Cultured Rat Cardiac Fibroblasts Research Articles

Angiotensin II Triggers NLRP3 Inflammasome Activation by a Ca2+ Signaling-Dependent Pathway in Rat Cardiac Fibroblast Ang-II by a Ca2+-Dependent Mechanism Triggers NLRP3 Inflammasome in CF

Angiotensin II (Ang-II) is a widely studied hypertensive, profibrotic, and pro-inflammatory peptide. In the heart, cardiac fibroblasts (CF) express type 1 angiotensin II receptors (AT1R), Toll-like receptor-4 (TLR4), and the NLRP3 inflammasome complex, which play important roles in pro-inflammatory processes. When activated, the NLRP3 inflammasome triggers proteolytic cleavage of pro-IL-1, resulting in its activation. However, in CF the mechanism by which Ang-II assembles and activates the NLRP3 inflammasome remains not fully known. To elucidate this important point, we stimulated TLR4 receptors in CF and evaluated the signaling pathways by which Ang-II triggers the assembly and activity. In cultured rat CF, pro-IL-1β levels, NLRP3, ASC, and caspase-1 expression levels were determined by Western blot. NLRP3 inflammasome complex assembly was analyzed by immunocytochemistry, whereas by ELISA, we analyzed NLRP3 inflammasome activity and [Formula: see text] release. In CF, Ang-II triggered NLRP3 inflammasome assembly and caspase-1 activity; and in LPS-pretreated CF, Ang-II also triggered [Formula: see text] secretion. These effects were blocked by losartan (AT1R antagonist), U73221 (PLC inhibitor), 2-APB (IP3R antagonist), and BAPTA-AM (Ca2+ chelator) indicating that the AT1R/PLC/IP3R/Ca2+ pathway is involved. Finally, bafilomycin A1 prevented Ang-II-induced [Formula: see text] secretion, indicating that a non-classical protein secretion mechanism is involved. These findings suggest that in CF, Ang-II by a Ca2+-dependent mechanism triggers NLRP3 inflammasome assembly and activation leading to [Formula: see text] secretion through a non-conventional protein secretion mechanism.

MicroRNA-122-5p Aggravates Angiotensin II-Mediated Myocardial Fibrosis and Dysfunction in Hypertensive Rats by Regulating the Elabela/Apelin-APJ and ACE2-GDF15-Porimin Signaling.

Hypertension is the leading risk factor for cardiovascular disorders. This study aimed to explore roles of microRNA (miR)-122-5p in hypertension. Angiotensin II (Ang II; 1.5 mg/kg/day) with an osmotic minipump was used to induce hypertensive rats pretreated by rAAV-miR-122-5p or rAAV-GFP, respectively. Notably, Ang II infusion caused marked increases in myocardial fibrosis, inflammation, oncosis, and oxidant injury in rats, which were aggravated by rAAV-miR-122-5p. RAAV-miR-122-5p exacerbated Ang II–mediated cardiac dysfunction and structural injury in hypertensive rats, with downregulated levels of apelin, elabela, ACE2, and GDF15, as well as upregulated expression of porimin and CTGF. In cultured rat cardiac fibroblasts, Ang II contributed to augmentation of cellular oncosis, migration, inflammation, and oxidative stress, with reduction of apelin, elabela, ACE2, and GDF15 levels, which were rescued by miR-122 inhibitor. In summary, miR-122-5p exacerbates myocardial fibrosis and dysfunction in hypertensive rats by modulating the elabela/apelin-ACE2-GDF15 signaling. MiR-122-5p has potential therapeutic significance for hypertension and hypertensive cardiac injury.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s12265-022-10214-3.

Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes.

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis; however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor–β (TGF-β). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-β receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-β-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.

MiR-146a mediates TLR-4 signaling pathway to affect myocardial fibrosis in rat constrictive pericarditis model.

BackgroundMyocardial fibrosis (MF) is thought to be associated with constrictive pericarditis (CP). miR-146a has been reported to be related to the survival of myocardial fibroblasts and related signal transduction pathways. The aim of this study was to investigate the expression of miR-146a in CP with MF and the activation of the Toll-like receptor 4 (TLR-4) signaling pathway, to understand the molecular mechanism of MF involvement in CP.MethodsThirty rats with different disease duration were randomly divided into three groups: an 8-week model group (CP-8W group), a 16-week model group (CP-16W group) model, and a normal control group (N group). After the CP model was established in the rats, the myocardial tissues were collected. The expression of miR-146a, the key factors of TLR-4 signaling pathway, including IL-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor-κB (NF-κB) and p-NF-κB, and the MF indicator α-SMA in myocardial tissue were detected. After treatment with lipopolysaccharide (LPS), primary cultured rat cardiac fibroblasts (CFs) were transfected with miR-146a. RT-PCR and western blot were used to detect the expression of downstream effectors to further verify the function of miRNA-146a in regulating MF via the TLR-4 signaling pathway.ResultsmiR-146a was increased in the CP-8W group but not in the CP-16W group. IRAK1 and TRAF6 in the CP-16W group were found to be higher than in the N group and CP-8W group. α-SMA in the model groups was higher than in the N group. Compared with the CP-8W group, α-SMA in the CP-16W model group was further increased. In the experiments using CFs, the expression of IRAK1, TRAF6, p-NF-κB and α-SMA increased in the LPS-treated group compared with the N group. After transfection of CFs with the miR-146a mimics, the expression of IRAK1, TRAF6, p-NF-κB and α-SMA decreased compared with the LPS-treated group. Following transfection of CFs with miR-146a inhibitors, the expression of IRAK1, TRAF6, p-NF-κB and α-SMA increased compared with the LPS-treated group.ConclusionsThe expression of miR-146a demonstrated a dynamic change in the CP model; it was increased at the early time point (CP-8W) and then decreased at the 16W time point. miR-146a suppressed MF by inhibiting the target genes TRAF6 and IRAK1 via the TLR-4 signaling pathway.

Cultured cardiac fibroblasts and myofibroblasts express Sushi Containing Domain 2 and assemble a unique fibronectin rich matrix

Cardiac fibroblasts and myofibroblasts assemble and maintain extracellular matrix during normal development and following injury. Culture expansion of these cells yield a bioengineered matrix that could lead to intriguing therapeutic opportunities. For example, we reported that cultured rat cardiac fibroblasts form a matrix that can be used to delivery therapeutic stem cells. Furthermore, we reported that matrix derived from cultured human cardiac fibroblasts/myofibroblasts converted monocytes into macrophages that express interesting anti-inflammatory and pro-angiogenic properties. Expanding these matrix investigations require characterization of the source cells for quality control. In these efforts, we observed and herein report that Sushi Containing Domain 2 (SUSD2) is a novel and consistent marker for cultured human cardiac fibroblast and myofibroblasts.

Role of IgE-FcεR1 in Pathological Cardiac Remodeling and Dysfunction.

Immunoglobulin E (IgE) belongs to a class of immunoglobulins involved in immune response to specific allergens. However, the roles of IgE and IgE receptor (FcεR1) in pathological cardiac remodeling and heart failure are unknown. Serum IgE levels and cardiac FcεR1 expression were assessed in diseased hearts from human and mouse. The role of FcεR1 signaling in pathological cardiac remodeling was explored in vivo by FcεR1 genetic depletion, anti-IgE antibodies, and bone marrow transplantation. The roles of the IgE-FcεR1 pathway were further evaluated in vitro in primary cultured rat cardiomyocytes and cardiac fibroblasts (CFs). RNA sequencing and bioinformatic analyses were used to identify biochemical changes and signaling pathways that are regulated by IgE/FcεR1. Serum IgE levels were significantly elevated in patients with heart failure as well as in 2 mouse cardiac disease models induced by chronic pressure overload via transverse aortic constriction and chronic angiotensin II infusion. Interestingly, FcεR1 expression levels were also significantly upregulated in failing hearts from human and mouse. Blockade of the IgE-FcεR1 pathway by FcεR1 knockout alleviated transverse aortic constriction- or angiotensin II-induced pathological cardiac remodeling or dysfunction. Anti-IgE antibodies (including the clinical drug omalizumab) also significantly alleviated angiotensin II-induced cardiac remodeling. Bone marrow transplantation experiments indicated that IgE-induced cardiac remodeling was mediated through non-bone marrow-derived cells. FcεR1 was found to be expressed in both cardiomyocytes and CFs. In cultured rat cardiomyocytes, IgE-induced cardiomyocyte hypertrophy and hypertrophic marker expression were abolished by depleting FcεR1. In cultured rat CFs, IgE-induced CF activation and matrix protein production were also blocked by FcεR1 deficiency. RNA sequencing and signaling pathway analyses revealed that transforming growth factor-β may be a critical mediator, and blocking transforming growth factor-β indeed alleviated IgE-induced cardiomyocyte hypertrophy and cardiac fibroblast activation in vitro. Our findings suggest that IgE induction plays a causative role in pathological cardiac remodeling, at least partially via the activation of IgE-FcεR1 signaling in cardiomyocytes and CFs. Therapeutic strategies targeting the IgE-FcεR1 axis may be effective for managing IgE-mediated cardiac remodeling.

MicroRNA-99b-3p promotes angiotensin II-induced cardiac fibrosis in mice by targeting GSK-3β.

Cardiac fibrosis is a typical pathological change in various cardiovascular diseases. Although it has been recognized as a crucial risk factor responsible for heart failure, there is still a lack of effective treatment. Recent evidence shows that microRNAs (miRNAs) play an important role in the development of cardiac fibrosis and represent novel therapeutic targets. In this study we tried to identify the cardiac fibrosis-associated miRNA and elucidate its regulatory mechanisms in mice. Cardiac fibrosis was induced by infusion of angiotensin II (Ang II, 2 mg·kg-1·d-1) for 2 weeks via osmotic pumps. We showed that Ang II infusion induced cardiac disfunction and fibrosis accompanied by markedly increased expression level of miR-99b-3p in heart tissues. Upregulation of miR-99b-3p and fibrotic responses were also observed in cultured rat cardiac fibroblasts (CFs) treated with Ang II (100 nM) in vitro. Transfection with miR-99b-3p mimic resulted in the overproduction of fibronectin, collagen I, vimentin and α-SMA, and facilitated the proliferation and migration of CFs. On the contrary, transfection with specific miR-99b-3p inhibitor attenuated Ang II-induced fibrotic responses. Similarly, intravenous injection of specific miR-99b-3p antagomir could prevent Ang II-infused mice from cardiac dysfunction and fibrosis. We identified glycogen synthase kinase-3 beta (GSK-3β) as a direct target of miR-99b-3p. In CFs, miR-99b-3p mimic significantly reduced the expression of GSK-3β, leading to activation of its downstream profibrotic effector Smad3, whereas miR-99b-3p inhibitor caused anti-fibrotic effects. GSK-3β knockdown ameliorated the anti-fibrotic role of miR-99b-3p inhibitor. These results suggest that miR-99b-3p contributes to Ang II-induced cardiac fibrosis at least partially through GSK-3β. The modulation of miR-99b-3p may provide a new approach for tackling fibrosis-related cardiomyopathy.

Colchicine prevents atrial fibrillation promotion by inhibiting IL-1β-induced IL-6 release and atrial fibrosis in the rat sterile pericarditis model

A few clinical trials have recently reported the potential effect of colchicine in preventing post-operative atrial fibrillation (POAF) and early atrial fibrillation (AF) recurrence after catheter pulmonary vein isolation. However, the molecular mechanisms through which colchicine inhibits AF remain unclear. We aim to assess the anti-AF effect of colchicine in the rat sterile pericarditis (SP) model and to investigate its molecular mechanisms. SP was induced in Sprague-Dawley rats by the epicardial application of sterile talc. Treatment with colchicine or vehicle began 1 d before pericardiotomy. AF was induced by transesophageal burst pacing on day 3 after surgery. Treatment with colchicine reduced the duration of AF and the probability of induction of AF in SP rats. The dose of 0.5 mg kg−1·day−1 had the best effect. Such treatment also reduced neutrophil infiltration, the mRNA expression of IL-6, TGF-β, and TNF-α, atrial fibrosis, fibrosis related genes, and signal molecules (STAT3, P38, and AKT). Meanwhile, the release of IL-1β (4−24 h) and IL-6 (4−72 h) in atria after surgery was significantly inhibited by colchicine. In cultured rat cardiac fibroblasts, colchicine treatment inhibited IL-1β-induced expression of IL-6, which was accompanied by significantly decreased phosphorylation of P38, AKT, JNK, and NFκB. Interestingly, the supplementation of IL-6 abolished the anti-AF effect of colchicine in SP rats. Colchicine prevents AF in SP rats through the inhibition of IL-1β-induced IL-6 release and subsequent atrial fibrosis. However, further studies are required to investigate whether colchicine inhibits POAF through other mechanisms.

In Vivo and In Vitro Effects of Vasopressin V2 Receptor Antagonism on Myocardial Fibrosis in Rats

BackgroundMyocardial fibrosis is a major pathophysiologic substrate of heart failure with preserved ejection fraction. Vasopressin is an important therapeutic target in heart failure with preserved ejection fraction since it can modulate fluid balance, and based on a few studies, myocardial matrix deposition. Hence we examined the role of vasopressin antagonism in modulating myocardial matrix metabolism in vivo and in vitro. Materials and MethodsIn vivo studies utilized an established model of hyperhomocysteinemia-induced myocardial fibrosis in Sprague-Dawley rats combined with high salt diet; in vivo studies also utilized the same profibrotic stimuli of homocysteine and NaCl in cultured rat cardiac fibroblasts. ResultsHyperhomocysteinemia combined with high-salt diet promoted myocardial fibrosis, profibrotic and matrix gene expression and tolvaptan attenuated all these in vivo effects. In cultured cardiac fibroblasts, combined treatment with homocysteine and NaCl increased profibrotic and matrix gene expression and activation of PI3/Akt pathway; all these effects were attenuated by tolvaptan Vasopressin levels, gene expression and V2 receptor expression were increased in vivo and in vitro on exposure to profibrotic stimuli, and tolvaptan attenuated these in vivo and in vitro effects. ConclusionsAntagonism of vasopressin V2 receptor, via direct actions on cardiac fibroblast, attenuates myocardial matrix deposition.

Anti-proliferative effect of the extract of Guangzao (Fructus Choerospondiatis) on cultured rat cardiac fibroblasts

ObjectiveTo ascertain if total flavonoids of Guangzao (Fructus Choerospondiatis) (TFFC) extracted from Guangzao (Fructus Choerospondiatis) can inhibit angiotensin II-induced proliferation of cardiac fibroblasts (CFs). MethodsCFs were cultured by the differential attachment method. A model of cell proliferation was established by stimulation with Ang II. Cardiac fibroblasts growth was determined using a hemocytometer. Cell proliferation was detected by methyl thiazole tetrazolium. Lactate dehydrogenase activity was measured by chemical colorimetric method. ResultsProliferation of TFFC-treated (25, 50, 100 mg/L) fibroblasts was significantly less than that of cells in the angiotensin II group (P < 0.01), and TFFC inhibited proliferation in a dose-dependent manner. These inhibitory effects were partly blocked by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) and 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ). ConclusionTFFC inhibited angiotensin II-induced proliferation of cardiac fibroblasts via a mechanism that probably involves activation of the NO-cyclic guanosine monophosphate signaling pathway.

Peptidomics for Studying Limited Proteolysis.

Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions.

A Food-Derived Flavonoid Luteolin Protects against Angiotensin II-Induced Cardiac Remodeling.

Oxidative stress has been implicated in cardiac remodeling (cardiac fibrosis and hypertrophy), which impairs cardiac function and metabolism; therefore, it is anticipated antioxidative compounds will have protective properties against cardiac remodeling. Luteolin (3’,4’,5,7-tetrahydroxyflavone), a widely distributed flavonoid found in many herbal extracts including celery, green pepper, perilla leaves and seeds, and chamomile, is a known to be a potent antioxidant and was previously demonstrated to exert an antifibrotic effect in the lungs and the liver. In this study, we clearly demonstrate that oral pretreatment with the higher-luteolin diet (0.035% (wt/wt)) protected against cardiac fibrosis and hypertrophy as well as a hyperoxidative state in Ang II-infused rats. In cardiac tissue, increased gene expression levels of TGFβ1, CTGF, Nox2, Nox4, ANP, and BNP induced by Ang II were restored by oral pretreatment of this high-luteolin diet. In cultured rat cardiac fibroblasts, H2O2-induced TGFβ1 expression and the phosphorylation of JNK were suppressed by luteolin pretreatment. In conclusion, food-derived luteolin has protective actions against Ang II-induced cardiac remodeling, which could be mediated through attenuation of oxidative stress.

Abstract 220: miR-486 Mediates the Benefits of Exercise in Attenuating Cardiac Fibrosis

MicroRNAs (miRNAs, miRs), a novel group of small non-coding RNAs, play important roles in cardiac fibrosis. Exercise-induced physiological cardiac growth is associated with hypertrophy and proliferation of cardiomyocytes. In addition, exercise has been shown to inhibit cardiac fibrosis. However, relative little is known about whether exercise could attenuating cardiac fibrosis via targeting miRNA. miR-486 is a muscle enriched miRNAs, however, its role in heart is relative unclear. The current study aimed to investigate the role of miR-486 in exercise-induced cardiac growth in a 3-week swimming training murine model as well as in the function of cardiac fibroblasts and production of extracellular matrix (ECM) using neonatal rat cardiac fibroblasts in primary culture. Our data showed that exercised mice displayed increased about three-fold expression of miR-486 in hearts as measured by microarray analysis and qRT-PCRs. EdU proliferation assays demonstrated that miR-486 mimics decreased (5.90%±0.57% vs 4.02%±0.27% in nc-mimics vs miR-486-mimics, respectively), while miR-486 inhibitor increased the proliferation of cardiac fibroblasts in vitro (5.87%±0.16% vs 9.60%±0.58% in nc-inhibitor vs miR-486-inhibitor, respectively). Although downregulation of miR-486 had no regulatory effect on α-sma and collagen-1 gene expression in cardiac fibroblasts, overexpression of miR-486 significantly reduced the mRNA level of α-sma (1.01±0.08 vs 0.28±0.04 in nc-mimics vs miR-486-mimics, respectively) and collagen-1(1.02±0.12 vs 0.58±0.09 in nc-mimics vs miR-486-mimics, respectively), indicative of attenuated activation of fibroblasts and reduced production of ECM. These data reveal that miR-486 is essentially involved in the proliferation and activation of cardiac fibroblasts, and might be a key regulator mediating the benefit of exercise in preventing cardiac fibrosis.

Abstract 370: MicroRNA-19b Contributes to Cardiac Fibrosis

Fibrosis is one of the most important characteristics of cardiac remodeling during heart failure. The accumulation of extracellular matrix (ECM) within myocardium is the major feature of cardiac fibrosis. microRNA (miR)-19b, a key functional member of miR-19-72 cluster family, has been suggested to be involved in aging-induced heart failure through regulating ECM-related proteins, such as connective tissue growth factor (CTGF), thrombospondin-1 (TSP-1), collagen-1A1, and collagen-3A1. In the current study, we aimed to investigate the role of miR-19b in cardiac fibroblast function and ECM production using neonatal rat cardiac fibroblasts in primary culture. We found that overexpression of miR-19b increased, while inhibition of miR-19b decreased the proliferation and migration of cardiac fibroblasts, using Cell Counting Kit-8 (CCK-8) (0.660±0.019 vs 0.720±0.014 in nc-mimic and miR-19b mimic, 0.506±0.009 vs 0.454±0.008 in nc-inhibitor and miR-19b inhibitor, respectively), EdU incorporation assay (0.059±0.002 vs 0.096±0.006 in nc-mimic and miR-19b mimic, 0.059±0.006 vs 0.040±0.003 in nc-inhibitor and miR-19b inhibitor, respectively), and wound healing assay (0.528±0.024 vs 0.896±0.027 in nc-mimic and miR-19b mimic,0.520±0.028 vs 0.174±0.019 in nc-inhibitor and miR-19b inhibitor, respectively), respectively. Meanwhile, the inhibition of miR-19b downregulated the mRNA levels of α-SMA (0.556±0.048 vs 1.038±0.137 in nc-inhibitor and miR-19b inhibitor, respectively) and collagen-1 (1.023±0.116 vs 0.551±0.033 in nc-inhibitor and miR-19b inhibitor, respectively) in cardiac fibroblasts, indicating a reduction in fibroblast activation and ECM production via miR-19b inhibition. Furthermore, we found that PTEN was negatively regulated by miR-19b in cardiac fibroblasts using western blot analysis. PTEN, a well-known tumor-suppressor gene, has been known to inhibit cell proliferation and migration. However, it remains to be further clarified whether PTEN could mediate the effect of miR-19b in the proliferation, migration and activation of fibroblasts. These data might provide important evidence suggesting that miR-19b could be a potential therapeutic target for cardiac fibrosis.

Suppression of TGF-β1/Smad signaling pathway by sesamin contributes to the attenuation of myocardial fibrosis in spontaneously hypertensive rats.

This study investigated the effect of sesamin on myocardial fibrosis in spontaneously hypertensive rats (SHRs) and the possible mechanisms involved. Twenty-eight male SHRs were randomly allocated to SHR group, Ses160 group (sesamin 160 mg/kg), Ses80 group (sesamin 80 mg/kg) and Cap30 group (captopril 30 mg/kg). Seven male WKY rats were used as control. Sesamin and captopril were administered intragastrically for 12 weeks. Captopril significantly reduced systolic blood pressure and angiotensin II (Ang II) levels in SHRs, accompanied by a marked attenuation of left ventricular hypertrophy (LVH) and collagen deposition (P <0.05 or P <0.01). Though sesamin had no significant influence on Ang II levels, and the hypotensive effect was also significantly inferior to that of captopril (P <0.05 or P <0.01), however, the improvement of LVH and collagen deposition was similar to that in captopril group. Sesamin markedly reduced transforming growth factor-β1 (TGF-β1) content in cardiac tissues, with Smad3 phosphorylation decreased and Smad7 protein expression increased notably (P <0.05 or P <0.01). Protein expression of type I collagen and type III collagen, target genes of Smad3, was down-regulated markedly by sesamin (P <0.05 or P <0.01). In addition, sesamin significantly increased total antioxidant capacity and superoxide dismutase protein in cardiac tissues (P <0.05 or P <0.01), while the expression of NADPH oxidase subunit p47phox and malondialdehyde content were reduced markedly (P <0.05 or P <0.01). In vitro studies also demonstrated that sesamin was able to suppress Ang II induced phosphorylation of Smad3 and secretion of TGF-β1 and type I and type III collagen in cultured rat cardiac fibroblasts. These data suggest that sesamin is capable of attenuating hypertensive myocardial fibrosis through, at least partly, suppression of TGF-β1/Smad signaling pathway.

Resveratrol inhibits high glucose induced collagen upregulation in cardiac fibroblasts through regulating TGF-β1–Smad3 signaling pathway

Cardiac fibrosis is a common pathological process presented in a variety of diseases, including hypertension and diabetes. Cardiac fibroblasts (CFs) have been identified as the most important participants in the development of cardiac fibrosis. Exposure of cultured CFs to high glucose (HG) or angiotensin II (Ang II) resulted in increased collagen synthesis. Resveratrol (Res) is a natural polyphenol exhibiting anti-fibrosis effects in a number of different organs fibrosis process, whether Res can prevent HG and Ang II induced fibrosis response in CFs remains unclear. The aim of this work was to evaluate the effects of Res in HG and Ang II induced fibrosis response in CFs. We cultured rat CFs in either normal glucose (5.6mM) or HG (25mM) media in the presence of Res or not and the changes in collagens synthesis and TGF-β1 production were assessed by Real-time PCR, Western blotting, and enzyme linked immunosorbent assay (ELISA). Furthermore, normal and diabetic mice (induced by single dose of streptozotocin (100mg/kg) via tail vein) receiving Res (10mg/kg) were used to explore the effects of Res on cardiac fibrosis in vivo. Masson staining and immunohistochemistry were performed to visualize cardiac collagen deposition. Results indicate that CFs exposed to HG condition shows enhanced proliferation rate. Furthermore, in the presence of HG or Ang II, CFs exhibited increased collagens synthesis and TGF-β1 production. And these effects were abolished by Res intervention. In vivo results show that diabetic mice exhibit increased collagen deposition in the cardiac compared with the normal mice. And this change was prevented by the treatment of Res. These results suggest that Res possesses a potential antifibrogenic effect in hypertension and diabetes-related cardiac fibrosis. Moreover, the action mechanism is probably associated with its ability to reduce TGF-β1 content in CFs.

Neuregulin-1 antagonizes myocardial fibrosis and diastolic dysfunction in angiotensin-II treated mice

Introduction: The neuregulin-1 (NRG-1)/ErbB system emerges as an endothelium-controlled cardioprotective system, indispensable for cardiac development and function. Treatment with NRG-1 of animal models with heart failure has been shown to impede LV remodeling and to reduce mortality. Phase III clinical trials in patients are currently performed. The underlying mechanisms of NRG-1 in heart failure are however incompletely understood. Here, we tested whether NRG-1 interferes with the cardiac actions of angiotensin II. Methods and results: Male C57Bl/6 mice (n=50) treated with angiotensin II (Ang II, osmotic mini-pumps, 1000 ng kg-1 day-1) or saline were daily treated with rhNRG-1 (20 μg.kg-1.day-1, i.p. injection) or placebo during 4 weeks. Mice were then analyzed by transthoracic echocardiography and invasive hemodynamic recordings, and sacrificed for (immuno-) histochemical and molecular myocardial analyses (collagen fraction, cross-sectional myocyte area, BNP mRNA, procollagen type III mRNA). In absence of NRG-1, Ang II induced arterial hypertension, concentric LV hypertrophy, interstitial myocardial fibrosis, and LV diastolic failure (increased slope of the end-diastolic pressure-volume relation, increased LV end diastolic pressure, concomitant with a reduced LV stroke volume and increased lung volumes). LV ejection fraction and hemodynamic parameters of LV systolic function remained normal. In presence of NRG-1, Ang II-induced arterial hypertension remained unaffected, whereas all echocardiographic, hemodynamic, histochemical and molecular parameters of LV hypertrophy, myocardial fibrosis and LV diastolic failure significantly decreased towards control levels. Subsequent experiments with cultured rat cardiac fibroblasts and myocytes (both expressing NRG-1 responsive-ErbB2/4 receptors) confirmed that NRG-1 (20 ng.ml-1, 15 min pre-treatment) directly attenuated Ang II-induced myocyte hypertrophy and fibroblast collagen synthesis. Also, NRG-1 significantly attenuated Ang II-induced phosphorylation of p38, extracellular signal-regulated kinases 1/2 (ERK1/2), and protein kinase C, pathways of Ang II mediated fibrosis. Conclusion: NRG-1 prevents Ang II-induced LV concentric remodeling and diastolic dysfunction, independently of effects on arterial blood pressure. These effects are explained by direct actions of NRG-1 on Ang II signaling in cardiomyocytes and fibroblasts. These effects help explaining the beneficial actions of NRG-1 in heart failure, and suggest that NRG-1 may provide a new treatment strategy in hypertensive heart disease.

TGF-β1 prevents simulated ischemia/reperfusion-induced cardiac fibroblast apoptosis by activation of both canonical and non-canonical signaling pathways

Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia–reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.

N-Acetyl–seryl–aspartyl–lysyl–proline inhibits ET-1-induced collagen production by preserving Src homology 2-containing protein tyrosine phosphatase-2 activity in cardiac fibroblasts

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits endothelin-1 (ET-1)-induced activation of p44/42 mitogen-activated protein kinase (p44/42 MAPK) and collagen production in cultured rat cardiac fibroblasts (RCFs). However, we do not know whether its inhibitory effect on p44/42 MAPK is due to the altered activity of protein tyrosine phosphatases (PTPs), which in turn downregulate the p44/42 MAPK signaling pathway. The activity of Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2) is downregulated by ET-1 in RCFs; thus, we hypothesized that Ac-SDKP inhibits ET-1-stimulated collagen production in part by preserving SHP-2 activity and thereby inhibiting p44/42 MAPK phosphorylation. When we stimulated RCFs with ET-1 in the presence or absence of Ac-SDKP, we found that (a) PTP activity was reduced by ET-1 and (b) this effect was counteracted by Ac-SDKP in a dose-dependent fashion. Next, we extracted SHP-2 from RCF lysates by immunoprecipitation and determined that (a) ET-1 inhibited SHP-2 by 40% and (b) this effect was prevented by Ac-SDKP. However, Ac-SDKP failed to inhibit ET-1-induced p44/42 MAPK phosphorylation in RCFs treated with SHP-2 short hairpin RNA (shRNA); in contrast, in cells transfected with control shRNA, Ac-SDKP's inhibitory effect on ET-1-induced p44/42 MAPK activation remained intact. Moreover, the inhibitory effect of Ac-SDKP on ET-1-stimulated collagen production was blunted in cells treated with the SHP-1/2 inhibitor NSC-87877. Thus, we concluded that the inhibitory effect of Ac-SDKP on ET-1-stimulated collagen production by RCFs is mediated in part by preserving SHP-2 activity and thereby preventing p44/42 MAPK activation. Ac-SDKP or its analogs could represent a new therapeutic tool to treat fibrotic diseases in the cardiovascular system.

Total Flavonoids of Fructus Chorspondiatis inhibits collagen synthesis of cultured rat cardiac fibroblasts induced by angiotensin II: Correlated with NO/cGMP signaling pathway

AimTo investigate the molecular mechanism of Total Flavonoids of Fructus Chorspondiatis (TFFC) on preventing cardiac fibroblasts collagen synthesis induced by angiotensin II. MethodsCollagen synthesis was determined by measuring 3H-proline incorporation cardiac fibroblasts and hydroxyproline content in the culture mediums. The expression of collagen types I and III mRNA and protein was measured by RT–PCR and western blot, respectively. NO level in the culture medium was measured by the Griess reagent. NOS level in the culture medium was measured by chemical colorimetric method. The cellular concentration of cyclic GMP (cGMP) was measured by radioimmunoassay. ResultsTFFC (25, 50, and 100mg/L) inhibited collagen synthesis in cardiac fibroblasts in a dose-dependent manner compared with angiotensin II group (P<0.01), and the inhibitory effects were blocked by pretreatment with NG-nitro-l-arginine methyl ester (L-NAME) and 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ). TFFC increased nitric oxide (NO) and nitric oxide synthase (NOS) levels in the culture medium, increased intracellular cGMP level in cardiac fibroblasts, decreased collagen types I and III protein level in cardiac fibroblasts. The mRNA expression of collagen type I and III was suppressed by TFFC. ConclusionsThese results suggested that TFFC inhibited collagen synthesis induced by angiotensin II in cardiac fibroblasts, and the inhibitory effect might associate with the activation of the NO/cGMP signaling pathway.