Role In Cardiac Fibrosis Research Articles

Serum markers of cardiac fibrosis suffering from heart failure with preserved left ventricular ejection fraction upon ST-segment elevation myocardial infarction

Aim. Currently, there is no method which accurately predicts an adverse outcome of heart failure with a preserved left ventricular ejection fraction (HFpEF) upon ST-segment elevation myocardial infarction (STEMI). Here we studied the prognostic significance of procollagen type I C-terminal propeptide (PICP) and procollagen type III N-terminal propeptide (PIIINP) in patients with post-STEMI HFpEF.Material and Methods. The study included 83 patients (60 men and 23 women) with post-STEMI HFpEF (left ventricular EF ≥ 50%) and 20 ageand gender-matched healthy controls. Serum concentrations of PICP and PIIINP were measured on the 1st day of hospitalization using enzyme-linked immunosorbent assay with the following calculation of PICP/PIIINP ratio.Results. Serum PICP and PIIINP on the 1st day of STEMI significantly (3.4-fold) exceeded the values of the control group and were as follows: PIIINP: 26.0 (18.9; 34.9) ng/mL (р = 0.047); PICP: 609.0 (583.0; 635.0) ng/mL (р = 0.049).Conclusion. Elevated values of procollagens indicate that cardiac fibrosis commences within the 24 hours after STEMI onset. The pivotal role of cardiac fibrosis in the formation of diastolic dysfunction suggests the usefulness of serum procollagens to predict the development of HFpEF in a long-term period upon STEMI.

Cardiac Fibrosis: Cellular Effectors, Molecular Pathways, and Exosomal Roles.

Cardiac fibrosis, a common pathophysiologic process in most heart diseases, refers to an excess of extracellular matrix (ECM) deposition by cardiac fibroblasts (CFs), which can lead to cardiac dysfunction and heart failure subsequently. Not only CFs but also several other cell types including macrophages and endothelial cells participate in the process of cardiac fibrosis via different molecular pathways. Exosomes, ranging in 30–150 nm of size, have been confirmed to play an essential role in cellular communications by their bioactive contents, which are currently a hot area to explore pathobiology and therapeutic strategy in multiple pathophysiologic processes including cardiac fibrosis. Cardioprotective factors such as RNAs and proteins packaged in exosomes make them an excellent cell-free system to improve cardiac function without significant immune response. Emerging evidence indicates that targeting selective molecules in cell-derived exosomes could be appealing therapeutic treatments in cardiac fibrosis. In this review, we summarize the current understandings of cellular effectors, molecular pathways, and exosomal roles in cardiac fibrosis.

Latent-transforming growth factor β-binding protein 2 accelerates cardiac fibroblast apoptosis by regulating the expression and activity of caspase-3.

Cardiac fibrosis is a core process in the development of heart failure. However, the underlying mechanism of cardiac fibrosis remains unclear. Recently, a study found that in an isoproterenol (ISO)-induced cardiac fibrosis animal model, there is high expression of latent-transforming growth factor β-binding protein 2 (LTBP2) in cardiac fibroblasts. Whether LTBP2 serves a role in cardiac fibrosis is currently unknown. In the present study, mouse cardiac fibroblasts (MCFs) were treated with 100 µM/l ISO for 24, 48, or 72 h, and small interfering RNAs (siRNAs) were used to knockdown LTBP2. Reverse transcription-quantitative PCR and western blotting were used to determine gene and protein expression levels, respectively. Caspase-3 serves a key role in cell apoptosis and is related to cardiac fibrosis-induced heart failure. Caspase-3 activity was therefore determined using a caspase-3 assay kit, CCK8 was used to determine the rate of cell proliferation and apoptosis rates were quantified using a cell death detection ELISA kit. The present study demonstrated that cell apoptosis and LTBP2 expression increased in MCFs treated with 100 µM/l ISO in a time-dependent manner. Expression and activity of caspase-3 also increased in MCFs treated with 100 µM/l ISO for 48 h compared with the control group. In addition, ISO stimulation-induced MCF apoptosis, along with the increased expression of caspase-3 were partly abolished when LTBP2 was knocked down. In conclusion, LTBP2 expression increased in ISO-treated MCFs and accelerated mouse cardiac fibroblast apoptosis by enhancing the expression and activity of caspase-3. LTBP2 may therefore be a potential therapeutic target for treating patients with cardiac fibrosis.

Ivabradine Ameliorates Cardiac Diastolic Dysfunction in Diabetic Mice Independent of Heart Rate Reduction.

Cardiac fibroblast (CF) proliferation and activation play important roles in cardiac fibrosis and diastolic dysfunction (DD), which are involved in fibrosis-associated cardiovascular diseases. A previous study showed that ivabradine, a specific heart rate (HR)-lowering agent, significantly ameliorated DD in diabetic db/db mice by reducing HR. Herein, we attempted to determine whether ivabradine has antifibrotic and cardioprotective effects in diabetic mice by directly suppressing CF proliferation and activation, independent of a reduction in HR. We found that knockdown of c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK), or treatment with ivabradine, reduced JNK and p38 MAPK phosphorylation and the protein expression of proliferating cell nuclear antigen, collagen I, collagen III, tissue inhibitor of matrix metalloproteinase 2, and α-smooth muscle actin, accompanied with upregulation of matrix metalloproteinase 2 both in high glucose-treated neonatal rat CFs and left ventricular CFs isolated from db/db mice. However, zatebradine (a HR-lowering agent) did not have these effects in vitro or in vivo. In addition, cardiac fibrosis and DD were ameliorated in db/db mice that were intravenously administered lentiviruses carrying short hairpin RNAs targeting JNK and p38 MAPK or administered ivabradine. Taken together, these findings demonstrate that the ivabradine-induced amelioration of cardiac fibrosis, and DD in db/db mice may be at least in part attributable to the suppression of CF proliferation and activation, through the inhibition of JNK and p38 MAPK.

B10 cells decrease fibrosis progression following cardiac injury partially by IL-10 production and regulating hyaluronan secretion.

B10 cells play negative roles in inflammatory disorders by producing IL-10. However, their effects on fibrosis have not been elucidated. Therefore, this study was conducted to examine the dynamic changes of B10 cell frequency and their potential role in cardiac fibrosis. We found that the frequency of B10 cells was significantly increased, and they participated in the regression of fibrosis via IL-10, particularly by accelerating hyaluronan secretion and inhibiting collagen deposition. In vivo, hyaluronan ablation or treatment significantly restricted cardiac fibrosis development. hyaluronan-induced conversion of M1/M2 Mc was dependent on the size of hyaluronan. Low molecular weight hyaluronan promoted the conversion to M1 Mϕ, whereas medium and high molecular weight hyaluronan accelerated Mϕ transdifferentiation into the M2 phenotype. Adoptive transfer of B10 cells significantly attenuated collagen deposition whereas CD19-/- mice with reduced B10 cells exacerbated fibrosis following cardiac injury. Our results provide new evidence suggesting that B10 cells exert antifibrotic effects by regulating the extracellular matrix composition during cardiac injury, and also highlight that B10 cells may serve as a promising therapeutic candidate for managing cardiac fibrosis-associated disorders.

SGLT2 promotes cardiac fibrosis following myocardial infarction and is regulated by miR-141.

Cardiac fibrosis is a primary event during myocardial infarction (MI) progression, which impairs cardiac function. The present study aimed to investigate the effect of SGLT2 on cardiac fibrosis following MI. To validate the role of SGLT2 in the regulation of cardiac fibrosis in vivo, an MI rat model was established. Echocardiography was performed to determine cardiac function at 4 weeks post-MI. MI model rats were transfected with short hairpin RNA (sh)-SGLT2 or sh-negative control lentiviruses to investigate the effect of SGLT2 on rat heart function post-MI. Subsequently, the effects of SGLT2 on the cardiac fibrosis of infarcted hearts were assessed by performing Masson's trichrome staining. To further clarify the effect of SGLT2 on cardiac fibroblast proliferation, TGFβ was used to stimulate primary cardiac fibroblasts in vitro. The results demonstrated that SGLT2 served a key role in cardiac fibrosis. SGLT2 expression levels in infarct tissues were significantly increased at week 1 post-MI compared with the sham group. Compared with the control group, SGLT2 knockdown attenuated cardiac fibrosis by inhibiting the expression of collagen I and collagen III in cardiac fibroblasts in vitro and in vivo. Furthermore, the results indicated that SGLT2 expression was modulated by miR-141 in cardiac fibroblasts. In summary, the present study indicated that upregulated SGLT2 expression in cardiac fibrosis following MI was regulated by miR-141 and SGLT2 that knockdown reduced cardiac fibrosis and improved cardiac function after MI.

TNAP is a novel regulator of cardiac fibrosis after myocardial infarction by mediating TGF-β/Smads and ERK1/2 signaling pathways.

Cardiac fibrosis is the most important pathogenesis leading to cardiac remodeling and heart failure after myocardial infarction (MI). Tissue nonspecific alkaline phosphatase (TNAP) has recently been recognized as a potential prognostic factor for MI. Nevertheless, the role of TNAP in cardiac fibrosis after MI has not been explicitly delineated. A systematic review and meta-analysis was conducted to assess the effect of serum TNAP levels on mortality in patients with ischemic heart disease (IHD). A correlation analysis was performed to investigate the relationship between serum levels of TNAP and biomarkers of fibrosis. Heart biopsies from patients with MI and a mouse model of MI were used to detect the expression and distribution of TNAP. Furthermore, we established adenovirus-mediated knockdown and overexpression of TNAP, using a combination of in vivo and in vitro studies in mice, to determine the role and mechanism of TNAP in cardiac fibrosis after MI. In the in vitro studies, cardiac fibroblasts were cultured on soft plates. After searching the main databases and performing a detailed assessment of the full-text articles, eight studies with 14,816 individuals were included in the quantitative analysis. We found that a high serum TNAP level was associated with an increased risk of mortality in patients with IHD and MI. The correlation analysis revealed a positive correlation between serum TNAP levels and the concentration of fibrosis biomarkers (PICP/PIIINP). The expression of TNAP was upregulated in the myocardium of patients with MI and in a mouse model of MI, accompanied by fibroblast activation and the deposition of collagen fibers. In the in vivo study, TNAP knockdown ameliorated cardiac fibrosis and improved cardiac function in mice. TNAP overexpression aggravated cardiac fibrosis and worsened cardiac function. In the in vitro study, TNAP promoted cardiac fibroblast differentiation, migration and proliferation. Mechanistically, the pro-fibrotic effect of TNAP on cardiac fibroblasts was at least partially achieved by activating the TGF-β1/Smads and ERK1/2 signaling pathways. Based on these findings, TNAP plays an important pro-fibrotic role in cardiac fibrosis after MI by activating TGF-β/Smads and ERK1/2 signaling, indicating that it functions as a potential regulator of and therapeutic target in cardiac fibrosis. This work was supported by the National Natural Science Foundation of China.

GENETIC SUSCEPTIBILITY TO EARLY ONSET OF TRANSFORMING GROWTH FACTOR-BETA DRIVEN CARDIAC REMODELING IN MICE IS ASSOCIATED WITH EGR1 AND STAT3

Objective: We have previously described that the severity of TGF-b induced cardiac remodeling was associated with genetic background differences in alb/TGF-b1 transgenic mice (with elevated plasma TGF-b1 levels) on B6 vs CBAxB6 strains. Early growth response factor-1 (EGR1) has been associated with kidney fibrosis, and we recently demonstrated non-canonical TGF-b induced STAT3 activation in vascular smooth muscle cells, but their role in cardiac fibrosis or hypertrophy was not yet elucidated. The present study investigated whether genetic susceptibility to TGF-b induced early cardiac remodeling is associated with transcription factors EGR1 and STAT3. Design and method: As the albumin promoter driven TGF-b1 transgene is expressed after birth only in males reaching early high plasma TGF-b1 levels, we investigated 14-day old male transgenic and wild type control mice. Blood pressure of TGF-b1 transgenic (B6-TGFb and CBAxB6-TGFb) mice, and wild type (B6 and CBAxB6) controls (n = 6–9/group) was assessed, then cardiac samples were harvested and analyzed for mRNA and protein expression. Data are presented as mean ± SD and evaluated using Kruskal-Wallis test with Dunn's post-hoc test. Results: Blood pressure was elevated in both transgenic strains as compared to controls (MAP mmHg, B6: 46 ± 3; CBAxB6: 48 ± 4; B6-TGFb:54 ± 9; CBAxB6-TGFb:68 ± 7, p < 0.01). Compared to B6-TGFb and wild type, CBAxB6-TGFb hearts showed 20-fold elevation in BNP mRNA relative expression (B6: 1.5 ± 0.9; CBAxB6: 2.3 ± 0.9; B6-TGFb:1.5 ± 1.0; CBAxB6-TGFb:23.5 ± 13,3 p < 0.001) associated with increased Collagen-I (B6: 1.3 ± 0.2; CBAxB6: 1.2 ± 0.1; B6-TGFb:1.2 ± 0.2; CBAxB6-TGFb:1.9 ± 0.6, p < 0.05) and EGR1 (B6: 0.9 ± 0.2; CBAxB6: 0.8 ± 0.3; B6-TGFb:0.9 ± 0.6; CBAxB6-TGFb:1.8 ± 0.5, p < 0.01) as well as STAT3 mRNA overexpression (B6: 0.8 ± 0.1; CBAxB6: 1.2 ± 0.2; B6-TGFb:1.1 ± 0.1; CBAxB6-TGFb:1.7 ± 0.6, p < 0.01) and STAT3 phosphorylation. Early cardiac remodeling was further associated with 12-fold and 2-fold overexpression of TIMP-1 (B6: 1.0 ± 0.4; CBAxB6: 0.8 ± 0.4; B6-TGFb:0.7 ± 0.3; CBAxB6-TGFb:12.2 ± 9.0, p < 0.01) and TIMP-2 (B6: 1.1 ± 0.1; CBAxB6: 1.2 ± 0.2; B6-TGFb:1.3 ± 0.4; CBAxB6-TGFb:2.1 ± 1.1, p < 0.05), respectively. Significant EGR1 immunoreactivity was detected only in cardiomyocytes of CBAxB6-TGFb hearts. Conclusions: We conclude that EGR1 and STAT3 might strongly contribute to the initiation and early progression of cardiac remodeling driven by high circulating TGF-b levels. We postulate that EGR1 and STAT3 could be potential targets to inhibit early cardiac remodeling.

CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42

Phenotype switching of cardiac fibroblasts into myofibroblasts plays important role in cardiac fibrosis following myocardial infarction (MI). Cellular repressor of E1A-stimulated genes (CREG) protects against vascular and cardiac remodeling induced by angiotensin-II. However, the effects and mechanisms of CREG on phenotype switching of cardiac fibroblasts after MI are unknown. This study aimed to investigate the role of CREG on the phenotype switching of cardiac fibroblasts following MI and its mechanism. Our findings demonstrated that, compared with littermate control mice, cardiac function was deteriorated in CREG+/− mice on day 14 post-MI. Fibrosis size, αSMA, and collagen-1 expressions were increased in the border regions of CREG+/− mice on day 14 post-MI. Conversely, exogenous CREG protein significantly improved cardiac function, inhibited fibrosis, and reduced the expressions of αSMA and collagen-1 in the border regions of C57BL/6J mice on day 14. In vitro, CREG recombinant protein inhibited αSMA and collagen-1 expression and blocked the hypoxia-induced proliferation and migration of cardiac fibroblasts, which was mediated through the inhibition of cell division control protein 42 (CDC42) expression. Our findings could help in establishing new strategies based on the clarification of the role of the key molecule CREG in phenotype switching of cardiac fibroblasts following MI.

Exosomes derived from human amniotic fluid mesenchymal stem cells alleviate cardiac fibrosis via enhancing angiogenesis in vivo and in vitro

Cardiac fibrosis is a pathological process characterized by excess extracellular matrix (ECM) deposition and plays a critical role in nearly all types of heart disease. The mechanism of cardiac fibrosis is still unclear and no effective medication treatment of cardiac fibrosis. Research showed that mesenchymal stem cell (MSC) derived exosomes may play a critical role in cardiac fibrosis. The effect of human amniotic fluid MSC (hAFMSC)-derived exosomes (hAFMSCExos) on cardiac fibrosis has remained unclear. The hAFMSCExos were extracted using a sequential centrifugation approach. The effects of hAFMSCExos on angiogenesis were analyzed both in human umbilical vein endothelial cells (HUVECs) after oxygen and glucose deprivation (OGD) in vitro, and in isoproterenol (ISO) induced-cardiac fibrosis in vivo. The hAFMSCExos remarkably up-regulate the motility and migration of HUVECs after OGD compared with phosphate-buffered saline (PBS). Meanwhile, total tube length, total branching points and total loops were significantly raised in HUVECs after OGD treated with hAFMSCExos. The hAFMSCExos alleviated the cardiac fibrosis degree tested by hematoxylin-eosin (H&E) and Masson staining. The protein levels of Collagen I and α-smooth muscle actin (α-SMA) were lower in exosomes group rats than PBS group. Immunofluorescence suggested that hAFMSCExos can promote the expression of CD31 in the rats. Meanwhile, the number of regenerated microvessels was significantly enhanced in rats administrated with exosomes by quantitative analysis of microvessel density. Furthermore, the micro-CT scanning evidenced that hAFMSCExos promote angiogenesis after cardiac fibrosis. The levels of hypoxia-inducible factor 1 α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in the left ventricle accepted HUVECs were higher than PBS treatment at 7 days post-treatment by Western blot analysis. The hAFMSCExos have proangiogenic effects on endothelial cells and enhanced angiogenesis in cardiac fibrosis. The hAFMSCExos may be a promising potential treatment strategy for cardiac fibrosis.

Bone Morphogenetic Protein 6 Alleviates Cardiac Fibrosis and Dysfunction in Heart Failure

Cardiac fibrosis, the main pathological process from ventricular remodeling to heart failure, has attracted much more attention in recent years. Transforming growth factor beta-1 (TGF-β1) plays a key role in the pathogenesis of cardiac fibrosis. Bone morphogenetic protein 6 (BMP6), a member of the TGF-β family, is reportedly involved in many fibrosis-related diseases, such as hepatic and renal fibrosis. Nevertheless, there are few reports about the specific role of BMP6 in cardiac fibrosis. Here, we demonstrated the protein levels of BMP6 were lower in patients with heart failure. In addition, BMP6 expression appeared lower in left ventricles of rats in a transverse aortic constriction (TAC) surgery group compared with the sham group. Next, we observed that silencing BMP6 using small interfering RNA remarkably aggravated TGF-β1-induced cardiac fibrosis, while overexpression of BMP6 by Adenovirus largely improved cardiac fibrosis. Moreover, we found that BMP6 played a protective role in cardiac fibrosis via inhibiting the TGF-β1/Smad3 pathway, that partially functioned via interferon regulatory factor 7 (IRF7) suppression. Finally, we introduced Adeno-associated virus-9 (AAV-9) carrying shBMP6 and found that TAC surgery-induced cardiac fibrosis was significantly aggravated, while administration of rh-BMP6 via tail vein injection markedly ameliorated cardiac fibrosis induced by knockdown of BMP6 in vivo. In conclusion, our study demonstrated that BMP6 may serve as a novel biomarker and a therapeutic target in heart failure related to cardiac fibrosis. Funding Statement: This study was supported by the National Natural Science Foundation of China [No. 81770242 to YaChen Zhang] and Natural Science Foundation of Ningbo (No:2019A610388). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The collection and use of human LV samples were approved by the local human research ethics committee of Shanghai Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine Animal studies were approved by the Animal care and Use Committee of Shanghai Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine.

P21‑activated kinase 1 mediates angiotensin II‑induced differentiation of human atrial fibroblasts via the JNK/c‑Jun pathway

Cardiac fibrosis is a common pathophysiological condition involved in numerous types of cardiovascular disease. The renin-angiotensin system, particularly angiotensin II (AngII), serves an important role in cardiac fibrosis and remodeling. Furthermore, p21-activated kinase 1 (PAK1) is a highly conserved serine/threonine protein kinase, which is abundantly expressed in all regions of the heart. However, the role of PAK1 in AngII-mediated activation of cardiac fibroblasts remains unknown. Therefore, the present study aimed to investigate the role of PAK1 in cardiac fibroblasts and its underlying mechanisms. Human cardiac fibroblasts (HCFs) were cultured and treated with PAK1 inhibitor IPA-3 or transduced with PAK1 short hairpin (sh)RNA by lentiviral particles to silence PAK1 expression levels. Subsequently, the cell proliferation and migration abilities of the HCFs were determined. Western blot analysis was used to detect the phosphorylation status of Janus kinase (JNK) and c-Jun. A Cell Counting Kit-8 assay showed that PAK1 inhibition following treatment of HCFs with 5 µM IPA-3 or PAK1-shRNA, significantly attenuated AngII-induced proliferation of fibroblasts. In addition, wound healing and Transwell migration assays demonstrated that inhibition of PAK1 significantly inhibited AngII-induced cell migration. Finally, decreased PAK1 expression levels downregulated AngII-mediated upregulation of α-smooth muscle actin (α-SMA), collagen I, phosphorylated (p)-JNK and p-c-Jun, a downstream molecule of JNK signaling. These findings indicate that PAK1 contributes to AngII-induced proliferation, migration and transdifferentiation of HCFs via the JNK/c-Jun pathway.

The potential regulatory role of hsa_circ_0004104 in the persistency of atrial fibrillation by promoting cardiac fibrosis via TGF-\u03b2 pathway

IntroductionThe progression of paroxysmal AF (PAF) to persistent AF (PsAF) worsens the prognosis of AF, but its underlying mechanisms remain elusive. Recently, circular RNAs (circRNAs) were reported to be associated with cardiac fibrosis. In case of the vital role of cardiac fibrosis in AF persistency, we hypothesis that circRNAs may be potential regulators in the process of AF progression.Materials and methods6 persistent and 6 paroxysmal AF patients were enrolled as derivation cohort. Plasma circRNAs expressions were determined by microarray and validated by RT-PCR. Fibrosis level, manifested by serum TGF-β, was determined by ELISA. Pathways and related non-coding RNAs involving in the progression of AF regulated were predicted by in silico analysis.ResultsPsAF patients showed a distinct circRNAs expression profile with 92 circRNAs significantly dysregulated (fold change ≥ 2, p < 0.05), compared with PAF patients. The validity of the expression patterns was subsequently validated by RT-PCR in another 60 AF patients (30 PsAF and PAF, respectively). In addition, all the 5 up and down regulated circRNAs were clustered in MAPK and TGF-beta signaling pathway by KEGG pathway analysis. Among the 5 circRNAs, hsa_circ_0004104 was consistently downregulated in PsAF group (0.6 ± 0.33 vs 1.46 ± 0.41, p < 0.001) and predicted to target several AF and/or cardiac fibrosis related miRNAs reported by previous studies. In addition, TGF-β1 level was significantly higher in the PsAF group (5560.23 ± 1833.64 vs 2236.66 ± 914.89, p < 0.001), and hsa_circ_0004104 showed a significant negative correlation with TGF-β1 level (r = − 0.797, p < 0.001).ConclusionCircRNAs dysregulation plays vital roles in AF persistency. hsa_circ_0004104 could be a potential regulator and biomarker in AF persistency by promoting cardiac fibrosis via targeting MAPK and TGF-beta pathways.

CRISPR Mutation of a Single Reactive Cysteine of the Na+/K+ Pump’s ß1-Subunit Abolishes Redox-Dependent Cardiac Hypertrophy and Fibrosis

Reactive oxygen species (ROS) play a critical role in cardiac fibrosis. Na+/K+-ATPase located on the cytomembrane, regulates ion transport. Increased ROS drives post-translational redox modification (glutathionylation) of the Na+/K+-ATPase β1 subunit (β1-GSS) on the cytomembrane as a molecular mechanism involved in angiotensin-induced oxidative fibrosis. Based on this, we created a genetically engineered mouse insensitive to β1-GSS.

The Downregulation of ADAM17 Exerts Protective Effects against Cardiac Fibrosis by Regulating Endoplasmic Reticulum Stress and Mitophagy.

Background A disintegrin and metalloproteinase 17 (ADAM17) is a transmembrane protein that is widely expressed in various tissues; it mediates the shedding of many membrane-bound molecules, involving cell-cell and cell-matrix interactions. We investigated the role of ADAM17 within mouse cardiac fibroblasts (mCFs) in heart fibrosis. Methods mCFs were isolated from the hearts of neonatal mice. Effects of ADAM17 on the differentiation of mCFs towards myofibroblasts and their fibrotic behaviors following induction with TGF-β1 were examined. The expression levels of fibrotic proteins, such as collagen I and α-SMA, were assessed by qRT-PCR analysis and western blotting. Cell proliferation and migration were measured using the CCK-8 and wound healing assay. To identify the target gene for ADAM17, the protein levels of the components of endoplasmic reticulum (ER) stress and the PINK1/Parkin pathway were assessed following ADAM17 silencing. The effects of ADAM17 silencing or treatment with thapsigargin, a key stimulator of acute ER stress, on mCFs proliferation, migration, and collagen secretion were also examined. In vivo, we used a mouse model of cardiac fibrosis established by left anterior descending artery ligation; the mice were administered oral gavage with a selective ADAM17 inhibitor (TMI-005) for 4 weeks after the operation. Results We found that the ADAM17 expression levels were higher in fibrosis heart tissues and TGF-β1-treated mCFs. The ADAM17-specific siRNAs decreased TGF-β1-induced increase in the collagen secretion, proliferation, and migration of mCFs. Knockdown of ADAM17 reduces the activation of mCFs by inhibiting the ATF6 branch of ER stress and further activating mitophagy. Moreover, decreased ADAM17 expression also ameliorated cardiac fibrosis and improved heart function. Conclusions This study highlights that mCF ADAM17 expression plays a key role in cardiac fibrosis by regulating ER stress and mitophagy, thereby limiting fibrosis and improving heart function. Therefore, ADAM17 downregulation, within the physiological range, could exert protective effects against cardiac fibrosis.

Long Non-Coding RNA 554 Promotes Cardiac Fibrosis via TGF-β1 Pathway in Mice Following Myocardial Infarction.

Rationale: Cardiac fibrosis is observed in nearly every form of myocardial disease. Long non-coding RNAs (lncRNAs) have been shown to play an important role in cardiac fibrosis, but the detailed molecular mechanism remains unknown. Object: We aimed at characterizing lncRNA 554 expression in murine cardiac fibroblasts (CFs) after myocardial infarction (MI) to identify CF-enriched lncRNA and investigate its function and contribution to cardiac fibrosis and function. Methods and Results: In this study, we identified lncRNA NONMMUT022554 (lncRNA 554) as a regulator of MI-induced cardiac fibrosis. We found that lncRNA 554 was significantly up-regulated in the mouse hearts following MI. Further study showed that lncRNA 554 was predominantly expressed in cardiac fibroblasts, indicating a potential role of lncRNA 554 in cardiac fibrosis. In vitro knockdown of lncRNA 554 by siRNA suppressed fibroblasts migration and expression of extracellular matrix (ECM); while overexpression of lncRNA 554 promoted expression of ECM genes. Consistently, lentivirus mediated in vivo knockdown of lncRNA 554 could inhibit cardiac fibrosis and improve cardiac function in mouse model of MI. More importantly, TGF-β1 inhibitor (TEW-7197) could reverse the pro-fibrotic function of lncRNA 554 in CFs. This suggests that the effects of lncRNA 554 on cardiac fibrosis is TGF-β1 dependent. Conclusion: Collectively, our study illustrated the role of lncRNA 554 in cardiac fibrosis, suggested that lncRNA 554 might be a novel target for cardiac fibrosis.

Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoforms: Possible Implications for New Antifibrotic Therapies.

Cardiac fibrosis is characterized by accumulation and activation of fibroblasts and excessive production of extracellular matrix, which results in myocardial stiffening and eventually leads to heart failure. Although previous work suggests that protein kinase C (PKC) isoforms play a role in cardiac fibrosis and remodeling, the results are conflicting. Moreover, the potential of targeting PKC with pharmacological tools to inhibit pathologic fibrosis has not been fully evaluated. Here we investigated the effects of selected PKC agonists and inhibitors on cardiac fibroblast (CF) phenotype, proliferation, and gene expression using primary adult mouse CFs, which spontaneously transdifferentiate into myofibroblasts in culture. A 48-hour exposure to the potent PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 nM concentration reduced the intensity of α-smooth muscle actin staining by 56% and periostin mRNA levels by 60% compared with control. The decreases were inhibited with the pan-PKC inhibitor Gö6983 and the inhibitor of classical PKC isoforms Gö6976, suggesting that classical PKCs regulate CF transdifferentiation. PMA also induced a 33% decrease in 5-bromo-2'-deoxyuridine-positive CFs, which was inhibited with Gö6983 but not with Gö6976, indicating that novel PKC isoforms (nPKCs) regulate CF proliferation. Moreover, PMA downregulated the expression of collagen-encoding genes Col1a1 and Col3a1 nPKC-dependently, showing that PKC activation attenuates matrix synthesis in CFs. The partial PKC agonist isophthalate derivative bis(1-ethylpentyl) 5-(hydroxymethyl)isophthalate induced parallel changes in phenotype, cell cycle activity, and gene expression. In conclusion, our results reveal distinct PKC-dependent regulation of CF transdifferentiation and proliferation and suggest that PKC agonists exhibit potential as an antifibrotic treatment. SIGNIFICANCE STATEMENT: Cardiac fibrosis is a pathological process that contributes to the development of heart failure. The molecular mechanisms regulating fibrosis in the heart are, however, not fully understood, which hinders the development of new therapies. Here, we demonstrate that classical and novel protein kinase C (PKC) isoforms distinctly regulate cardiac fibroblast transdifferentiation and proliferation, the two central processes in fibrosis. Our results indicate that pharmacological PKC activation may be a promising strategy to inhibit myocardial fibrosis.

INTRUSION OF GLYCOGEN SYNTHASE KINASE-3Β TO COPE VARIOUS CARDIAC DISORDERS AT MOLECULAR LEVEL

All eukaryotes consist of kinases with a serine/threonine residue called glycogen synthase kinase 3 (GSK-3) which mediates cellular functions by causing phosphorylation of glycogen synthase and regulating glucose metabolism. It establishes disease mechanisms through cell signalling and different transcription factors. Glycogen synthase kinase-3β (GSK-3β) has pharmacological role in cardiac fibrosis, hyperlipidaemia, hyperglycaemia, hyperhomocysteinemia and in case of myocardial reperfusion injury and estrogen deficiency on the heart. The lead compounds were discovered from natural products possessing GSK-3β inhibitory activity. New signalling pathways involving mitochondrion have been investigated for ischemic preconditioning. GSK-3β may bind with mitochondrial protein and mediate mitochondrion function by binding with PI3K-Akt, PGC-1α, HK-II, PKCε subunits of mPTP. The present study explores the structural functionalities of GSK-3β and their contributory role in cardiac disorders and various other diseases. Therefore, GSK-3β is believed to be an imperative target for the discovery and development of newer drugs.

Activation of activin/Smad2 and 3 signaling pathway and the&amp;nbsp;potential involvement of endothelial‑mesenchymal transition in the valvular damage due to rheumatic heart disease

Rheumatic heart disease (RHD) is an autoimmune disease caused by rheumatic fever following group A hemolytic streptococcal infection and primarily affects the mitral valve. RHD is currently a major global health problem. However, the exact pathological mechanisms associated with RHD-induced cardiac valve damage remain to be elucidated. The endothelial-mesenchymal transition (EndMT) serves a key role in a number of diseases with an important role in cardiac fibrosis and the activin/Smad2 and 3 signaling pathway is involved in regulating the EndMT. Nevertheless, there are no studies to date, to the best of the authors' knowledge, investigating the association between RHD and EndMT. Thus, the aim of the current study was to investigate the potential role of EndMT in cardiac valve damage and assess whether activin/Smad2 and 3 signaling was activated during RHD-induced valvular injury in a rat model of RHD induced by inactivated Group A streptococci and complete Freund's adjuvant. Inflammation and fibrosis were assessed by hematoxylin and eosin and Sirius red staining. Serum cytokine and rheumatoid factor levels were measured using ELISA kits. Expression levels of activin/Smad2 and 3 signaling pathway-related factors [activin A, Smad2, Smad3, phosphorylated (p-)Smad2 and p-Smad3], EndMT-related factors [lymphoid enhancer factor-1 (LEF-1), Snail1, TWIST, zinc finger E-box-binding homeobox (ZEB)1, ZEB2, α smooth muscle actin (α-SMA) and type I collagen α 1 (COL1A1)], apoptosis-related markers (BAX and cleaved caspase-3) and valvular inflammation markers (NF-κB and p-NF-κB) were detected using reverse transcription-quantitative PCR and western blot analyses. Compared with the control group, the degree of valvular inflammation and fibrosis, serum levels of IL-6, IL-17, TNF-α and expression of apoptosis-related markers (BAX and cleaved caspase-3) and valvular inflammation marker (p-NF-κB), activin/Smad2 and 3 signaling pathway-related factors (activin A, p-Smad2 and p-Smad3), EndMT-related factors (LEF-1, Snail1, TWIST, ZEB 1, ZEB2, α-SMA and COL1A1) were significantly increased in the RHD group. These results suggested that the activin/Smad2 and 3 signaling pathway was activated during the development of valvular damage caused by RHD and that the EndMT is involved in RHD-induced cardiac valve damage.

Nur77 deficiency exacerbates cardiac fibrosis after myocardial infarction through promoting endothelial to mesenchymal transition

Abstract Background Cardiac fibrosis is a reparative process after myocardial infarction (MI), which leads to cardiac remodeling and finally heart failure. Endothelial-to-mesenchymal transition (EndMT) is induced after MI and contributes to cardiac fibrosis after MI. Orphan nuclear receptor Nur77 is a key regulator of inflammation, angiogenesis, proliferation, and apoptosis in vascular endothelial cells. Here, we investigated the role of orphan nuclear receptor Nur77 in EndMT and cardiac fibrosis after MI. Methods and results Cardiac fibrosis was induced through MI by ligation of the left anterior descending coronary artery. Results suggested that Nur77 knockout aggravated cardiac dysfunction and cardiac fibrosis 30 days after MI. Moreover, Nur77 deficency resulted in enhanced EndMT as shown by increased expression of FSP-1, SM22α, Snail and decreased expression of PECAM-1 and eNOS compared with WT mice after MI. Then we found overexpression Nur77 in HCAECs significantly inhibited IL-1β and TGFβ2 induced EndMT, as shown by reduced transition to a fibroblast-like phenotype and preserved angiogenesis potential. Mechanistically, we demonstrated that Nur77 downregulated EndMT through inhibiting NF-κB-dependent pathway Conclusion Nur77 plays a role in cardiac fibrosis through inhibition of EndMT, and may be a promising target for therapy of cardiac fibrosis after MI. Nur77 inhibited EndMT Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Natural Science Foundation of China