Adult Rat Cardiac Fibroblasts Research Articles

Cyanidin 3-O-glucoside prevents the development of maladaptive cardiac hypertrophy and diastolic heart dysfunction in 20-week-old spontaneously hypertensive rats.

The present study investigated the effects of cyanidin 3-O-glucoside (C3G) in cardiomyocytes (CM) and fibroblasts exposed to endothelin 1 (ET1), as well as in the spontaneously hypertensive rat (SHR) model, alone or in combination with hydrochlorothiazide (HCT). Adult rat CM and cardiac fibroblasts (CF) were pretreated with C3G and co-incubated with ET1 (10-7 M) for 24 hours. Five-week-old male SHR and their normotensive controls, Wistar-Kyoto rats (WKY), received one of 4 treatments via oral gavage daily for 15 weeks: (1) water (control); (2) C3G (10 mg per kg per day); (3) HCT (10 mg per kg per day); (4) C3G + HCT (10 mg per kg per day each). Blood pressure (BP) was measured at 1, 8 and 15 weeks. Echocardiography measurements were performed at 15 weeks. C3G prevented ET1-induced CM death and hypertrophy. Stimulating CF with ET1 did not induce their phenoconversion; nevertheless, C3G inhibited un-stimulated CF differentiation. HCT slowed the rise of systolic BP (SBP) in the SHR over time (week 1: SHRs control = 161 ± 6.3 mmHg, SHRs HCT = 129 ± 6.3 mmHg; week 15: SHRs control = 201 ± 7.3 mmHg, SHRs HCT = 168 ± 7.3 mmHg), but C3G had no effect on SBP (week 1: SHRs control = 161 ± 6.3 mmHg, SHRs C3G = 126 ± 6.3 mmHg; week 15: SHRs control = 201 ± 7.3 mmHg, SHRs C3G = 186 ± 7.3 mmHg). SHRs treated with C3G, HCT, and C3G + HCT had lower left ventricular mass and shorter isovolumetric relaxation time compared to control SHRs. C3G ameliorated cardiac hypertrophy and diastolic dysfunction in SHRs.

T3 peptide, a fragment of tumstatin, stimulates proliferation and migration of cardiac fibroblasts through activation of Akt signaling pathway.

Proliferation and migration of cardiac fibroblasts are important in early stage of wound-healing after myocardial infarction. The effects of tumstatin, a cleaved fragment of collagen type IV α3 chain, on these functions of cardiac fibroblasts have not been clarified. In this study, we examined it by using T3 peptide, an active fragment of tumstatin. Cardiac fibroblasts were isolated from ventricles of adult male Wistar rats. Proliferation was examined by a cell counting assay. Boyden chamber assay was performed to examine migration. Expression and phosphorylation of proteins were determined by Western blotting. T3 peptide (300ng/ml, 24h) significantly increased proliferation and migration of cardiac fibroblasts. T3 peptide (300ng/ml, 30min) significantly increased Akt (Ser473) phosphorylation. LY294002 (10μM, 30min pretreatment), a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, significantly inhibited the T3 peptide-induced proliferation, migration, and activation of Akt signaling pathway in cardiac fibroblasts. Cilengitide, an inhibitor of integrin αvβ3/αvβ5, suppressed Akt phosphorylation and proliferation of cardiac fibroblasts. Expression of tumstatin decreased in the infarcted area of rat model of myocardial infarction. We for the first time demonstrated that T3 peptide stimulates proliferation and migration at least partly through the activation of PI3K/Akt signaling pathway via binding integrin αvβ3/αvβ5 in adult rat cardiac fibroblasts. These results indicate that tumstatin promotes the initial stage of wound-healing through activation of cardiac fibroblasts after myocardial infarction.

Evodiamine attenuates TGF-β1-induced fibroblast activation and endothelial to mesenchymal transition.

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.

Lipopolysaccharide Activates Toll-Like Receptor 4 and Prevents Cardiac Fibroblast-to-Myofibroblast Differentiation.

Bacterial lipopolysaccharide (LPS) is a known ligand of Toll-like receptor 4 (TLR4) which is expressed in cardiac fibroblasts (CF). Differentiation of CF to cardiac myofibroblasts (CMF) is induced by transforming growth factor-β1 (TGF-β1), increasing alpha-smooth muscle actin (α-SMA) expression. In endothelial cells, an antagonist effect between LPS-induced signaling and canonical TGF-β1 signaling was described; however, it has not been studied whether in CF and CMF the expression of α-SMA induced by TGF-β1 is antagonized by LPS and the mechanism involved. In adult rat CF and CMF, α-SMA, ERK1/2, Akt, NF-κβ, Smad3, and Smad7 protein levels were determined by western blot, TGF-β isoforms by ELISA, and α-SMA stress fibers by immunocytochemistry. CF and CMF secrete the three TGF-β isoforms, and the secretion levels of TGF-β2 was affected by LPS treatment. In CF, LPS treatment decreased the protein levels of α-SMA, and this effect was prevented by TAK-242 (TLR4 inhibitor) and LY294002 (Akt inhibitor), but not by BAY 11-7082 (NF-κβ inhibitor) and PD98059 (ERK1/2 inhibitor). TGF-β1 increased α-SMA protein levels in CF, and LPS prevented partially this effect. In addition, in CMF α-SMA protein levels were decreased by LPS treatment, which was abolished by TAK-242. Finally, in CF LPS decreased the p-Smad3 phosphorylation and increased the Smad7 protein levels. LPS treatment prevents the CF-to-CMF differentiation and reverses the CMF phenotype induced by TGF-β1, through decreasing p-Smad3 and increasing Smad7 protein levels.

Canstatin stimulates migration of rat cardiac fibroblasts via secretion of matrix metalloproteinase-2

Type IV collagen, a nonfibrillar type, is ubiquitously expressed in the basement membrane around cardiomyocytes. Canstatin, a cleaved product of α2 chain of type IV collagen, is an antiangiogenic factor. Because it has not been clarified whether canstatin exerts other biological activities in heart, we investigated the effects of canstatin on adult rat cardiac fibroblasts. Cell migration was determined by Boyden chamber assay. Western blotting was performed to detect secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9 and phosphorylation of extracellular signal-regulated kinase (ERK). Localization of MMP-2 was detected by immunofluorescence staining. Canstatin (250 ng/ml) significantly increased migration, secretion, and activity of MMP-2 but not MMP-9. CTTHWGFTLC peptide, an MMP inhibitor and small interfering RNA (siRNA) against MMP-2 suppressed the canstatin-induced (250 ng/ml, 24 h) migration. Canstatin (250 ng/ml, 30 min) significantly increased phosphorylation of ERK. PD98059, a MEK inhibitor, significantly suppressed the canstatin-induced (250 ng/ml, 24 h) migration but not secretion of MMP-2. An increase in MMP-2 expression was observed in cytoplasm of the canstatin-treated (250 ng/ml) cardiac fibroblasts (within 30 min). Canstatin induced actin stress fiber formation, which was inhibited by Y-27632, a Rho-associated kinase inhibitor. Y-27632 also suppressed the canstatin-induced (250 ng/ml, 24 h) MMP-2 secretion. Canstatin (250 ng/ml, 30 min) failed to induce ERK phosphorylation in MMP-2 siRNA-treated cardiac fibroblasts. In conclusion, this study revealed a novel function of canstatin for inducing cell migration of adult rat cardiac fibroblasts at least in part by ERK phosphorylation through MMP-2 secretion, possibly via actin cytoskeletal change.

Cardiac fibroblast cytokine profiles induced by proinflammatory or profibrotic stimuli promote monocyte recruitment and modulate macrophage M1/M2 balance in vitro

Macrophage polarization plays an essential role in cardiac remodeling after injury, evolving from an initial accumulation of proinflammatory M1 macrophages to a greater balance of anti-inflammatory M2 macrophages. Whether cardiac fibroblasts themselves influence this process remains an intriguing question. In this work, we present evidence for a role of cardiac fibroblasts (CF) as regulators of macrophage recruitment and skewing. Adult rat CF, were treated with lipopolysaccharide (LPS) or TGF-β1, to evaluate ICAM-1 and VCAM-1 expression using Western blot and proinflammatory/profibrotic cytokine secretion using LUMINEX. We performed in vitro migration and adhesion assays of rat spleen monocytes to layers of TGF-β1- or LPS-pretreated CF. Finally, TGF-β1- or LPS-pretreated CF were co-cultured with monocyte, to evaluate their effects on macrophage polarization, using flow cytometry and cytokine secretion.There was a significant increase in monocyte adhesion to LPS- or TGF-β1-stimulated CF, associated with increased CF expression of ICAM-1 and VCAM-1. siRNA silencing of either ICAM-1 or VCAM-1 inhibited monocyte adhesion to LPS-pretreated CF; however, monocyte adhesion to TGF-β1-treated CF was dependent on only VCAM-1 expression. Pretreatment of CF with LPS or TGF-β1 increased monocyte migration to CF, and this effect was completely abolished with an MCP-1 antibody blockade. LPS-treated CF secreted elevated levels of TNF-α and MCP-1, and when co-cultured with monocyte, LPS-treated CF stimulated increased macrophage M1 polarization and secretion of proinflammatory cytokines (TNF-α, IL-12 and MCP-1). On the other hand, CF stimulated with TGF-β1 produced an anti-inflammatory cytokine profile (high IL-10 and IL-5, low TNF-α). When co-cultured with monocytes, the TGF-β1 stimulated fibroblasts skewed monocyte differentiation towards M2 macrophages accompanied by increased IL-10 and decreased IL-12 levels. Taken together, our results show for the first time that CF can recruit monocytes (via MCP-1-mediated chemotaxis and adhesion to ICAM-1/VCAM-1) and induce their differentiation to M1 or M2 macrophages (through the CF cytokine profile induced by proinflammatory or profibrotic stimuli).

Aristoyunnolin H attenuates extracellular matrix secretion in cardiac fibroblasts by inhibiting calcium influx.

Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca2+, we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca2+/calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

Activin A stimulates the proliferation and differentiation of cardiac fibroblasts via the ERK1/2 and p38-MAPK pathways

Activin A is a key regulator of cardiac fibrosis. However, little is known about the mechanisms by which it contributes to cardiac fibrosis. Our study explored the effects of activin A on proliferation and differentiation of adult rat cardiac fibroblasts (CFs) via the activin A receptor, activin receptor-like kinase 4 (ALK4). CF proliferation was measured by CCK8 and EdU assays, while differentiation, fibrosis and signaling were measured by western blot analysis of α-smooth muscle actin, collagen type I, phosphorylated extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (p38-MAPK) expression. Activin A levels were measured by ELISA and western blot analysis. We demonstrated that CFs express activin A and its expression was significantly enhanced by angiotensin II (Ang II), but follistatin (activin A inhibitor) significantly reversed Ang II-induced activin A upregulation, CF proliferation, differentiation, collagen type I expression as well as ERK1/2 and p38-MAPK pathways activation. Conversely, recombinant activin A largely increased these parameters in both the presence and absence of Ang II. Interestingly, p38-MAPK (SB203580) and ALK4 (SB431542) inhibitors significantly reduced all activin A-mediated responses; however, an ERK1/2 inhibitor (PD98059) could only significantly reduce CF proliferation and collagen type I expression but not differentiation. Importantly, the most significant effects were observed in the presence vs. absence of Ang II. Thus, activin A promotes basal and Ang II-induced CF proliferation and differentiation via ALK4, and the effects are partly mediated through the ERK1/2 and p38-MAPK pathways. These data suggest that activin A is a potential therapeutic target for cardiac fibrosis.

Abstract 89: 17beta-Estradiol-activated Estrogen Receptors Attenuate Cardiac Fibrosis Through Inhibition of Collagen I and III Expression in Female Hearts

Objectives: Female pressure-overloaded hearts show less fibrosis compared with males. 17β-Estradiol (E2) attenuates cardiac fibrosis in female mice. Whether this is mediated by direct E2-effects on collagen synthesis is still unknown. Therefore, we investigated the role of E2 and estrogen receptors (ER) on collagen I and III expression and analyzed involved mechanisms. Methods: Female C57BL/6J mice (7 weeks) underwent sham operation, ovariectomy (OVX), OVX with E2-supplementation (390mg E2-containing pellets) or placebo. After 2 weeks, animals underwent transverse aortic constriction (TAC) or sham surgery. Mice were sacrificed after 9 weeks. Collagen amount, collagen I and III protein in left ventricular tissue were detected by Sirius Red and antibody staining, respectively. Gene and protein expression were determined by quantitative Real-Time PCR and Western blot. Adult female rat cardiac fibroblasts were treated with E2 (10 -8 M), vehicle, ERα- and β-agonists (10 -7 M) for 24h or pre-treated with PD98059 for 1h. ER binding to the collagen I and III promoter was analyzed by chromatin immunoprecipitation assays. Findings: In female OVX mice, undergoing TAC surgery, E2-supplementation significantly reduced collagen deposition, collagen I and III mRNA and protein levels in comparison with mice without E2. In female rat cardiac fibroblasts, E2 significantly down-regulated collagen I and III mRNA and protein level. Specific ER-agonist-treatment showed that E2-mediated regulation of collagen I and III expression was mediated via activation of ERα, but not ERβ. Further, upon E2-treatment, ERα was phosphorylated at Ser118, which occurred by E2-induced activation of ERK1/2 signaling. Furthermore, we could show that ERα and ERβ bind to two putative half-palindromic estrogen response elements within the collagen I and III promoter in female cardiac fibroblasts. Conclusion: E2 inhibits cardiac collagen I and III mRNA and protein in female mice under pressure overload. Data from rat female cardiac fibroblasts suggest that this is mediated via E2-activated ERK1/2 signaling and ERα, which binds with ERβ to the collagen I and III promoter. Understanding of how E2/ER attenuate collagen I and III expression in pathological hypertrophy may improve therapy.

Store-Operated Ca2+ Entry (SOCE) contributes to angiotensin II-induced cardiac fibrosis in cardiac fibroblasts

Store-operated Ca2+ entry (SOCE) is an important mechanism of extracellular Ca2+ entry into cells. It has been proved that SOCE is involved in many pathologic and physiological processes. Two key participants of SOCE, stromal interaction molecule1 (STIM1) and Orai1, have been identified. But their function in cardiac fibroblasts remains elusive. In present study, our findings suggested the expression of STIM1 and Orai1 were increased followed by angiotensin II (Ang II) stimulation in vivo and in vitro. In cultured adult rat cardiac fibroblasts, Ang II led to STIM1 interact with Orai1 and Ca2+ release from intracellular calcium store. In addition, the upregulation of fibronectin (FN), connective tissue growth factor (CTGF) and smooth muscle α-actin (α-SMA) induced by Ang II were attenuated by SOCE inhibitor SKF-96365, similar results were observed by knocking down STIM1 and Orai1. Furthermore, we found that silencing Orai1 by RNA interference also suppressed the translocation of Nuclear Factor of Activated T-cells (NFAT) Isoforms NFATc4 and decreased the phosphorylation of Smad2 and Smad3 induced by Ang II. These results unraveled a novel role of SOCE as a key modulator in the Ang II-induced cardiac fibrosis by mediating Ca2+ influx.

Rapid atrial pacing induces myocardial fibrosis by down-regulating Smad7 via microRNA-21 in rabbit.

Tachycardia-induced atrial fibrosis is a hallmark of the structural remodeling of atrial fibrillation (AF). The mechanisms underlying tachycardia-induced atrial fibrosis remain unclear. In our previous study, we found that Smad7-downregulation promoted the development of atrial fibrosis in AF. Fibroblasts are enriched in microRNA-21 (miR-21), which contributes to the development of fibrosis and heart failure in the cardiovascular system. Our study was designed to test the hypothesis that miR-21 reinforces the TGF-β1/Smad signaling pathway in AF-induced atrial fibrosis by down-regulating Smad7. Rapid atrial pacing (RAP, 1000 ppm) was applied to the left atrium of the rabbit heart to induce atrial fibrillation and fibrosis. qRT-PCR and northern blot analysis revealed that RAP caused a marked increase in the expression of miR-21. Transfection with a miR-21 inhibitor significantly increased the expression of Smad7, while the expression of collagen I/III significantly decreased. These changes were implicated in the AF-induced release of miR-21 and down-regulation of Smad7. Adult rat cardiac fibroblasts treated with TGF-β1 showed increased miR-21 expression and decreased Smad7 expression. Pretreatment with a TGF-β1 inhibitor reduced the TGF-β1-induced up-regulation of miR-21. Pretreatment with pre-miR-21 and a miR-21 inhibitor significantly decreased and increased Smad7 expression, respectively. This result was negatively correlated with the expression of collagen I/III in fibroblasts. Moreover, the results of a luciferase activity assay suggest that Smad7 is a validated miR-21 target in CFs. Our results provide compelling evidence that the miR-21 specific degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β1/Smad signaling and serves as a new insight of the mechanism of atrial fibrosis in atrial fibrillation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00380-016-0808-z) contains supplementary material, which is available to authorized users.

Na/K-ATPase signaling regulates collagen synthesis through microRNA-29b-3p in cardiac fibroblasts.

Chronic kidney disease (CKD) is accompanied by cardiac fibrosis, hypertrophy, and dysfunction, which are commonly referred to as uremic cardiomyopathy. Our previous studies found that Na/K-ATPase ligands or 5/6th partial nephrectomy (PNx) induces cardiac fibrosis in rats and mice. The current study used in vitro and in vivo models to explore novel roles for microRNA in this mechanism of cardiac fibrosis formation. To accomplish this, we performed microRNA profiling with RT-qPCR based arrays on cardiac tissue from rats subjected to marinobufagenin (MBG) infusion or PNx. The analysis showed that a series of fibrosis-related microRNAs were dysregulated. Among the dysregulated microRNAs, microRNA (miR)-29b-3p, which directly targets mRNA of collagen, was consistently reduced in both PNx and MBG-infused animals. In vitro experiments demonstrated that treatment of primary cultures of adult rat cardiac fibroblasts with Na/K-ATPase ligands induced significant increases in the fibrosis marker, collagen protein, and mRNA expression compared with controls, whereas miR-29b-3p expression decreased >50%. Transfection of miR-29b-3p mimics into cardiac fibroblasts inhibited cardiotonic steroids-induced collagen synthesis. Moreover, a specific Na/K-ATPase signaling antagonist, pNaKtide, prevented ouabain-induced increases in collagen synthesis and decreases in miR-29b-3p expression in these cells. In conclusion, these data are the first to indicate that signaling through Na/K-ATPase regulates miRNAs and specifically, miR-29b-3p expression both in vivo and in vitro. Additionally, these data indicate that miR-29b-3p expression plays an important role in the formation of cardiac fibrosis in CKD.

Levosimendan inhibits interleukin-1β-induced apoptosis through activation of Akt and inhibition of inducible nitric oxide synthase in rat cardiac fibroblasts

Levosimendan, a calcium sensitizer, known as an inotropic agent has a cytoprotective effect against apoptosis in cardiomyocytes. However, the cytoprotective effect of levosimendan on cardiac fibroblasts has not been clarified. The aim of this study was to examine whether levosimendan modulates interleukin (IL)-1β-induced apoptosis in adult rat cardiac fibroblast. Cardiac fibroblasts were isolated from adult male Wistar rats. Apoptosis of cardiac fibroblasts was evaluated by 4′,6-diamidino-2-phenylindole staining and TdT-mediated d-UTP nick end labeling (TUNEL)-based staining. Expression of inducible nitric oxide (NO) synthase (iNOS) and phosphorylation of Akt (Ser473) were determined by Western blotting. Assessment of NO production in the culture medium was performed by using 4,5-diaminofluorescein as a fluorescent probe. Immunofluorescence staining was performed to examine cytochrome-c translocation. Levosimendan (3–100μM) concentration-dependently inhibited IL-1β (4ng/ml, 24h)-induced apoptotic changes in cardiac fibroblasts, such as cellular shrinkage, nuclear condensation and the increase of TUNEL-positive cells. Levosimendan inhibited IL-1β-induced iNOS expression and subsequent NO production. 1400W, an iNOS inhibitor, suppressed the IL-1β-induced apoptosis. Levosimendan alone-treatment concentration-dependently increased phosphorylation of Akt (Ser473). LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, reversed the protective effect of levosimendan on IL-1β-induced apoptosis in TUNEL assay. In addition, levosimendan and 1400W inhibited the IL-1β-induced cytosolic translocation of cytochrome-c. LY294002 reversed the suppressive effects of levosimendan. The present study for the first time demonstrated that levosimendan inhibits IL-1β-induced apoptosis via the activation of PI3K/Akt pathway and the inhibition of iNOS expression in adult rat cardiac fibroblasts.

Erythropoietin Decreases the Occurrence of Myocardial Fibrosis by Inhibiting the NADPH-ERK-NF-κB Pathway

Objectives: The aim of this study was to investigate the protective role of erythropoietin (EPO) against myocardial fibrosis (MF). Methods: Pressure-overloaded rats were established by abdominal aortic constriction, the rats were randomly divided in a double-blind manner into 3 groups (n = 12 for each group): sham-operated rats (sham), operated rats receiving physiological saline (vehicle) and operated rats receiving 4,000 U/kg rhEPO (EPO group). The vehicle and drugs were administered to rats by intraperitoneal injection. In addition, cultured adult rat cardiac fibroblasts (CFs) were utilized to investigate the role of EPO in CF proliferation and collagen secretion. Results: After 4 weeks, besides an increase in blood pressure, myocardial hypertrophy, collagen deposition in the myocardium and decreased cardiac function were observed in the pressure-overloaded rats. The expression of NADPH oxidase (Nox2 and Nox4) and inflammatory cytokines (CD45, F4/80 and MCP-1) was also significantly increased. All these alterations were prevented by EPO. TGF-ß promoted CF proliferation, collagen secretion, ROS production and Nox2/Nox4 expression, which was inhibited by EPO. In addition, the TGF-ß-induced increase of ERK1/2 phosphorylation and NF-κB expression were attenuated by EPO. Conclusion: EPO inhibited rat MF induced by pressure overload and improved myocardial function by decreasing CF proliferation and differentiation via inhibition of the NADPH-ERK-NF-κB pathway.

CTRP3 attenuates post-infarct cardiac fibrosis by targeting Smad3 activation and inhibiting myofibroblast differentiation.

C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel adipokine with modulation effects on metabolism, inflammation, and cardiovascular system. This study aimed to investigate the effect of CTRP3 on cardiac fibrosis and its underlying mechanism. The myocardial expression of CTRP3 was significantly decreased after myocardial infarction (MI). Adenovirus-delivered CTRP3 supplement attenuated myocardial hypertrophy, improved cardiac function, inhibited interstitial fibrosis, and decreased the number of myofibroblasts post-MI. In cultured adult rat cardiac fibroblasts (CFs), CTRP3 attenuated cell proliferation; migration; and the expression of connective tissue growth factor, collagen I, and collagen III induced by transforming growth factor (TGF)-β1. Moreover, CTRP3 inhibited whereas CTRP3 small interfering RNA (siRNA) facilitated the expression of α-SMA and profibrotic molecules induced by TGF-β1. CTRP3 also attenuated TGF-β1-induced Smad3 phosphorylation, nuclear translocation, and interaction with p300. CTRP3 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in both rat hearts and CFs. Adenine 9-β-D-arabinofuranoside (AraA), an AMPK inhibitor, abolished the protective effect of CTRP3 against TGF-β1-induced profibrotic response and Smad3 activation. Taken together, CTRP3 attenuates cardiac fibrosis by inhibiting myofibroblast differentiation and the subsequent extracellular matrix production. AMPK is required for the anti-fibrotic effect of CTRP3 through targeting Smad3 activation and inhibiting myofibroblast differentiation. CTRP3 alleviates cardiac fibrosis in a rat post-MI model and in cardiac fibroblasts. CTRP3 inhibits fibroblast-to-myofibroblast differentiation. CTRP3 exerts anti-fibrotic effect through targeting Smad3 activation. AMPK mediates the anti-fibrotic effect of CTRP3 by inhibition of Smad3 activation.

Losartan reverses the down-expression of long noncoding RNA-NR024118 and Cdkn1c induced by angiotensin II in adult rat cardiac fibroblasts

Angiotensin II (Ang II) plays a pivotal role in the pathogenesis of cardiac fibrosis and long noncoding RNAs (lncRNAs) have been found to be involved in human diseases. The roles of Ang II receptors (AT1 and AT2) have been controversial. Our previous studies revealed that Ang II dynamically down-regulated the expression of lncRNA-NR024118 and Cdkn1c in adult rat cardiac fibroblasts. However, up to now, whether the decrease of lncRNA-NR024118 and Cdkn1c induced by Ang II is mediated by AT1 or AT2 has never been illustrated. In order to reveal which subtype of Ang II receptors mediate the decrease of lncRNA-NR024118 and Cdkn1c induced by Ang II, we studied the expression of NR024118 and Cdkn1c with different receptor blockers in Ang II–treated adult rat cardiac fibroblasts. In this study, we found that losartan (AT1 blocker) nearly completely reversed the decrease of lncRNA-NR024118 and partly reversed the decrease of Cdkn1c induced by Ang II in adult rat cardiac fibroblasts, while AT2 blocker (PD123319) did not show effect to the level of lncRNA-NR024118 and Cdkn1c. In conclusion, our current studies showed that the decrease of lncRNA-NR024118 and Cdkn1c induced by Ang II is mediated by AT1 receptor-dependent not AT2 receptor-dependent, which is helpful to understand the molecular mechanism of Ang II receptors in adult rat cardiac fibroblasts.

C1q/tumor necrosis factor-related protein-6 attenuates post-infarct cardiac fibrosis by targeting RhoA/MRTF-A pathway and inhibiting myofibroblast differentiation.

C1q/tumor necrosis factor-related protein-6 (CTRP6) is a newly identified adiponectin paralog with modulation effects on metabolism and inflammation. However, the cardiovascular function of CTRP6 remains unknown. This study aimed to determine its role in cardiac fibrosis and explore the possible mechanism. Myocardial infarction (MI) was induced by left anterior descending coronary artery ligation in rats. CTRP6 was mainly expressed in the cytoplasm of adult rat cardiomyocytes and significantly decreased in the border and infarct zones post-MI. Adenovirus-mediated CTRP6 delivery improved cardiac function, attenuated cardiac hypertrophy, alleviated cardiac fibrosis, and inhibited myofibroblast differentiation as well as the expression of collagen I, collagen III, and connective tissue growth factor post-MI. In cultured adult rat cardiac fibroblasts (CFs), exogenous or cardiomyocyte-secreted CTRP6 inhibited, whereas knockdown of CTRP6 facilitated transforming growth factor-β1 (TGF-β1)-induced expression of α-smooth muscle actin, smooth muscle 22α, and profibrotic molecules. CTRP6 had no effect on CFs proliferation but attenuated CFs migration induced by TGF-β1. CTRP6 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in CFs and post-MI hearts. Pretreatment with adenine 9-β-D-arabinofuranoside (AraA), an AMPK inhibitor, or LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, abolished the protective effect of CTRP6 on TGF-β1-induced profibrotic response. Furthermore, CTRP6 had no effect on TGF-β1-induced Smad3 phosphorylation and nuclear translocation, whereas significantly decreased TGF-β1-induced RhoA activation and myocardin-related transcription factor-A (MRTF-A) nuclear translocation, and these effects were blocked by AMPK or Akt inhibition. In conclusion, CTRP6 attenuates cardiac fibrosis via inhibiting myofibroblast differentiation. AMPK and Akt activation are responsible for the CTRP6-mediated anti-fibrotic effect by targeting RhoA/MRTF-A pathway.

Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease: A NOVEL MECHANISM FOR GPCR-EGFR TRANSACTIVATION

Agonist stimulation of G protein-coupled receptors (GPCRs) can transactivate epidermal growth factor receptors (EGFRs), but the precise mechanisms for this transactivation have not been defined. Key to this process is the protease-mediated "shedding" of membrane-tethered ligands, which then activate EGFRs. The specific proteases and the events involved in GPCR-EGFR transactivation are not fully understood. We have tested the hypothesis that transactivation can occur by a membrane-delimited process: direct increase in the activity of membrane type-1 matrix metalloprotease (MMP14, MT1-MMP) by heterotrimeric G proteins, and in turn, the generation of heparin-binding epidermal growth factor (HB-EGF) and activation of EGFR. Using membranes prepared from adult rat cardiac myocytes and fibroblasts, we found that MMP14 activity is increased by angiotensin II, phenylephrine, GTP, and guanosine 5'-O-[γ-thio]triphosphate (GTPγS). MMP14 activation by GTPγS occurs in a concentration- and time-dependent manner, does not occur in response to GMP or adenosine 5'-[γ-thio]triphosphate (ATPγS), and is not blunted by inhibitors of Src, PKC, phospholipase C (PLC), PI3K, or soluble MMPs. This activation is specific to MMP14 as it is inhibited by a specific MMP14 peptide inhibitor and siRNA knockdown. MMP14 activation by GTPγS is pertussis toxin-sensitive. A role for heterotrimeric G protein βγ subunits was shown by using the Gβγ inhibitor gallein and the direct activation of recombinant MMP14 by purified βγ subunits. GTPγS-stimulated activation of MMP14 also results in membrane release of HB-EGF and the activation of EGFR. These results define a previously unrecognized, membrane-delimited mechanism for EGFR transactivation via direct G protein activation of MMP14 and identify MMP14 as a heterotrimeric G protein-regulated effector.

Endostatin stimulates proliferation and migration of adult rat cardiac fibroblasts through PI3K/Akt pathway

Endostatin, a non-collagenous fragment of type XVIII collagen, has anti-angiogenic roles. Although the expression level of endostatin increases in some experimental models of cardiac diseases, its effects on cardiac remodeling have not been clarified. In this study, we investigated the effect of endostatin on proliferation, migration and collagen synthesis of cardiac fibroblasts, which are activated during the cardiac remodeling following myocardial infarction. Cardiac fibroblasts were isolated from adult male Wistar rats and treated with recombinant endostatin for 20min to 24h. Cell counting assay was used to determine a cell proliferation. Boyden chamber assay was performed to determine a cell migration. Wound-healing assay was performed for detecting a wound-induced migration. Expression of collagen type I and phosphorylation of Akt, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were detected by Western blotting. Endostatin (100–3000ng/ml) stimulated cell proliferation, migration and wound-induced migration but not collagen type I expression. Endostatin stimulated phosphorylation of Akt (Ser473) but not ERK, JNK and p38 MAPK. LY294002 (10μM), a PI3K/Akt pathway inhibitor, significantly inhibited the endostatin-induced proliferation and migration. N-acetyl-l-cysteine (NAC) (5mM), an antioxidant, significantly inhibited the endostatin-induced Akt phosphorylation. This study for the first time demonstrated that endostatin stimulates cell proliferation, migration and wound-induced migration of adult rat cardiac fibroblasts at least partly through the reactive oxygen species-dependent activation of Akt. Our findings suggest a novel function of endostatin except for an anti-angiogenic activity during the wound-healing process following myocardial infarction.

CALCIUM/CALMODULIN DEPENDENT PROTEIN KINASE II δ MODULATES INTRACELLULAR Ca2+ RELEASE AND CELL PROLIFERATION IN ADULT CARDIAC FIBROBLASTS

Calcium/calmodulin dependent protein kinase II δ (CaMKIIδ) is a well-recognised mediator of cardiomyocyte function and dysfunction, however little is known of its role in the cardiac fibroblast (CF). [Ca2+]i plays a crucial role in cell proliferation, yet the mechanism by which this is regulated in adult CFs remains elusive. We hypothesised that CaMKIIδ can regulate intracellular Ca2+ release channel activity and proliferation in adult CFs. Using Fura-2 loaded cells, we initially confirmed agonist-mediated intracellular Ca2+ release using Angiotensin II (1 µM). We demonstrated that CaMKIIδ and Inositol 1,4,5-triphosphate receptor type 2 (IP3R2) are both highly expressed in adult rat CFs but the ryanodine receptor (RyR2) is not. This identifies IP3R2 as the main intracellular Ca2+ release channel in these cells. To investigate whether CaMKIIδ modulates IP3R activity in CFs, cells were pre-treated with inhibitors autocamtide inhibitory peptide (AIP) (1 µM) and KN-93 (5 µM) prior to Angiotensin II application. Ca2+ release was significantly inhibited in the presence of both inhibitors (81±6.5% (AIP) vs 123±6% (control), p