Ang II-stimulated Cardiac Fibroblasts Research Articles

Exosomes derived from cardiac fibroblasts with Ang-II stimulation provoke myocardial hypertrophy via miR-15b-5p/PTEN-L axis.

This study aimed to examine the impact of exosomes derived from Ang II-stimulated cardiac fibroblasts (CFs) on myocardial hypertrophy. Neonatal rat CFs were isolated and identified using Vimentin immunofluorescence. Following Ang II stimulation, exosomes were collected, characterized, and subjected to miRNA sequencing. Myocardial hypertrophy models were induced both in vitro and in vivo using Ang II. CFs were transfected with miR-15b-5p mimics or inhibitors, and their exosomes were co-cultured with rat cardiomyocytes (H9C2). Changes in cell viability, myocardial hypertrophy, and the expression levels of PTEN-L, PINK1, and Parkin proteins were assessed using the CCK-8 assay, cell surface area evaluation, and Western blot analysis. Cardiac tissue pathology and myocardial hypertrophy were evaluated through HE and WAG staining, respectively, while PTEN-L expression was detected by immunohistochemistry. The results demonstrated successful isolation of CFs and their exosomes, with miR-15b-5p significantly enriched in the exosomes derived from Ang II-stimulated CFs (Ang II-CFs-Exos). Ang II-CFs-Exos inhibited cell viability, exacerbated myocardial hypertrophy, and activated mitophagy via miR-15b-5p in the in vitro myocardial hypertrophy model. PTEN-L was identified as a downstream target of miR-15b-5p, with its overexpression reversed the effects of miR-15b-5p mimic on myocardial hypertrophy and mitophagy. Additionally, mitochondrial inhibitors also countered the effects of the miR-15b-5p mimic on myocardial hypertrophy. Furthermore, Ang II-CFs-Exos exacerbated myocardial hypertrophy in rats, while knockout of miR-15b-5p in Ang II-CFs-Exos mitigated this effect. To sum up, Ang II-CFs-Exos promote myocardial hypertrophy by modulating PINK1/Parkin signaling -mediated mitophagy through the miR-15b-5p/PTEN-L axis.

Ershen Zhenwu Decoction Suppresses Myocardial Fibrosis of Chronic Heart Failure with Heart–Kidney Yang Deficiency by Down-Regulating the Ras Homolog Gene Family Member A/Rho-Associated Coiled-Coil Kinases Signaling Pathway

Ethnopharmacological significanceThe therapeutic efficacy of Ershen Zhenwu Decoction (ESZWD)—a famous formulation from Xin’an for patients with chronic heart failure heart–kidney Yang deficiency (CHF-HKYD)—is well established. Still, the underlying molecular mechanism is not clear. Aim of the studyThis study investigated mechanisms by which ESZWD suppresses cardiac pathology, including myocardial fibrosis, in CHF-HKYD model rats and Ang II-stimulated cardiac fibroblasts (CFs). Materials and methodsThe components in ESZWD were analyzed by ultra-high-performance liquid chromatography coupled with Quadrupole Time-Of-Flight mass spectrometry (UHPLC-Q-TOF-MS). CHF-HKYD model was established in the male Sprague–Dawley rats through bilateral thyroidectomy and intraperitoneal administration of 0.02% doxorubicin (DOX), twice weekly for 3 weeks. Subsequently, the CHF-HKYD model rats were randomly categorized into the Model, ESZWD-L (3.96 g/kg/d ESZWD), ESZWD-M (7.92 g/kg/d ESZWD), ESZWD-H (15.84 g/kg/d ESZWD), and Sac/Val (68 mg/kg/d sacubitril/valsartan) groups and treated daily for 4 weeks. As a control, the sham surgery group (Sham) was used. Primary cardiac fibroblasts (CFs) were categorized into Control, Model, ESZWD, and Sac/Val groups. Then, the CFs were stimulated with Ang-II. The ESZWD and Sac/Val groups were incubated with different concentrations of drug-containing sera and their effects on CF viability were assessed via the CCK-8 assay. The ESZWD and Sac/Val groups received drug-containing serum concentrations determined by CCK-8 assay results. The cardioprotective effects of ESZWD were determined using echocardiography, Hematoxylin & Eosin (H&E) staining, Masson staining, and Sirius red staining, and the Enzyme Linked Immunosorbent Assay (ELISA). ESZWD's effects on the Ras Homolog Gene Family Member A (RhoA) / Rho-Associated Coiled-Coil Kinases (ROCKs) signaling pathway and myocardial fibrosis were assessed by Western blotting and Quantitative Real-Time PCR (qRT-PCR) analyses. Immunofluorescence was used to observe fibrotic markers in CFs. ResultsESZWD treatment improved cardiac function in the CHF-HKYD rats by significantly reducing myocardial fibrosis and ventricular remodeling. ESZWD treatment increased the rats' body temperature (Tb) and 24-hour urine volume, left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS), and decreased LV internal systolic diameter (LVIDs), LV internal diastolic diameter (LVIDd), and heart weight/ body weight (HW/BW) compared to the Model group. In comparison to the model rats, ESZWD treatment decreased serum levels of B-type natriuretic peptide precursor (NT-proBNP), tumor necrosis factor-alpha (TNF-α), interleukin-11 (IL-11), and IL-17A. ESZWD treatment significantly down-regulated the protein and mRNA expression levels of collagen I A1, α-SMA, RhoA, ROCK1, and ROCK2 in the heart tissues of the CHF-HKYD rats and the Ang II-stimulated CFs. ConclusionESZWD significantly improved cardiac function and attenuated myocardial fibrosis and inflammation in the CHF-HKYD rats by inhibiting the RhoA/ROCKs signaling pathway.

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.

Ghrelin ameliorates cardiac fibrosis after myocardial infarction by regulating the Nrf2/NADPH/ROS pathway

To evaluate the role of ghrelin in cardiac fibrosis after myocardial infarction (MI) and to investigate the underlying mechanisms of ghrelin-regulated Nrf2/NADPH/ROS pathway-mediated cardioprotection, the profile of Nrf2, fibrosis markers, and oxidative stress markers were characterized in a rat model of MI and Angiotensin II (Ang II)-stimulated cardiac fibroblasts (CFs). The effects of ghrelin on cardiac function, fibrosis and oxidative stress were investigated after MI in vivo. The role of ghrelin in CF migration and proliferation was evaluated in Ang II-stimulated CFs in vitro. Inhibition of ghrelin receptors using the antagonist, d-Lys3-GHRP-6, in addition to ghrelin was employed in MI and CFs to investigate the direct effect of ghrelin on cardiac fibrosis. Loss function of Nrf2 in CFs was performed to investigate the effect of ghrelin-regulated Nrf2 on oxidative stress and cardiac fibrosis. Ghrelin improved the post-MI cardiac function and reduced cardiac fibrosis. This phenotype is associated with the upregulation of Nrf2 and downregulation of fibrotic proteins, NADPH oxidase and ROS production. In line with in vivo findings, ghrelin attenuated Ang II-stimulated CF migration, proliferation, and oxidative stress in vitro. Inhibition of the ghrelin receptor or knockdown of Nrf2 abolished the beneficial effects of ghrelin on MI or Ang II-stimulated cardiac fibroblasts. In conclusion, ghrelin ameliorates post-MI and Ang II-induced cardiac fibrosis by activating Nrf2, which in turn inhibits the NADPH/ROS pathway.

Hydrogen Sulfide Attenuates Angiotensin II-Induced Cardiac Fibroblast Proliferation and Transverse Aortic Constriction-Induced Myocardial Fibrosis through Oxidative Stress Inhibition via Sirtuin 3.

Sirtuin 3 (SIRT3) is critical in mitochondrial function and oxidative stress. Our present study investigates whether hydrogen sulfide (H2S) attenuated myocardial fibrosis and explores the possible role of SIRT3 on the protective effects. Neonatal rat cardiac fibroblasts were pretreated with NaHS followed by angiotensin II (Ang II) stimulation. SIRT3 was knocked down with siRNA technology. SIRT3 promoter activity and expression, as well as mitochondrial function, were measured. Male wild-type (WT) and SIRT3 knockout (KO) mice were intraperitoneally injected with NaHS followed by transverse aortic constriction (TAC). Myocardium sections were stained with Sirius red. Hydroxyproline content, collagen I and collagen III, α-smooth muscle actin (α-SMA), and dynamin-related protein 1 (DRP1) expression were measured both in vitro and in vivo. We found that NaHS enhanced SIRT3 promoter activity and increased SIRT3 mRNA expression. NaHS inhibited cell proliferation and hydroxyproline secretion, decreased collagen I, collagen III, α-SMA, and DRP1 expression, alleviated oxidative stress, and improved mitochondrial respiration function and membrane potential in Ang II-stimulated cardiac fibroblasts, which were unavailable after SIRT3 was silenced. In vivo, NaHS reduced hydroxyproline content, ameliorated perivascular and interstitial collagen deposition, and inhibited collagen I, collagen III, and DRP1 expression in the myocardium of WT mice but not SIRT3 KO mice with TAC. Altogether, NaHS attenuated myocardial fibrosis through oxidative stress inhibition via a SIRT3-dependent manner.

Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II–stimulated cardiac fibroblasts

Excessive collagen deposition by myofibroblasts during adverse cardiac remodeling leads to myocardial fibrosis that can compromise cardiac function. Unraveling the mechanisms underlying collagen gene expression in cardiac myofibroblasts is therefore an important clinical goal. The collagen receptors, discoidin domain receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, and integrin-β1, are reported to mediate tissue fibrosis. Here, we probed the role of DDR2-integrin-β1 cross-talk in the regulation of collagen α1(I) gene expression in angiotensin II (Ang II)-stimulated cardiac fibroblasts. Results from gene silencing/overexpression approaches, electrophoretic mobility shift assays, and ChIP revealed that DDR2 acts via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK)-dependent transforming growth factor-β1 (TGF-β1) signaling to activate activator protein-1 (AP-1) that in turn transcriptionally enhances the expression of collagen-binding integrin-β1 in Ang II-stimulated cardiac fibroblasts. The DDR2-integrin-β1 link was also evident in spontaneously hypertensive rats and DDR2-knockout mice. Further, DDR2 acted via integrin-β1 to regulate α-smooth muscle actin (α-SMA) and collagen type I expression in Ang II-exposed cardiac fibroblasts. Downstream of the DDR2-integrin-β1 axis, α-SMA was found to regulate collagen α1(I) gene expression via the Ca2+ channel, transient receptor potential cation channel subfamily C member 6 (TRPC6), and the profibrotic transcription factor, Yes-associated protein (YAP). This finding indicated that fibroblast-to-myofibroblast conversion is mechanistically coupled to collagen expression. The observation that collagen receptor cross-talk underlies α-SMA-dependent collagen type I expression in cardiac fibroblasts expands our understanding of the complex mechanisms involved in collagen gene expression in the heart and may be relevant to cardiac fibrogenesis.

Kaempferol Prevents Against Ang II-induced Cardiac Remodeling Through Attenuating Ang II-induced Inflammation and Oxidative Stress.

:Heart failure characterized by cardiac remodeling is a global problem. Angiotensin II (Ang II) induces cardiac inflammation and oxidative stress, which also is implicated in the pathophysiology of adverse collagen accumulation–induced remodeling. Kaempferol (KPF), a kind of flavonoid compounds, is capable of anti-inflammatory and antioxidant activities. However, the target of KPF still remains blurred. In this study, we investigated the effect of KPF on Ang II-induced collagen accumulation and explored the underlying mechanisms. Our results suggested that KPF prevented Ang II-induced cardiac fibrosis and dysfunction, in mice challenged with subcutaneous injection of Ang II. In culture cells, KPF significantly reduced Ang II-induced collagen accumulation. Furthermore, KPF remarkably decreased inflammation and oxidative stress in Ang II-stimulated cardiac fibroblasts by modulating NF-κB/mitogen‐activated protein kinase and AMPK/Nrf2 pathways.

Ellagic acid inhibits proliferation and migration of cardiac fibroblasts by down-regulating expression of HDAC1.

Cardiac fibroblasts (CFs) could be activated after myocardial infarction (MI). Thus, it is necessary to explore effective drugs to suppress the activation of CFs following MI. This study was designed to investigate the impacts of ellagic acid on CFs and the underlying mechanisms. The expression of histone deacetylases (HDACs) and fibrosis-related genes was detected by qRT-PCR and western blot. The Masson's Trichrome Staining assay was used to evaluate the area of cardiac fibrosis. The proliferation and migration of CFs were measured by CCK8 Kit and Transwell assay, respectively. Our results showed that ellagic acid significantly reduced protein expression of HDAC1, mRNA expression of collagen I, collagen III, MMP-2 and MMP-9 and the area of cardiac fibrosis in MI rats. In Ang II-stimulated CFs, ellagic acid (60 μmol/L) decreased the protein expression of HDAC1, collagen I, collagen III, MMP-2 and MMP-9, and inhibited cell proliferation and migration. Further, HDAC1 over-expression reversed the inhibitor effects of ellagic acid on proteins expression (collagen I, collagen III, MMP-2 and MMP-9) and proliferation and migration of CFs. The present results suggested that ellagic acid suppressed proliferation and migration of CFs by down-regulating expression of HDAC1.

Piperlongumine inhibits angiotensin II-induced extracellular matrix expression in cardiac fibroblasts.

Piperlongumine (PL), a single component isolated from Piper longum, has been reported to possess anti-inflammatory, antibacterial, antiangiogenic, antioxidant, antitumor, and antidiabetic activities. However, its role in cardiac fibrosis remains to be clarified. Therefore, we determined the effects of PL on cardiac fibroblasts (CFs) proliferation, and extracellular matrix (ECM) production under angiotensin II (Ang II) conditions, and further investigated the underlying molecular mechanism. Our data revealed that PL inhibited the proliferation and migration of CFs induced by Ang II. In addition, PL blocked the transformation of CFs to myofibroblasts induced by Ang II, as well as decreased cellular reactive oxygen species (ROS) production and malondialdehyde level in Ang II-stimulated CFs. Furthermore, PL significantly suppressed the Ang II-increased phosphorylation of extracellular regulated protein kinase 1/2 (ERK1/2) in CFs. Taken together, the results of the current study demonstrated that PL inhibits Ang II-induced cell proliferation, migration, and ECM expression in CFs through the inactivation of the ERR1/2 signaling pathway. These data strongly suggest that PL may be a promising therapeutic candidate for the treatment of cardiac fibrosis.

Scoparone attenuates angiotensin II-induced extracellular matrix remodeling in cardiac fibroblasts

Scoparone is a biologically active constituent isolated from Artemisia capillaris and possesses a variety of pharmacological activities, such as anti-inflammatory, anti-tumor, anti-allergic and anti-cardiovascular activities. However, there are no studies focusing on the effects of scoparone against cardiac fibrosis. Therefore, the aim of this study was to investigate the effects of scoparone on Angiotensin II (Ang II)-induced extracellular matrix (ECM) remodeling and its possible mechanism in cardiac fibroblasts (CFs). Our results demonstrated that scoparone effectively attenuated CFs proliferation in Ang II-stimulated CFs. Scoparone also prevented the differentiation of CFs to myofibroblasts and ECM proteins (type I collagen and fibronectin) expression in Ang II-stimulated CFs. Furthermore, scoparone prevented Ang II-induced the activation of TGF-β1/Smad signalling in CFs. Taken together, these studies indicated that scoparone attenuated Ang II-induced ECM remodeling in CFs, at least in part, by inhibiting TGF-β1/Smad signalling. These findings suggest that scoparone may be used a novel therapeutic agent against cardiac fibrosis.

Flavonoid Extract from Propolis Inhibits Cardiac Fibrosis Triggered by Myocardial Infarction through Upregulation of SIRT1.

The flavonoid extract from propolis (FP) has been shown to protect against heart injury induced by isoproterenol. However, the effect of FP on cardiac fibrosis after myocardial infarction (MI) as well as the underlying mechanisms is not known. In the present study, we used biochemical and histological approaches to examine the effects of FP on MI-induced cardiac fibrosis and the related mechanisms in a rat MI model and in angiotensin II- (Ang II-) treated rat cardiac fibroblasts (CFs). In vivo, MI was generated by ligation of the left anterior descending coronary artery of rats, which remained for 4 weeks. Rats were randomly divided into the sham, MI, FP (12.5 mg/kg/d), and MI+FP groups. We found that FP treatment improved heart function, reduced cardiac fibrosis, and downregulated the expression of fibrosis-related factors including collagen I, collagen III, matrix metalloproteinase-2 (MMP-2), MMP-9, transforming growth factor-β1 (TGF-β1), and p-Smad2/3, which coincided with the upregulated expression of silent information regulator 1 (SIRT1) in the hearts of MI rats. Our in vitro experiments showed that FP inhibited the proliferation and migration of primary cultured rat CFs and downregulated the expression of the above-mentioned fibrosis-related factors in Ang II-stimulated CFs. In addition, FP can decrease ROS production induced by MI and Ang II in vivo and vitro. Notably, silencing SIRT1 counteracted the FP-induced effects on CFs treated with Ang II. We conclude that FP inhibits MI-induced cardiac fibrosis through SIRT1 activation and that FP represents a potential promising drug for the treatment of MI patients in the clinic.

Inhibitory effects of low decibel infrasound on the cardiac fibroblasts and the involved mechanism.

Introduction:Infrasound is a mechanical vibration wave with frequency between 0.0001 and 20 Hz. It has been established that infrasound of 120 dB or stronger is dangerous to humans. However, the biological effects of low decibel infrasound are largely unknown. The purpose of this study was to investigate the effects of low decibel infrasound on the cardiac fibroblasts.Materials and Methods:The cardiac fibroblasts were isolated and cultured from Sprague–Dawley rats. The cultured cells were assigned into the following four groups: control group, angiotensin II (Ang II) group, infrasound group, and Ang II+infrasound group. The cell proliferation and collagen synthesis rates were evaluated by means of [3H]-thymidine and [3H]-proline incorporation, respectively. The levels of TGF-β were determined by enzyme-linked immunosorbent assay. Moreover, RNAi approaches were used for the analysis of the biological functions of miR-29a, and the phosphorylation status of Smad3 was detected using western blotting analysis.Results:The results showed that low decibel infrasound significantly alleviated Ang II-induced enhancement of cell proliferation and collagen synthesis.Discussion:Compared with the control, Ang II markedly decreased the expression of miR-29a levels and increased the secretion of TGF-β and phosphorylation of Smad3, which was partly reversed by the treatment with low decibel infrasound. Importantly, knockdown of miR-29a diminished the effects of infrasound on the cardiac fibroblasts. In conclusion, low decibel infrasound inhibits Ang II-stimulated cardiac fibroblasts via miR-29a targeting TGF-β/Smad3 signaling.

Rivaroxaban Inhibits Angiotensin II-Induced Activation in Cultured Mouse Cardiac Fibroblasts Through the Modulation of NF-κB Pathway.

Cell migration, proliferation, and differentiation of cardiac fibroblasts (CFs) play a central role in cardiac fibrosis. Factor Xa (FXa)-dependent protease-activated receptor (PAR)-1 and PAR-2 have been reported as important targets in proinflammatory and fibroproliferative diseases. From this viewpoint, we aimed to investigate whether treatment of rivaroxaban, an approved oral direct FXa inhibitor, attenuates functional changes in angiotensin (Ang) II-induced mouse CFs.Confluent cultured mouse CFs were pretreated with or without rivaroxaban. Ang II-induced cell migration was decreased by 73% in rivaroxaban induced cells. Rivaroxaban inhibited Ang II-induced cell proliferation by 27% at 0.01 μg/ mL, 69% at 0.1 μg/mL, 71% at 1 μg/mL, and 69% at 5 μg/mL. In mouse cytokine array measuring 40 cytokines, the productions of interleukin-16, TIMP-1, and tumor necrosis factor-α (TNF-α) were significantly reduced with 0.1 μg/mL of rivaroxaban pretreatment (all P < 0.05). TIMP-1 levels in the culture supernatant measured by ELISA were also decreased by rivaroxaban pretreatment in Ang II-induced CFs (35% decrease at 0.01 μg/mL, 47% at 0.1 μg/mL, 47% at 1 μg/mL, and 57% at 5 μg/mL). In the dual reporter assay analysis, rivaroxaban inhibited various inflammatory signal pathways, including the nuclear factor-kappa B (NF-κB), active protein-1 (AP-1), and mitogen-activated protein kinase (MAPK) pathways (decreases of 82%, 78%, and 75%, respectively).These data suggest that rivaroxaban inhibits Ang II-induced functional activation in cultured mouse CFs via inhibiting NF-κB and MAPK/AP-1 signaling pathways, which may be a possible target of heart failure, through the antifibrotic and anti-inflammatory efficacy of rivaroxaban in Ang II-stimulated cardiac fibroblasts.

Inhibition of farnesyl pyrophosphate synthase prevents angiotensin II-induced cardiac fibrosis in vitro

Farnesyl pyrophosphate synthase (FPPS)-catalysed isoprenoid intermediates are important for the activation of Ras homologue gene family, member A (RhoA) in angiotensin (Ang) II-induced cardiac fibrosis. This study was designed to investigate the specific role of FPPS in the development of cardiac fibrosis. We demonstrated that FPPS expression was elevated in both in-vivo and in-vitro models of Ang II-mediated cardiac fibrosis. FPPS inhibition by zolendronate and FPPS knock-down by a silencing lentivirus decreased the expression of cardiac fibrosis marker genes, including collagen I, collagen III and transforming growth factor (TGF)-β1. FPPS inhibition was reversed by geranylgeraniol (GGOH) and mimicked by RhoA knock-down with siRhoA. The antagonistic effect of GGOH on the zolendronate-mediated modulation of RhoA activation in Ang II-stimulated cardiac fibroblasts was demonstrated by a pull-down assay. Furthermore, FPPS knock-down also prevented RhoA activation by Ang II in vitro. In conclusion, FPPS and RhoA may be part of a signalling pathway that plays an important role in Ang II-induced cardiac fibrosis in vitro.

Inhibition of NADPH oxidase 4-related signaling by sodium hydrosulfide attenuates myocardial fibrotic response

Myocardial fibrosis plays a pivotal role in the development of heart failure. Hydrogen sulfide (H2S) is an endogenous gasotransmitter with potent cardioprotective properties; however, whether H2S is involved in fibrotic process remains unknown. This study aimed to explore the role of H2S in the process of cardiac fibrosis and the underlying mechanisms. Myocardial infarction (MI) was established in rats by ligation of coronary artery. Activation of rat neonatal cardiac fibroblasts was induced by angiotensin II (Ang II). Fibrotic responses in ischemic myocardium and in Ang II-stimulated cardiac fibroblasts were examined. The effects of sodium hydrosulfide (NaHS, an exogenous H2S donor) on NADPH oxidase 4 (Nox4), reactive oxygen species (ROS) production, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, heme oxygenase-1 (HO-1), and cystathionine γ-lyase (CSE) were tested to elucidate the protective mechanisms of H2S on fibrotic response. NaHS treatment inhibited Ang II-induced expression of α-smooth muscle actin, connective tissue growth factor (CTGF), and type I collagen and upregulated expression of HO-1 in cardiac fibroblasts. Ang II-induced Nox4 expression in cardiac fibroblasts was quenched by NaHS and this was associated with a decreased ROS production and reduced ERK1/2 phosphorylation and CTGF expression. In vivo studies using MI model indicated that NaHS administration attenuated Nox4 expression and fibrotic response. Moreover, NaHS therapy also prevented cardiac inflammatory response accompanied by increases in HO-1 and CSE expression. The beneficial effect of H2S, at least in part, was associated with a decrease of Nox4-ROS-ERK1/2 signaling axis and an increase in HO-1 expression.

Effect of angiotensin II on primary cardiac fibroblast matrix metalloproteinase activities

Left ventricular dysfunction is associated with reperfusion injury occurring during open-heart surgery. There is an increased secretion of angiotensin II (Ang II) and increased matrix metalloproteinases (MMPs) activities associated with open-heart surgery that may affect the cardiac extracellular matrix (ECM). The goal of this study was to determine the effects of Ang II and selective angiotensin II receptor (AT1-R and AT2-R) blockers on the enzymatic activities of MMPs in primary adult murine cardiac fibroblasts (CF). Our hypothesis is that Aug II, with and without a selective receptor blocker, differentially affects CF MMPs activities. The CF were treated with Ang II (10(-6) M) and doses of AT1-R and AT2-R blockers (losartan and PD123319, respectively) at doses of 10(-7) to 10(-5) M for 48 hours. The Ang II-stimulated CF reduced collagenase activities by only 24% (p = 0.004); however, the MMP-2 and MMP-9 gelatinase activities were reduced by 42% and 39%, respectively (p = 0.022). The losartan dose dependently increased MMP-2 (p = 0.02) and MMP-9 (ns). PD123319 at 10(-5) M significantly reduced MMP-2 and MMP-9 activities compared with the Ang II group (p = 0.014 and p = 0.02, respectively). The doses of PD123319 at 10(-6) and 10(-7) M increased the MMP-2 and MMP-9 enzymatic activities significantly above the Ang II only group. Thus, Ang II and AT1-R and AT2-R differentially affect the collagenase and gelatinase MMPs activities released by cardiac fibroblasts.