vs. f/f 46.79 ± 0.76, p&lt;0.001). CKO mice also exhibited diastolic dysfunction (IVRT: CKO 20.23 ± 0.30 vs. f/f 16.83 ± 0.23, p&lt;0.0001), which was exacerbated after HFD feeding (IVRT, CKO 24.75 ± 0.50 vs. f/f 19.94 ± 0.45, p&lt;0.0001). The observed cardiac dysfunction was associated with severe cardiac fibrosis in 8‐month old CKO mice, and HFD feeding leads to further exacerbation in cardiac fibrosis. Cardiac fibrosis was validated with elevated protein expression of connective tissue growth factor (CTGF), and significant upregulations of mRNA expression of CTGF, α smooth muscle actin (α‐SMA), and collagen 1 in CKO HFD mice.ConclusionsThis study elucidates that cardiomyocyte β2AR is essential to maintain normal cardiac function and plays a protective role against the development of cardiac fibrosis in aging and HFD feeding in vivo.Support or Funding InformationNIH HL113413, HL147264, VABX002900

DNMT1 Methylation of LncRNA GAS5 Leads to Cardiac Fibroblast Pyroptosis via Affecting NLRP3 Axis.

Cell death and inflammation play critical roles in cardiac fibrosis. During the fibrosis process, inflammation and tissue injury were triggered; however, the mechanisms initiating cardiac fibrosis and driving fibroblast pyroptosis remained largely unknown. In this study, we identified long non-coding RNA (LncRNA)-GAS5 as the key onset of cardiac fibroblast pyroptosis and cardiac fibrosis. Here, we detected ISO-induced cardiac fibrosis models and cardiac fibroblast pyroptosis model by stimulating with LPS. We found that the expression of pyroptosis-related proteins such as caspase 1, NLRP3, and DNMT1 was increased in cardiac fibrosis tissue, while the expression of GAS5 was decreased. The overexpressing of LncRNA GAS5 was shown to increase and inhibit cardiac fibroblast pyroptosis, as well as attenuate caspase 1 and NLRP3 expression in cardiac fibroblast. However, the silencing of GAS5 was also observed; it shows the opposite situation. Furthermore, further studies revealed that treatment of DNMT inhibitor, 5-aza-2-deoxycytidine, or downregulation of DNMT1 led to increased GAS5 expression by reversion of promoter hypermethylation in cardiac fibroblast. Importantly, we have demonstrated that DNMT1 methylation of LncRNA GAS5 leads to cardiac fibroblast pyroptosis via affecting NLRP3 axis. Our findings indicate a new regulatory mechanism for cardiac fibroblast pyroptosis under LPS stress, providing a novel therapeutic target for cardiac fibrosis. Graphical Abstract.

Deficiency of MicroRNA miR-1954 Promotes Cardiac Remodeling and Fibrosis.

BackgroundCardiac fibrosis occurs because of disruption of the extracellular matrix network leading to myocardial dysfunction. Angiotensin II (AngII) has been implicated in the development of cardiac fibrosis. Recently, microRNAs have been identified as an attractive target for therapeutic intervention in cardiac pathologies; however, the underlying mechanism of microRNAs in cardiac fibrosis remains unclear. Next‐generation sequencing analysis identified a novel characterized microRNA, miR‐1954, that was significantly reduced in AngII‐infused mice. The finding led us to hypothesize that deficiency of miR‐1954 triggers cardiac fibrosis.Methods and ResultsA transgenic mouse was created using α‐MHC (α‐myosin heavy chain) promoter and was challenged with AngII infusion. AngII induced cardiac hypertrophy and remodeling. The in vivo overexpression of miR‐1954 showed significant reduction in cardiac mass and blood pressure in AngII‐infused mice. Further analysis showed significant reduction in cardiac fibrotic genes, hypertrophy marker genes, and an inflammatory gene and restoration of a calcium‐regulated gene (Atp2a2 [ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2]; also known as SERCA2), but no changes were observed in apoptotic genes. THBS1 (thrombospondin 1) is indicated as a target gene for miR‐1954.ConclusionsOur findings provide evidence, for the first time, that miR‐1954 plays a critical role in cardiac fibrosis by targeting THBS1. We conclude that promoting the level of miR‐1954 would be a promising strategy for the treatment of cardiac fibrosis.

P1626MicroRNA-21 deteriorates left ventricular reverse remodeling by promoting cardiac fibrosis in non-ischemic cardiomyopathy

Abstract Background Left ventricular reverse remodeling (LVRR) contributes to better outcomes in patients with non-ischemic cardiomyopathy (NICM). It is reported that LVRR is associated with progression of cardiac fibrosis. MicroRNAs (miRs) have emerged as powerful regulators of post-transcriptional gene expression. We focused on miR-21, which plays a key role in pathogenesis of fibrosis in multiple organs. The aim of this study was to clarify the effect of miR-21 on cardiac fibrosis and LVRR in patients with NICM. Methods We measured plasma miR-21 levels in 16 patients with NICM. LVRR was defined as increased LVEF by ≥10% and decreased LV end-diastolic diameter index by ≥10% from baseline data after optimal medication treatment at 1-year of follow-up. Further, we examined miR-21 expression and its potential role in cardiac fibrosis induced by transverse aortic constriction (TAC) in mice and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes (NRCMs). Results There were 12 patients without LVRR and 4 patients with LVRR. Plasma miR-21 levels were significantly higher in patients without LVRR compared with those with LVRR. In TAC mice heart, miR-21 levels were significantly increased and programmed cell death 4 (PDCD4), a main target of miR-21, was decreased. In vitro, miR-21 levels were significantly increased and its upstream transcriptional factor, activator protein 1 (AP-1), was activated by Ang II stimulation in NRCMs. After transfection of miR-21 specific inhibitor, PDCD4 levels were upregulated. Furthermore, AP-1 activity, expression of collagen type I, and α-smooth muscle actin levels were significantly decreased after miR-21 inhibition. Conclusions These findings suggested that miR-21/PDCD4/AP-1 feedback loop pathway was involved in LVRR in patients with NICM by promoting cardiac fibrosis. MiR-21 can be the therapeutic target in NICM.

The axis of local cardiac endogenous Klotho-TGF-β1-Wnt signaling mediates cardiac fibrosis in human

BackgroundCardiovascular 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 resultsHuman 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-β1. 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-β1. Klotho knockdown or overexpression aggravates or mitigates TGF-β1-induced fibrosis and canonical Wnt signaling in HCMs, respectively. Furthermore, co-culture of HCMs with HCFs increases TGF-β1-induced fibrogenic proteins in HCFs, but overexpression of endogenous Klotho in HCMs mitigates this effect, suggesting functional crosstalk between HCMs and HCFs. ConclusionsOur 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-β1 signaling which enables more vigorous cardiac fibrosis through upregulated Wnt signaling. Upregulation of endogenous Klotho inhibits pathogenic Wnt/β-catenin signaling and may offer a novel strategy for prevention and treatment of cardiac fibrosis in CKD patients.

Abstract 518: Pharmacological Inhibition of Human Antigen R (HuR) Blunts fibroblast Activation and Cardiac Fibrosis

Myocardial fibrosis is associated with many cardiac diseases and often leads to a worsened prognosis. In a healthy heart, cardiac fibroblasts are responsible for maintaining the extracellular matrix (ECM). In response to stress or injury, however, the fibroblasts activate to myofibroblasts and begin proliferating, migrating, and depositing excess ECM. This leads to decreased compliance and contractility of the myocardium, furthering the progression to heart failure. Dampening the chronic activation of fibroblasts, therefore, is an attractive target for future cardiac therapeutics. Human Antigen R (HuR) is an RNA binding protein known to stabilize mRNA through binding to AU rich regions in the 3’UTR. Our lab has previously utilized a myocyte-specific knockout of HuR to show that HuR plays an important role in cardiac fibrosis through mediating TGFβ gene expression in the myocytes. Additionally, pharmacological inhibition of HuR markedly reduced interstitial cardiac fibrosis in an 11-week transverse aortic constriction (TAC) mouse model of heart failure, quantifying the fibrosis with Masson’s Trichrome Stains, western blots, and qPCR of profibrotic markers. The goal of this work is to identify the functional role of HuR in mediating pro-fibrotic gene expression and ECM remodeling activity in cardiac fibroblasts. Our results demonstrate that HuR inhibition blunts the activation and wound healing activity of primary adult cardiac fibroblasts. Specifically, utilizing an in vitro scratch/wound healing assay we show that HuR inhibition significantly prolongs wound closure time and blunts the expression of profibrotic genes (e.g. collagen1a1, periostin, TGFb, fibronectin). Ongoing studies will determine the effect of HuR inhibition in a two-week isoproterenol pump (30mg/kg/day) model. Quantification of fibrosis and upstream signaling of fibroblast activation, such as TGFβ-dependent phosphorylation of SMAD2/3, will be assessed to determine the role of HuR in the initiation of fibrotic signaling.

Regulation of Cardiac Mast Cell Maturation and Function by the Neurokinin-1 Receptor in the Fibrotic Heart

Cardiac fibrosis is an underlying cause of diastolic dysfunction, contributing to heart failure. Substance P (SP) activation of the neurokinin-1 receptor (NK-1R) contributes to cardiac fibrosis in hypertension. However, based on in vitro experiments, this does not appear to be via direct activation of cardiac fibroblasts. While numerous cells could mediate the fibrotic effects of SP, herein, we investigate mast cells (MC) as a mechanism mediating the fibrotic actions of SP, since MCs are known to play a role in cardiac fibrosis and respond to SP. Spontaneously hypertensive rats (SHR) were treated with the NK-1R antagonist L732138 (5 mg/kg/d) from 8 to 12 weeks of age. L732138 prevented increased MC maturation of resident immature MCs. NK-1R blockade also prevented increased cardiac MC maturation in angiotensin II-infused mice. MC-deficient mice were used to test the importance of MC NK-1Rs to MC activation. MC-deficient mice administered angiotensin II did not develop fibrosis; MC-deficient mice reconstituted with MCs did develop fibrosis. MC-deficient mice reconstituted with MCs lacking the NK-1R also developed fibrosis, indicating that NK-1Rs are not required for MC activation in this setting. In conclusion, the NK-1R causes MC maturation, however, other stimuli are required to activate MCs to cause fibrosis.

CARD9 inhibits mitochondria-dependent apoptosis of cardiomyocytes under oxidative stress via interacting with Apaf-1

Cardiomyocyte apoptosis is known to contribute to myocardial ischemia/reperfusion (I/R) injury. Caspase recruitment domain-containing protein 9 (CARD9) play a role in cardiac fibrosis and dysfunction. However, the role of CARD9 in apoptosis of cardiomyocytes in myocardial I/R injury and its underlying mechanisms are still unclear. In this study, CARD9 expression was found to increase in H9c2 cells in response to hydrogen peroxide. Loss of CARD9 significantly increased caspase-3 activation and cardiomyocyte death following oxidative stress in vitro. Conversely, CARD9 overexpression decreased apoptosis as evidenced by a reduction in caspase-3 activation and the apoptotic rate. The caspase recruitment domain (CARD) of CARD9 was necessary for the protective effect of CARD9 against oxidative stress in cardiomyocytes. CARD9 suppressed the activation of caspase-9 by interacting with Apaf-1 via its CARD domain in H9c2 cells exposed to H2O2. Ablation of caspase-9 activity by z-lehd-fmk effectively prevented the detrimental effect of CARD9 deficiency on cardiomyocytes. Wild-type (WT) and CARD9−/− mice were subjected to 30 min of left ascending coronary (LAD) ischemia and 12 h of reperfusion. TdT-mediated dUTP nick end labeling (TUNEL) staining analysis showed that CARD9−/− mice exhibited a significantly higher number of apoptotic-positive cells after myocardial I/R injury than the WT mice. These results suggest that CARD9 protects cardiomyocytes from apoptosis by interacting with Apaf-1 and interfering with apoptosome formation following myocardial I/R injury in vivo and in vitro.

Improvements in left ventricular regional and global systolic function following treatment with S100A4-shRNA after myocardial infarction in mice.

S100A4 plays a vital role in cardiac fibrosis after myocardial infarction (MI), but its effects on myocardial mechanics and remodeling are unknown. We hypothesized that regional and global left ventricular (LV) systolic function as determined by speckle tracking echocardiography (STE) would be improved with inhibition of S100A4 using short hairpin (sh) RNA. This study aimed to investigate whether STE can delineate the efficacy and safety of S100A4-shRNA in MI. A total of 48 mice were randomly assigned to sham+S100A4-shRNA, sham+scrambled (Scr) sequence-shRNA, MI+S100A4-shRNA, and MI+Scr-shRNA groups (n=12 per group) and underwent intramyocardial injection of target agents after MI was produced by left anterior descending ligation. Two-dimensional STE and M-mode echocardiography were performed at baseline and at day 7, 14, and 28 post-MI by GE Vivid 7 ultrasound (il3L linear probe, 10.0-14.0 MHz) and Echopac PC software. Radial strain was analyzed from 6 segments of the mid short axis images with 20-30 frames per cardiac cycle. Post-mortem western blotting, immunohistochemistry, and Masson's trichrome stain were performed to quantify infarct size and detect suppression of S100A4. STE detected a statistically significant improvement in peak radial strain (pRS) and time to peak radial strain (pRSt) by day 14 post-MI in the MI+S100A4-shRNA group (P<0.05), especially in the LV anteroseptal wall (pRS: 23.83%±1.12% vs. 20.25%±1.02%, pRSt: 76.75±3.18 vs. 92.00±3.69 ms, P<0.05). After 1 month of S100A4-shRNA administration, cardiac function improved in the MI+S100A4-shRNA group according to both STE and M-mode tracing in mice. Additionally, both biochemical and histopathological examinations found reduced cardiac fibrosis in the MI+S100A4-shRNA group. S100A4-shRNA can be utilized as a therapeutic target to improve regional deformation and attenuate cardiac fibrosis following MI. Two-dimensional STE is useful in the early and comprehensive assessment of LV systolic function in mice.

Serum endotrophin levels in patients with heart failure with reduced and mid-range ejection fraction

BackgroundEndotrophin, a type VI collagen cleavage product, has fibrosis, and insulin resistance effects. Type VI collagen also plays a role in cardiac fibrosis. In this study, we aimed to investigate the role of endotrophin in the pathogenesis of cardiac fibrosis by determining its levels in patients with heart failure with reduced and mid-range ejection fraction (EF). We also aimed to determine the possible association between endotrophin and treatment that prevents ventricular fibrosis. MethodsSixty patients with heart failure with reduced and mid-range EF and 27 volunteers with no cardiac failure were included in this study. In both groups, biochemical tests, EF, and endotrophin levels were measured. ELISA was performed for the determination of endotrophin levels. ResultsWhen compared with the control group, there was no significant difference for endotrophin levels in the patient group (p = .35). Participants in the study were divided into two groups according to their EFs, 40% and less, and 40–49%. They were classified according to their use of renin-angiotensin-aldosterone system (RAAS) blocking drugs. Endotrophin levels were significantly lower in patients with mid-range EFs between 40 and 49% (p = .03) using RAAS blockers. ConclusionThis study is the first to evaluate the relationship between endotrophin and heart failure. Endotrophin levels were found to be low in patients with heart failure with mid-range EF who were using RAAS blockers. This suggests that RAAS blockers may influence endotrophin levels and thus could have a role in the prevention of remodelling.

ERK1/2 mediates the lipopolysaccharide-induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts

Myocardial fibrosis is a critical event during septic shock. Upregulation in the fibrosis signaling cascade proteins such as fibroblast growth factor (FGF), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and activation of matrix metalloproteinases (MMPs) are widely associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac fibrosis and heart failure. However, evidences suggest that the common upstream mediators of fibrosis cascade play little role in cardiac fibrosis induced by LPS; further, it is unknown if LPS directly triggers the expressions and/or activity of FGF-2, uPA, tPA, MMP-2 and MMP-9 in cardiac fibroblasts. In the present study, we treated primary cultures of cardiac fibroblasts with LPS to explore whether LPS upregulates FGF-2, uPA, tPA, MMP-2, MMP-9 and enhance cellular migration. Further the precise molecular and cellular mechanisms behind these LPS induced responses were identified. Inhibition assays on MAPKs using U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor) and QNZ (NFκB inhibitor) show that LPS-induced upregulation of FGF-2, uPA, MMP-2 and MMP-9 in cardiac fibroblasts was mediated through ERK1/2 signaling. Collectively, our results provide a link between LPS-induced cardiac dysfunction and ERK1/2 signaling pathway and thereby implies ERK1/2 as a possible target to regulate LPS induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac fibroblasts.

Inhibition of circHIPK3 prevents angiotensin II-induced cardiac fibrosis by sponging miR-29b-3p

BackgroundCircular RNAs (circRNAs) are emerging as powerful regulators of cardiac development and disease. Nevertheless, detailed studies describing circRNA-mediated regulation of cardiac fibroblasts (CFs) biology and their role in cardiac fibrosis remain limited. MethodsPCR and Sanger sequencing were performed to identify the expression of circHIPK3 in CFs. Edu corporation assays, Transwell migration assays, and immunofluorescence staining assays were conducted to detect the function of circHIPK3 in CFs in vitro. Bioinformatics analysis, dual luciferase activity assays, RNA immunoprecipitation, and fluorescent in situ hybridization experiments were conducted to investigate the mechanism of circHIPK3-mediated cardiac fibrosis. Echocardiographic analysis, Sirius Red staining and immunofluorescence staining were performed to investigate the function of circHIPK3 in angiotensin II (Ang II) induced cardiac fibrosis in vivo. ResultscircHIPK3 expression markedly increased in CFs and heart tissues after the treatment of Ang II. circHIPK3 silencing attenuates CFs proliferation, migration and the upregulation of a-SMA expression levels induced by Ang II in vitro. circHIPK3 acted as a miR-29b-3p sponge and overexpression of circHIPK3 effectively reverses miR-29b-3p-induced inhibition of CFs proliferation and migration and alters the expression levels of miR-29b-3p targeting genes (a-SMA, COL1A1, COL3A1) in vitro. Combination of circHIPK3 silencing and miR-29b-3p overexpression had a stronger effect on cardiac fibrosis suppression in vivo than did circHIPK3 silencing or miR-29b-3p overexpression alone. ConclusionsOur data suggest that circHIPK3 serves as a miR-29b-3p sponge to regulate CF proliferation, migration and development of cardiac fibrosis, revealing a potential new target for the prevention of Ang II-induced cardiac fibrosis.

Silencing of c-Ski augments TGF-b1-induced epithelial-mesenchymal transition in cardiomyocyte H9C2 cells.

The shRNA lentiviral vector was constructed to silence c-Ski expression in cardiac mus- cle cells, with the aim of exploring the role of c-Ski in transforming growth factor b1 (TGF-b1)-induced epithelial-mesenchymal transitions (EMT) in H9C2 cells. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect c-Ski ex- pression at protein and messenger ribonucleic acid (mRNA) levels in 5 different cell lines. Then, lentiviral vector was constructed to silence or overexpress c-Ski in H9C2 cells. MTT and/or soft agar assay and tran- swell assay were used to detect cell proliferation and migration, respectively. The expression levels of c-Ski under different concentrations of TGF-b1 stimulation were detected by RT-qPCR and immunocytochemi- cal analysis. In the presence or absence of TGF-b1 stimulation, the proteins' expression levels of a-SMA, FN and E-cadherin, which are closely correlated with the process of EMT, were measured by western blot after c-Ski silencing or overexpression. Meanwhile, the effect of c-Ski on Samd3 phosphorylation with TGF-b1 stimulation was investigated. There is a high expression of c-Ski at protein and mRNA levels in H9C2 cell line, which first demonstrated the presence of c-Ski expression in H9C2 cells. Overexpression of c-Ski significantly increased H9C2 cell proliferation. The ability of c-Ski gene silencing to suppress cell proliferation was gradually enhanced, and inhibition efficiency was the highest after 6 to 7 d of transfection. Moreover, H9C2 cells with c-Ski knockdown gained significantly aggressive invasive potential when compared with the control group. TGF-b1 stimulation could dose-independently reduce c-Ski expression in H9C2 cells and lead to obvious down-regulated expression of E-cadherin. Interestingly, c-Ski could restore E-cadherin expression while suppressing a-SMA and/or FN expression stimulated by TGF-b1. How- ever, shRNA-induced c-Ski knockdown aggravated only the TGF-b1-induced EMT. Moreover, c-Ski- -shRNA also promoted the phosphorylation of Samd3 induced by TGF-b1. c-Ski expression in cardiac muscle cells could be down-regulated by TGF-b1. Silencing of c-Ski gene was accompanied by down-regulation of E-cadherin, up-regulation of a-SMA and/or FN and Smad3 phosphorylation induced by TGF-b1, promoting EMT process. Therefore, c-Ski may be closely associated with TGF-b1-induced EMT and play an important role in cardiac fibrosis develop- ment and progression.

The Role of Pathological Aging in Cardiac and Pulmonary Fibrosis.

Aging promotes a range of degenerative pathologies characterized by progressive losses of tissue and/or cellular function. Fibrosis is the hardening, overgrowth and scarring of various tissues characterized by the accumulation of extracellular matrix components. Aging is an important predisposing factor common for fibrotic heart and respiratory disease. Age-related processes such as senescence, inflammaging, autophagy and mitochondrial dysfunction are interconnected biological processes that diminish the regenerative capacity of the aged heart and lung and have been shown to play a crucial role in cardiac fibrosis and idiopathic pulmonary fibrosis. This review focuses on these four processes of aging in relation to their role in fibrosis. It has long been established that the heart and lung are linked both functionally and anatomically when it comes to health and disease, with an ever-expanding aging population, the incidence of fibrotic disease and therefore the number of fibrosis-related deaths will continue to rise. There are currently no feasible therapies to treat the effects of chronic fibrosis therefore highlighting the importance of exploring the processes of aging and its role in inducing and exacerbating fibrosis of each organ. The focus of this review may help to highlight potential avenues of therapeutic exploration

DNA methylation regulates α-smooth muscle actin expression during cardiac fibroblast differentiation.

Cardiac fibroblast (CF) differentiation to myofibroblasts expressing α-smooth muscle actin (α-SMA) plays a key role in cardiac fibrosis. Therefore, a study of the mechanism regulating α-SMA expression is a means to understanding the mechanism of fibroblast differentiation and cardiac fibrosis. Previous studies have shown that DNA methylation is associated with gene expression and is related to the development of tissue fibrosis. However, the mechanisms by which CF differentiation is regulated by DNA methylation remain unclear. Here, we explored the epigenetic regulation of α-SMA expression and its relevance in CF differentiation. In this study, we demonstrated that α-SMA was overexpressed and DNMT1 expression was downregulated in the infarct area after myocardial infarction. Treatment of CFs with transforming growth factor-β1 (TGF-β1 ) in vitro upregulated α-SMA expression via epigenetic modifications. TGF-β1 also inhibited DNMT1 expression and activity during CF differentiation. In addition, α-SMA expression was regulated by DNMT1. Conversely, increasing DNMT1 expression levels rescued the TGF-β1 -induced upregulation of α-SMA expression. Finally, TGF-β1 regulated α-SMA expression by inhibiting the DNMT1-mediated DNA methylation of the α-SMA promoter. Taken together, our research showed that inhibition of the DNMT1-mediated DNA methylation of the α-SMA promoter plays an essential role in CF differentiation. In addition, DNMT1 may be a new target for the prevention and treatment of myocardial fibrosis.