Ang II-induced Cardiac Fibroblasts Research Articles

Morroniside improves AngII-induced cardiac fibroblast proliferation, migration, and extracellular matrix deposition by blocking p38/JNK signaling pathway through the downregulation of KLF5.

Myocardial fibrosis (MF), which is an inevitable pathological manifestation of many cardiovascular diseases in the terminal stage, often contributes to severe cardiac dysfunction and sudden death. Morroniside (MOR) is the main active component of Cornus officinalis with a variety of biological activities. This study was designed to explore the efficacy of MOR in MF and to investigate its pharmacological mechanism. The viability of MOR-treated human cardiac fibroblast (HCF) cells with or without Angiotensin II (AngII) induction was assessed with Cell Counting Kit-8 (CCK-8). The migration of AngII-induced HCF cells was appraised with a transwell assay. Gelatin zymography analysis was adopted to evaluate the activities of MMP2 and MMP9, while immunofluorescence assay was applied for the estimation of Collagen I and Collagen III. By means of western blot, the expressions of migration-, fibrosis-, and p38/c-Jun N-terminal kinase (JNK) signal pathway-related proteins were resolved. The transfection efficacy of oe-Kruppel-like factor 5 (KLF5) was examined with reverse transcription-quantitative PCR (RT-qPCR) and western blot. In this study, it was found that MOR treatment inhibited AngII-induced hyperproliferation, migration, and fibrosis of HCF cells, accompanied with decreased activities of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), connective tissue growth factor (CTGF), Fibronectin, and α-SMA, which were all reversed by KLF5 overexpression. Collectively, MOR exerted protective effects on MF by blocking p38/JNK signal pathway through the downregulation of KLF5.

Riboflavin ameliorates pathological cardiac hypertrophy and fibrosis through the activation of short-chain acyl-CoA dehydrogenase

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid β-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. FAD, a coenzyme of SCAD, participates in the electron transfer of SCAD-catalyzed fatty acid β-oxidation, which plays a crucial role in maintaining the balance of myocardial energy metabolism. Insufficient riboflavin intake can lead to symptoms similar to short-chain acyl-CoA dehydrogenase (SCAD) deficiency or flavin adenine dinucleotide (FAD) gene abnormality, which can be alleviated by riboflavin supplementation. However, whether riboflavin can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of riboflavin on pathological cardiac hypertrophy and fibrosis. In vitro experiments, riboflavin increased SCAD expression and the content of ATP, decreased the free fatty acids content and improved PE-induced cardiomyocytes hypertrophy and AngⅡ-induced cardiac fibroblasts proliferation by increasing the content of FAD, which were attenuated by knocking down the expression of SCAD using small interfering RNA. In vivo experiments, riboflavin significantly increased the expression of SCAD and the energy metabolism of the heart to improve TAC induced pathological myocardial hypertrophy and fibrosis in mice. The results demonstrate that riboflavin improves pathological cardiac hypertrophy and fibrosis by increasing the content of FAD to activate SCAD, which may be a new strategy for treating pathological cardiac hypertrophy and fibrosis.

ANGPTL4 suppresses the profibrogenic functions of atrial fibroblasts induced by angiotensin II by up-regulating PPARγ.

The present study's objective was to investigate the association between angiopoietin-like 4 (ANGPTL4) levels and the prognosis of Atrial fibrillation (AF), the causative effect in angiotensin II- (Ang II) induced AF, and its underlying mechanisms. Baseline serum ANGPTL-4 concentrations were measured in 130 patients with AF. Rat atrial fibroblasts were isolated from 14-day-old SD rats and transfected with Ang II treatment. Transfected cells were divided into: The control group, ANGPTL4-OE group, Ang II group, and Ang II+ANGPTL4-OE group. Transfected cells were used to analyze fibroblasts' proliferation, migration, and collagen production at the cellular level. RT-qPCR and western blotting evaluated the ANGPTL4-targeted gene and PPARγ-Akt pathway. In patients with AF, serum ANGPTL4 concentrations decreased significantly compared with the healthy group. ANGPTL4 mRNA and protein expressions were significantly down-regulated in Ang II-induced cardiac fibroblasts. ANGPTL4 overexpression potentially attenuated Ang IIinduced fibroblast proliferation, migration, and collagen production in atrial tissue. ANGPTL4 inhibited the signaling proteins, such as PPARγ, α-SMA, and Akt. Our experimental data speculate that ANGPTL4 is a key factor in regulating AF progression. Therefore, increasing ANGPTL4 expression could be an effective strategy for AF treatment.

Integrin subunit β-like 1 mediates angiotensin II-induced myocardial fibrosis by regulating the forkhead box Q1/Snail axis

Cardiac fibrosis is a severe condition with limited therapeutic options and often occurs in chronic cardiovascular diseases such as hypertension and myocardial infarction. There is currently a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of more effective therapeutic strategies targeting cardiac fibrosis. Integrin subunit β-like 1 (ITGBL1), an extracellular matrix protein, has previously been implicated in various fibrotic diseases. However, the precise role of ITGBL1 in regulating myocardial fibrosis remains unknown. The present study was designed to investigate whether ITGBL1 regulates angiotensin II (Ang II)-induced myocardial fibrosis in vitro and in vivo and the possible mechanism of action. It was found that the protein expressions of ITGBL1, Forkhead box Q1 (FOXQ1), and Snail were all increased significantly in fibrotic heart tissues from Ang II-infused mice and Ang II-stimulated cardiac fibroblasts, all of which were inhibited by the Ang II type I (AT1) receptor antagonist losartan. Silencing the ITGBL1/FOXQ1/Snail axis with specific siRNAs reversed Ang II-induced fibrotic effects and upregulation of FOXQ1 and Snail expressions in cardiac fibroblasts. FOXQ1 siRNA inhibited Snail expression in Ang II-induced cardiac fibroblasts. Furthermore, ITGBL1/FOXQ1 interacted with the TGF-β1 signaling to form a positive feedback loop. Our findings suggest that the extracellular matrix protein ITGBL1 mediates Ang II-induced cardiac fibrosis via the FOXQ1/Snail axis, which identifies ITGBL1 as a novel mediator of cardiac fibrosis and represents a potential therapeutic target for cardiac fibrosis.

Liraglutide inhibits AngII-induced cardiac fibroblast proliferation and ECM deposition through regulating miR-21/PTEN/PI3K pathway.

Cardiac fibrosis characterized with the aberrant proliferation of cardiac fibroblasts and extracellular matrix (ECM) deposition is a major pathophysiological feature of atrial fibrillation (AF). Liraglutide has exerted an alleviative role in various cardiovascular diseases, and can also regulate the level of microRNAs (miRNAs). It has been reported that miR-21 modulated cardiac fibrosis in AF. However, the regulative effect of liraglutide on atrial fibrosis via miR-21 and the underlying mechanism are still unclear. The atrial fibroblasts were isolated from the heart of C57BL/6 mice, and treated with Angiotensin II (AngII) and liraglutide. The proliferation, migration, and ECM deposition were determined by cell counting Kit-8 (CCK-8), Brdu, transwell assay, cell scratch, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot and immunofluorescence. The underlying mechanism was explored after transfection of miR-21 mimics into cells. Liraglutide inhibited proliferation, migration, invasion of fibroblast cell and ECM deposition in AngII-stimulated cardiac fibroblasts. Additionally, liraglutide decreased the AngII-induced increase in the expression level of miR-21, but enhanced the expression of phosphatase and tensin homolog (PTEN), a target of miR-21, thereby suppressing the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Rescue assay confirmed that overexpression of miR-21 counteracted the ameliorative effect of liraglutide on the proliferation, migration, invasion and ECM deposition in fibroblasts stimulated by AngII. Liraglutide dampened AngII-induced proliferation and migration, and ECM deposition of cardiac fibroblast via modulating miR-21/PTEN/PI3K pathway.

Brown Adipocyte ADRB3 Mediates Cardioprotection via Suppressing Exosomal iNOS.

The ADRB3 (β3-adrenergic receptors), which is predominantly expressed in brown adipose tissue (BAT), can activate BAT and improve metabolic health. Previous studies indicate that the endocrine function of BAT is associated with cardiac homeostasis and diseases. Here, we investigate the role of ADRB3 activation-mediated BAT function in cardiac remodeling. BKO (brown adipocyte-specific ADRB3 knockout) and littermate control mice were subjected to Ang II (angiotensin II) for 28 days. Exosomes from ADRB3 antagonist SR59230A (SR-exo) or agonist mirabegron (MR-exo) treated brown adipocytes were intravenously injected to Ang II-infused mice. BKO markedly accelerated cardiac hypertrophy and fibrosis compared with control mice after Ang II infusion. In vitro, ADRB3 KO rather than control brown adipocytes aggravated expression of fibrotic genes in cardiac fibroblasts, and this difference was not detected after exosome inhibitor treatment. Consistently, BKO brown adipocyte-derived exosomes accelerated Ang II-induced cardiac fibroblast dysfunction compared with control exosomes. Furthermore, SR-exo significantly aggravated Ang II-induced cardiac remodeling, whereas MR-exo attenuated cardiac dysfunction. Mechanistically, ADRB3 KO or SR59230A treatment in brown adipocytes resulted an increase of iNOS (inducible nitric oxide synthase) in exosomes. Knockdown of iNOS in brown adipocytes reversed SR-exo-aggravated cardiac remodeling. Our data illustrated a new endocrine pattern of BAT in regulating cardiac remodeling, suggesting that activation of ADRB3 in brown adipocytes offers cardiac protection through suppressing exosomal iNOS.

Knockdown of HIPK2 Attenuates Angiotensin II-Induced Cardiac Fibrosis in Cardiac Fibroblasts.

Homeodomain-interacting protein kinase-2 (HIPK2), a member of an evolutionary conserved family of serine/threonine kinases, has been observed to be involved in the pathogenesis of fibrotic diseases. However, its role in cardiac fibrosis remains unclear. In this study, we assessed the effect of HIPK2 on cardiac fibroblasts (CFs) in response to angiotensin II (Ang II) stimulation. The results indicated that HIPK2 expression was significantly increased in Ang II-induced CFs in a dose-dependent manner. Then, HIPK2 was knocked down in CFs to evaluate the roles of HIPK2. Knockdown of HIPK2 suppressed cell proliferation and migration in Ang II-induced CFs. The Ang II-caused increase in expression of α-smooth muscle actin, a hallmark of myofibroblast differentiation, was decreased by knockdown of HIPK2. HIPK2 knockdown also reduced extracellular matrix production including type I collagen and connective tissue growth factor. Furthermore, knockdown of HIPK2 blocked the activation of TGF-β1/Smad pathway in Ang II-induced CFs. These data suggested that HIPK2 knockdown prevented the Ang II-induced activation of CFs through inhibiting TGF-β1/Smad pathway, indicating HIPK2 might be an antifibrosis target for the treatment of cardiac fibrosis.

Intermedin1-53 Inhibits NLRP3 Inflammasome Activation by Targeting IRE1α in Cardiac Fibrosis.

Intermedin (IMD), a paracrine/autocrine peptide, protects against cardiac fibrosis. However, the underlying mechanism remains poorly understood. Previous study reports that activation of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cardiac fibrosis. In this study, we aimed to investigate whether IMD mitigated cardiac fibrosis by inhibiting NLRP3. Cardiac fibrosis was induced by angiotensin II (Ang II) infusion for 2weeks in rats. Western blot, real-time PCR, histological staining, immunofluorescence assay, RNA sequencing, echocardiography, and hemodynamics were used to detect the role and the mechanism of IMD in cardiac fibrosis. Ang II infusion resulted in rat cardiac fibrosis, shown as over-deposition of myocardial interstitial collagen and cardiac dysfunction. Importantly, NLRP3 activation and endoplasmic reticulum stress (ERS) were found in Ang II-treated rat myocardium. Ang II infusion decreased the expression of IMD and increased the expression of the receptor system of IMD in the fibrotic rat myocardium. IMD treatment attenuated the cardiac fibrosis and improved cardiac function. In addition, IMD inhibited the upregulation of NLRP3 markers and ERS markers induced by Ang II. In vitro, IMD knockdown by small interfering RNA significantly promoted the Ang II-induced cardiac fibroblast and NLRP3 activation. Moreover, silencing of inositol requiring enzyme 1 α (IRE1α) blocked the effects of IMD inhibiting fibroblast and NLRP3 activation. Pre-incubation with PKA pathway inhibitor H89 blocked the effects of IMD on the anti-ERS, anti-NLRP3, and anti-fibrotic response. In conclusion, IMD alleviated cardiac fibrosis by inhibiting NLRP3 inflammasome activation through suppressing IRE1α via the cAMP/PKA pathway.

Discovery of Potent and Selective 2-(Benzylthio)pyrimidine-based DCN1-UBC12 Inhibitors for Anticardiac Fibrotic Effects.

DCN1, a co-E3 ligase, interacts with UBC12 and activates cullin-RING ligases (CRLs) by catalyzing cullin neddylation. Although DCN1 has been recognized as an important therapeutic target for human diseases, its role in the cardiovascular area remains unknown. Here, we first found that DCN1 was upregulated in isolated cardiac fibroblasts (CFs) treated by angiotensin (Ang) II and in mouse hearts after pressure overload. Then, structure-based optimizations for DCN1-UBC12 inhibitors were performed based on our previous work, yielding compound DN-2. DN-2 specifically targeted DCN1 at molecular and cellular levels as shown by molecular modeling studies, HTRF, cellular thermal shift and co-immunoprecipitation assays. Importantly, DN-2 effectively reversed Ang II-induced cardiac fibroblast activation, which was associated with the inhibition of cullin 3 neddylation. Our findings indicate a potentially unrecognized role of DCN1 inhibition for anticardiac fibrotic effects. DN-2 may be used as a lead compound for further development.

Ginsenoside Re inhibits myocardial fibrosis by regulating miR-489/myd88/NF-κB pathway

BackgroundMyocardial fibrosis (MF) is an advanced pathological manifestation of many cardiovascular diseases, which can induce heart failure and malignant arrhythmias. However, the current treatment of MF lacks specific drugs. Ginsenoside Re has anti-MF effect in rat, but its mechanism is still not clear. Therefore, we investigated the anti-MF effect of ginsenoside Re by constructing mouse acute myocardial infarction (AMI) model and AngⅡ induced cardiac fibroblasts (CFs) model. MethodsThe anti-MF effect of miR-489 was investigated by transfection of miR-489 mimic and inhibitor in CFs. Effect of ginsenoside Re on MF and its related mechanisms were investigated by ultrasonographic, ELISA, histopathologic staining, transwell test, immunofluorescence, Western blot and qPCR in the mouse model of AMI and the AngⅡ-induced CFs model. ResultsMiR-489 decreased the expression of α-SMA, collagenⅠ, collagen Ⅲ and myd88, and inhibited the phosphorylation of NF-κB p65 in normal CFs and CFs treated with AngⅡ. Ginsenoside Re could improve cardiac function, inhibit collagen deposition and CFs migration, promote the transcription of miR-489, and reduce the expression of myd88 and the phosphorylation of NF-κB p65. ConclusionMiR-489 can effectively inhibit the pathological process of MF, and the mechanism is at least partly related to the regulation of myd88/NF-κB pathway. Ginsenoside Re can ameliorate AMI and AngⅡ induced MF, and the mechanism is at least partially related to the regulation of miR-489/myd88/NF-κB signaling pathway. Therefore, miR-489 may be a potential target of anti-MF and ginsenoside Re may be an effective drug for the treatment of MF.

Fus knockdown inhibits the profibrogenic effect of cardiac fibroblasts induced by angiotensin II through targeting Pax3 thereby regulating TGF-β1/Smad pathway

ABSTRACT The Angiotensin II/transforming growth factor-β1 (AngII/TGF-β1) signal axis is an important regulatory pathway for atrial fibrosis, which can contribute to atrial fibrillation (AF). Fused in sarcoma (FUS) was recently found to regulate cardiac diseases. This study aimed to investigate whether FUS could regulate AngII induced fibrosis and uncover the possible mechanisms. The expression of FUS in AF patients and AngII-induced cardiac fibroblasts was measured by RT-qPCR and western blot assays. Fus was silenced in cells using short hairpin RNA (shRNA), then cell proliferation, migration, collagen synthesis and TGF-β1/Smad signaling were detected by CCK-8, wound healing and western blot assays, respectively. The possible target for Fus was predicted by searching Starbase database and verified by RNA-binding protein immunoprecipitation (RIP) and RNA pull down. Cells were overexpressed with Pax3 in the presence of Fus silence and AngII stimulation, then the above cellular processes were further evaluated. Results showed that FUS was upregulated in AF patients and AngII-induced cardiac fibroblasts. Fus knockdown inhibited AngII-enhanced cell proliferation, migration, collagen synthesis and TGF-β1/Smad signaling activation. Furthermore, Fus functions as an RNA-binding protein to bind to Pax3 mRNA and positively regulate its expression. Further studies demonstrated that Pax3 overexpression canceled the above effects of Fus knockdown on cell proliferation, migration, collagen synthesis, and TGF-β1/Smad signaling activation in AngII-induced cells. In conclusion, Fus could target Pax3 to increase the pro-fibrotic effect of AngII in cardiac fibroblasts via activating TGF-β1/Smad signaling. Knockdown of Fus/Pax3 axis may provide a potential therapy for relieving AF.

Mep1a contributes to Ang II-induced cardiac remodeling by promoting cardiac hypertrophy, fibrosis and inflammation

Pathological cardiac remodeling, characterized by excessive deposition of extracellular matrix proteins and cardiac hypertrophy, leads to the development of heart failure. Meprin α (Mep1a), a zinc metalloprotease, previously reported to participate in the regulation of inflammatory response and fibrosis, may also contribute to cardiac remodeling, although whether and how it participates in this process remains unknown. Here, in this work, we investigated the role of Mep1a in pathological cardiac remodeling, as well as the effects of the Mep1a inhibitor actinonin on cardiac remodeling-associated phenotypes. We found that Mep1a deficiency or chemical inhibition both significantly alleviated TAC- and Ang II-induced cardiac remodeling and dysfunction. Mep1a deletion and blocking both attenuated TAC- and Ang II-induced heart enlargement and increases in the thickness of the left ventricle anterior and posterior walls, and reduced expression of pro-hypertrophic markers, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and myosin heavy chain beta (β-MHC). In addition, Mep1a deletion and blocking significantly inhibited TAC- and Ang II-induced cardiac fibroblast activation and production of extracellular matrix (ECM). Moreover, in Mep1a−/− mice and treatment with actinonin significantly reduced Ang II-induced infiltration of macrophages and proinflammatory cytokines. Notably, we found that in vitro, Mep1a is expressed in cardiac myocytes and fibroblasts and that Mep1a deletion or chemical inhibition both markedly suppressed Ang II-induced hypertrophy of rat or mouse cardiac myocytes and activation of rat or mouse cardiac fibroblasts. In addition, blocking Mep1a in macrophages reduced Ang II-induced expression of interleukin (IL)-6 and IL-1β, strongly suggesting that Mep1a participates in cardiac remodeling processes through regulation of inflammatory cytokine expression. Mechanism studies revealed that Mep1a mediated ERK1/2 activation in cardiac myocytes, fibroblasts and macrophages and contributed to cardiac remodeling. In light of our findings that blocking Mep1a can ameliorate cardiac remodeling via inhibition of cardiac hypertrophy, fibrosis, and inflammation, Mep1a may therefore serve as a strong potential candidate for therapeutic targeting to prevent cardiac remodeling.

Flavine adenine dinucleotide inhibits pathological cardiac hypertrophy and fibrosis through activating short chain acyl-CoA dehydrogenase

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid β-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. Furthermore, flavin adenine dinucleotide (FAD) can enhance the expression and enzyme activity of SCAD. However, whether FAD can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of FAD on pathological cardiac hypertrophy and fibrosis. FAD significantly inhibited PE-induced cardiomyocyte hypertrophy and AngII-induced cardiac fibroblast proliferation. In addition, FAD ameliorated pathological cardiac hypertrophy and fibrosis in SHR. FAD significantly increased the expression and enzyme activity of SCAD. Meanwhile, ATP content was increased, the content of free fatty acids and reactive oxygen species were decreased by FAD in vivo and in vitro. In addition, molecular dynamics simulations were also used to provide insights into the structural stability and dynamic behavior of SCAD. The results demonstrated that FAD may play an important structural role on the SCAD dimer stability and maintenance of substrate catalytic pocket to increase the expression and enzyme activity of SCAD. In conclusion, FAD can inhibit pathological cardiac hypertrophy and fibrosis through activating SCAD, which may be a novel effective treatment for pathological cardiac hypertrophy and fibrosis, thus prevent them from developing into heart failure.

FOXF1 ameliorates angiotensin II-induced cardiac fibrosis in cardiac fibroblasts through inhibiting the TGF-β1/Smad3 signaling pathway

Cardiac fibrosis is a pathological feature common to a variety of heart diseases such as myocardial infarction, arrhythmias, cardiomyopathies and heart failure. The molecular mechanism underlying the cardiac fibrosis is still unclear. Forkhead box F1 (FOXF1), a member of the forkhead transcription factor superfamily, plays critical roles in the development of hepatic fibrosis. However, whether FOXF1 is involved in the pathogenesis of cardiac fibrosis remains to be elucidated. The present study aimed to investigate the role of FOXF1 and its mechanisms in regulating cardiac fibrosis. The results demonstrated that FOXF1 was downregulated in Ang II-induced CFs. Overexpression of FOXF1 inhibited angiotensin II (Ang II)-induced proliferation, migration and oxidative stress in cardiac fibroblasts (CFs). Overexpression of FOXF1 also reduced the expression of alpha-smooth muscle actin (a-SMA) in Ang II-induced CFs, suggesting that overexpression of FOXF1 prevented the differentiation of CFs to myofibroblasts. Furthermore, the production of extracellular matrix (ECM) components including type I collagen and fibronectin were reduced by overexpression of FOXF1 in Ang II-induced CFs. Furthermore, overexpression of FOXF1 prevented Ang II-induced activation of transforming growth factor beta 1 (TGF-β1)/Smad3 pathway in CFs. In conclusion, the results of the present study indicated that FOXF1 acted as a key regulator of pathological cardiac fibrosis, and overexpression of FOXF1 ameliorated cardiac fibrosis by inhabiting the TGF-β1/Smad3 signaling pathway. These results indicated that FOXF1 may be a novel target for attenuating cardiac fibrosis.

MiR-142-5p and miR-212-5p cooperatively inhibit the proliferation and collagen formation of cardiac fibroblasts by regulating c-Myc/TP53INP1.

The aim of this study was to investigate the effect and mechanism of miR-142-5p/212-5p on the proliferation and collagen formation of cardiac fibroblasts (CFs) after myocardial infarction (MI). The mouse MI model was established by ligation of the left anterior descending coronary artery. CFs were induced by transforming growth factor-beta 1 (TGF-β1) or angiotensin (Ang II). The molecule expressions were measured by qRT-PCR and Western blot. CF proliferation was detected by an MTT assay. The effect of miR-142-5p/212-5p on the luciferase activity of c-Myc 3'UTR was assessed by the luciferase reporter assay. miR-142-5p and miR-212-5p were downregulated in cardiac tissues of MI mice and in TGF-β1- or Ang II-induced CFs, while the protein levels of collagen I and III were upregulated. Moreover, simultaneous overexpression of miR-142-5p/212-5p inhibited the proliferation and collagen formation of TGF-β1- or Ang II-stimulated CFs to a greater extent than either miR-142-5p or miR-212-5p overexpression alone. MiR-142-5p/212-5p targeted c-Myc and negatively regulated its expression. The effects of miR-142-5p/212-5p overexpression on the TP53INP1 protein level and the proliferation and collagen formation of CFs were reversed by c-Myc overexpression. Moreover, overexpression of miR-142-5p/212-5p improved cardiac function and collagen formation of MI mice. Overexpression of miR-142-5p/212-5p cooperatively suppresses the proliferation and collagen formation after MI by regulating c-Myc/TP53INP1.

Protocatechuic acid attenuates angiotensin II-induced cardiac fibrosis in cardiac fibroblasts through inhibiting the NOX4/ROS/p38 signaling pathway.

Cardiac fibrosis plays a crucial role in the pathogenesis of myocardial infarction (MI). It has been found that differentiation of cardiac fibroblasts (CFs) into myofibroblasts is a major event in the process of cardiac fibrosis. In the present study, we aimed to investigate the effects of protocatechuic acid (PCA), a cardiac protective agent, on the CFs differentiation in vitro. The results showed that PCA exhibited inhibitory effects on the cell proliferation and migration in angiotensin II (Ang II)-induced CFs. PCA treatment suppressed the Ang II-induced expression of α-smooth muscle actin (α-SMA), which is a hallmark of myofibroblasts. In addition, the production of extracellular matrix (ECM) proteins, including type I collagen (Col I) and connective tissue growth factor (CTGF), were significantly decreased in the PCA-treated CFs. The Ang II-induced increased levels of matrix metalloproteinase (MMP)-2, and MMP-9 were reduced by PCA. Furthermore, PCA resulted in decrease in reactive oxygen species (ROS) generation, as well as the expressions of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 (NOX4) and p-p38 in Ang II-induced CFs. These findings showed that PCA treatment prevented the Ang II-induced cardiac fibrosis by inhibiting the NOX4/ROS/p38 signaling pathway in vitro, suggesting that PCA might be a therapeutic agent for MI.

IMM-H007 improves heart function via reducing cardiac fibrosis

Cardiac dysfunction is a pathological state characterized by damaged ability of the left ventricle (LV) to either eject or fill blood accompanied by cardiac hypertrophy and fibrosis. IMM-H007, an adenosine derivative, is an activator of AMP-Activated Protein Kinase (AMPK). AMPK can decrease the transforming growth factor-β1 (TGF-β1) expression during fibrosis. Therefore, we hypothesized that IMM-H007 contributed to cardiac dysfunction by mediating cardiac fibrosis. To test this hypothesis, we used angiotensin II (AngII)-induced cardiac remodeling model treated with IMM-H007 or vehicle. Echocardiography measurements showed that IMM-H007 significantly improved heart function indicated by increased LV ejection fraction (%LVEF) and LV fractional shortening (%LVFS). Histological staining and qRT-PCR analysis revealed that IMM-H007 markedly reduced AngII-induced cardiac fibroblast activation (α-smooth muscle actin and periostin) and matrix protein production (Collagen I and Collagen III). However, IMM-H007 did not affect AngII-induced cardiac hypertrophy. Immunoblotting analysis revealed that IMM-H007 activated AMPK, decreased the expression of TGF-β1, and inhibited the activation of Smad2 in heart tissues. In mouse primary cultured cardiac fibroblasts, pharmacological activation of AMPK by IMM-H007 significantly reduced AngII-induced TGF-β1 expression as well. Consistently, in human cardiac fibroblasts-adult ventricular (HCF-av), IMM-H007 activated AMPK and markedly suppressed AngII-induced TGF-β1 expression. These results together reveal that IMM-H007 improves heart function, and alleviates AngII-induced cardiac fibrosis by regulating AMPK-TGF-β1 signaling. These findings suggest IMM-H007 as a potential drug for treating cardiac dysfunction.

HOTAIR promotes myocardial fibrosis through regulating URI1 expression via Wnt pathway.

To investigate whether HOX transcript antisense RNA (HOTAIR) regulates myocardial fibrosis via promoting proliferation of cardiac fibroblasts (CFs) and upregulating the expression levels of fibrotic proteins through activating Wnt signaling pathway. The expression level of HOTAIR in Ang II-induced cardiac fibroblasts was detected by quantitative Real-time-polymerase chain reaction (qRT-PCR). Overexpression or knockdown of HOTAIR expression was achieved by lentivirus transfection. The effects of HOTAIR on regulating cell proliferation, migration and apoptosis were measured by cell counting kit-8 (CCK-8) test, transwell assay and flow cytometry, respectively. Western blot and qRT-PCR experiments were performed to detect expressions of fibrosis-related genes and Wnt pathway-related genes. Target gene of HOTAIR was predicted by bioinformatics analysis. Rescue assays were conducted to assess whether HOTAIR could regulate cell proliferation and fibrosis by activating Wnt signaling pathway via URI1. QRT-PCR results showed that HOTAIR expression in Ang II-induced CF cells was significantly higher than that in control. HOTAIR overexpression in CF cells can promote cell proliferation and migration, inhibit apoptosis, and promote the expressions of fibrosis-related genes. Western blot results indicated that HOTAIR could upregulate URI1 expression and activate Wnt signaling pathway. In addition, rescue assay demonstrated that overexpression of URI1 reversed the inhibitory effect of HOTAIR knockdown on Wnt pathway. Highly expressed HOTAIR promoted proliferation and migration of cardiac fibroblasts. HOTAIR remarkably upregulated fibrosis-related genes in CF cells. The mechanism of HOTAIR in regulating myocardial fibrosis might be related to the activation of Wnt signaling pathway through targeting URI1 expression.

Glaucocalyxin A attenuates angiotensin II-induced cardiac fibrosis in cardiac fibroblasts

Glaucocalyxin A (GLA) is a natural ent-Kaurane diterpenoid that possesses cardioprotective effect. Recently, it has been reported that GLA inhibits liver and pulmonary fibrosis, whereas its role in cardiac fibrosis remains unknown. In the present study, we evaluated the effect of GLA on the pathogenesis of cardiac fibrosis in vitro. The results showed that GLA inhibited angiotensin II (Ang II)-induced proliferation and migration in cardiac fibroblasts (CFs). GLA reduced the expression of alpha-smooth muscle actin (α-SMA) in Ang II-induced CFs, suggesting that GLA prevented the differentiation of CFs to myofibroblasts. Furthermore, the production of extracellular matrix (ECM) components including type I collagen (Col-I) and fibronectin, as well as the matrix metalloproteinases (MMPs) including MMP-2 and MMP-9 were reduced by GLA in Ang II-induced CFs. In addition, GLA prevented the Ang II-induced activation of transforming growth factor beta 1 (TGF-β1)/Smad3 pathway in CFs. Collectively, our results demonstrated that GLA acted as an anti-fibrotic agent in cardiac fibrosis, which might be mediated by the regulation of TGF-β1/Smad3 pathway. GLA might be an attractive candidate for improving the prognosis of acute myocardial infarction (AMI) by controlling the cardiac fibrosis.

Long Noncoding RNA H19 Acts as a Competing Endogenous RNA to Mediate CTGF Expression by Sponging miR-455 in Cardiac Fibrosis.

Cardiac fibrosis is closely related to multiple cardiovascular system diseases, and noncoding RNAs (ncRNAs), including long noncoding RNA (lncRNA) and microRNA (miRNA), have been reported to play a vital role in fibrogenesis. The present study aims to investigate the potential regulatory mechanism of lncRNA H19 and miR-455 on fibrosis-associated protein synthesis in cardiac fibroblasts (CFs). miRNA microarray assay revealed 34 significantly dysregulated miRNAs, including 13 upregulated miRNAs and 21 downregulated miRNAs. Among these aberrantly expressed miRNAs, we paid attention to miR-455, which was significantly downregulated in diabetic mouse myocardium and Ang II-induced CFs. Loss- and gain-of-function experiments showed that miR-455 expression levels were negatively correlated with collagen I and III expression in Ang II-induced CFs. Bioinformatic prediction programs (TargetScan, miRanda, starBase) predicted that miR-455 targeted connective tissue growth factor (CTGF) and H19 with complementary binding sites at the 3'-untranslated region, which was validated by luciferase reporter assay. Functional validation assay demonstrated that H19 knockdown could enhance the antifibrotic role of miR-455 and attenuate the CTGF expression and further decrease fibrosis-associated protein synthesis (collagen I, III, and α-SMA). The present study reveals a novel function of the H19/miR-455 axis targeting CTGF in cardiac fibrosis, suggesting its potential therapeutic role in cardiac diseases.