Ruizhen Chen

Astragaloside IV attenuates myocardial fibrosis by inhibiting TGF-β1 signaling in coxsackievirus B3-induced cardiomyopathy

Myocardial fibrosis plays an important role in coxsackievirus B3 (CVB3) induced dilated cardiomyopathy. Excessive transforming growth factor (TGF)-β1 contributes to a pathologic excess of tissue fibrosis. We investigated the effect of astragaloside IV on myocardial fibrosis in CVB3-induced dilated cardiomyopathy. BALB/c mice were inoculated with CVB3 to induce acute viral myocarditis on day 7 (acute VMC group), monthly for 3 months to induce chronic myocarditis (chronic VMC group), and monthly for 9 months to induce dilated cardiomyopathy (DCM group). The same method was used for the DCM+Astra group as that of the DCM group, but former group was given with astragaloside IV-containing drinking water. Compared to DCM group, astragaloside IV treatment significantly increased the survival rate. Histological findings and the collagen volume fraction showed that astragaloside IV decreased fibrosis in heart tissues. Astragaloside IV decreased the level of the serum carboxy-terminal propeptide of procollagen type I (PICP) and the ratio of PICP/ N-terminal type I procollagen propeptide (PINP). Ameliorated myocardial fibrosis was consistent with the downregulated expression of TGF-β1 and its downstream pSmad2/3 and Smad4 in the myocardium of the DCM+Astra group compared to the DCM group. The level of type I collagen was lower in the DCM+Astra group than the DCM group. The same effect was found in the in vitro study. These findings showed that astragaloside IV had a potent preventive effect on myocardial fibrosis in CVB3-induced dilated cardiomyopathy that might be due to downregulation of TGF-β1-Smad signaling.

The role of Th17 cells and regulatory T cells in Coxsackievirus B3-induced myocarditis.

IL-17-producing (Th17) and regulatory T (Treg) cells have been well established in the pathogenesis of many inflammatory diseases. To assess whether Th17 and Treg were altered in acute virus-induced myocarditis (AVMC) mice, we assessed Th17/Treg functions on different levels in AVMC. It was shown that the expression of splenic Th17 cells and Th17-related cytokines (IL-17A, IL-21) markedly increased. Interestingly, the expression of splenic Treg cells and Treg-related cytokines (TGF-β, IL-10) also significantly increased. Using neutralization of IL-17 in the AVMC, we found that Treg cells roughly decreased compared with isotype control mice. However, T cells and perforin dramatically increased, followed by a marked reduction in CVB3 replication. The results suggested that Th17 cells possibly contributed to viral replication through the action of Treg cells in mediating T cells and perforin response in AVMC.

Effect of astragaloside on cardiomyocyte apoptosis in murine coxsackievirus B3 myocarditis

Astragaloside is the major component of Astragalus membranaceus, one of the Chinese medical herbs, and has several pharmacological actions on cardiovascular system, including positive inotropic, anti-arrhythmia and anti-oxidant activities. We have investigated the effect of astragaloside on cardiomyocyte apoptosis and expression of apoptosis-associated genes in mice with coxsackievirus B3 (CVB3)-induced myocarditis, the former of which has been detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay and the latter determined by using a focused microarray. Results showed that cardiomyocyte apoptosis and expression of various apoptosis associated genes are inhibited after treatment with astragaloside. The anti-apoptotic activity of astragaloside may contribute to the improvement of clinical outcomes in treating myocarditis with pharmaceutics of Astragalus membranaceus.

In Vivo Delivery of Adenoviral Vector Containing Interleukin-17 Receptor A Reduces Cardiac Remodeling and Improves Myocardial Function in Viral Myocarditis Leading to Dilated Cardiomyopathy

Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM.

Preexposure to PM2.5 exacerbates acute viral myocarditis associated with Th17 cell.

BACKGROUND It is increasingly recognized that exposure to ambient fine particles (PM(2.5)) is a risk factor for the development of cardiovascular events. This study was to explore the link between PM(2.5) exposure and viral myocarditis in the functional mechanism of Th17 cells. METHODS Male BALB/c mice were administered an intratracheal (i.t.) instillation of 10 mg/kg b.w. PM(2.5) particles. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100 μl of coxsackievirus B3 (CVB3) diluted in Eagles minimal essential medium (EMEM). Seven days after the treatment, pulmonary and cardiac tissues were examined. RESULTS The results showed that preexposure to PM(2.5) increased the cardiac and pulmonary injuries and viral replication in the heart of CVB3-infected mice along with an increase in CD4(+) IL-17(+) cells in the spleen and heart. The mRNA expressions of interleukin-17A (IL-17A), perforin, transforming growth factor-β (TGF-β) and RORγt were up-regulated in PM(2.5)-pretreated mice than that in the virus-treated mice. Additionally, compared to virus-treated mice, the cardiac protein expressions of IL-17A and matrix metalloproteinases-2 (MMP-2) were increased, but interferon-γ (IFN-γ) and metalloproteinases-1 (TIMP-1) were decreased in PM(2.5)-pretreated mice. Interestingly, PM(2.5) caused IFN-γ decreased, whereas CVB3 caused a dramatic increase in IFN-γ. Subsequently, preexposure to PM(2.5) induced a slight increase of IFN-γ in the sera of CVB3-infected mice. CONCLUSIONS These results demonstrated that PM(2.5) exposure exacerbated virus-induced myocarditis possibly through the increase in Th17-mediated viral replication, perforin response and imbalance of MMP-2/TIMP-1. These findings provided supportive evidence for the epidemiological research that ambient particles could increase the occurrence and development of cardiovascular diseases.

Coxsackievirus B3-induced calpain activation facilitates the progeny virus replication via a likely mechanism related with both autophagy enhancement and apoptosis inhibition in the early phase of infection: an in vitro study in H9c2 cells.

Calpain is a family of neutral cysteine proteinase involved in many physiological and pathological processes including virus replication, autophagy and apoptosis. Previous study has indicated the involvement of calpain in pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis. Besides, many studies demonstrated that host cell autophagy and apoptosis mechanisms participate in virus life cycle. However, role of calpain in CVB3 replication via autophagy/apoptosis mechanisms has not been reported, which was discussed here in H9c2 cardiomyocytes. The data demonstrated that calpain was activated following CVB3 infection. Calpain inhibition decreased autophagy, indicating role of calpain in enhancing autophagy during CVB3 infection. Both calpain activity and autophagy were involved in facilitating CVB3 replication demonstrated by virus titer and CVB3 capsid protein VP1 expression alterations resulting from calpain inhibitor ALLN and autophagy inhibitor 3MA intervention. We also found that both calpain activity and autophagy suppressed caspase3 activity and host cell apoptosis 5-10h post-infection (p.i.). In summary, the present study shows that CVB3 infection of H9c2 cells hinders caspase3 activity provocation and cell apoptosis at least in the early phase of infection (5-10h p.i.) via calpain-induced autophagy enhancement, which might be a mechanism facilitating CVB3 replication in host cells.

Blockade of interleukin‐17A protects against coxsackievirus B3‐induced myocarditis by increasing COX‐2/PGE2 production in the heart

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2.

Coxsackievirus B3 infection promotes generation of myeloid dendritic cells from bone marrow and accumulation in the myocardium

Myocarditis is associated with increased number of CD4+ T cells in the myocardium after coxsackievirus B3 (CVB3) infection. Previous studies show that CD11c+ myeloid dendritic cells (mDC) loaded with myosin could induce myocarditis. This study aims to investigate the generation and accumulation of mDC in CVB3-induced myocarditis. The presence of mDC in myocardium was detected by immunohistochemisty. Bone marrow-derived mDC were generated from uninfected and CVB3-infected mice. The percentage of CD11c+ mDC on cultured cells and mean fluorescence index (MFI) of double positive cells (CD11c+CD40+, CD11c+CD80+) were measured by flow cytometry. The expression of chemokine receptors (CCR5, CCR7) on mDC and chemokines (CCL4, CCL19) in the myocardium was detected. The migration of mDC in response to CCL4 or CCL19 was measured by chemotaxis assay. Mature mDC were elevated in the myocardium from CVB3-infected mice. The percentage of mDC generated from CVB3-infected group was increased. The MFI of CD11c+CD40+ and CD11c+CD80+ was increased in CVB3-infected group. The mDC showed a down-regulation of CCR5 and unaffected CCR7 mRAN levels associated with elevated CCL4 and CCL19 in the myocardium in CVB3-infected group. Numbers of migrating bone marrow-derived mDC from CVB3-infected mice were increased in vitro. We conclude that CVB3 infection induced the greater generation of mDC from bone marrow and accumulation of mature mDC in myocardial tissues. This migration was associated with increased levels of both CCL4 and CCL19 in the heart tissue. These suggest that blocking the migration of mDC may provide a novel therapy for myocarditis.

Regulation of p53 by Jagged1 Contributes to Angiotensin II-Induced Impairment of Myocardial Angiogenesis

Angiotensin II (AngII) is a major contributor to the development of heart failure, however, the molecular and cellular mechanisms still remain elucidative. Inadequate angiogenesis in myocardium leads to transition from cardiac hypertrophy to dysfunction, this study was therefore conducted to examine the effects of AngII on myocardial angiogenesis and the underlying mechanisms. AngII treatment significantly impaired angiogenetic responses, which were determined by counting the capillaries either in matrigel formed by cultured cardiac microvascular endothelial cells (CMVECs) or in myocardium of mice and by measuring the in vitro and in vivo production of VEGF proteins, and stimulated accumulation and phosphorylation of cytosolic p53 which led to increases in phosphorylated p53 and decreases of hypoxia inducible factor (Hif-1) in nucleus. All of these cellular and molecular events induced by AngII in CEMCs and hearts of mice were largely reduced by a p53 inhibitor, pifithrin-α (PFT-α). Interestingly, AngII stimulated the upregulation of Jagged1, a ligand of Notch, but it didn’t affect the expression of Delta-like 4 (Dll-4), another ligand of Notch. Inhibition of p53 by PFT-α partly abolished this effect of AngII. Further experiments showed that knockdown ofJagged1 by addition of siRNA to cultured CMVECs dramatically declined AngII-stimulated accumulation and phosphorylation of p53 in cytosol, upregulation of phosphorylated p53 and downregulation of Hif-1 expression in nucleus, decrease of VEGF production and impairment of capillary-like tube formation by the cells. Our data collectively suggest that AngII impairs myocardial angiogenetic responses through p53-dependent downregulation of Hif-1 which is regulated by Jagged1/Notch1 signaling.

Impaired cardiac microvascular endothelial cells function induced by Coxsackievirus B3 infection and its potential role in cardiac fibrosis.

CVB3 virus tropism and tissue access are modulated by cardiac microvascular endothelial cells (CMVECs) in the context of microvasculature. This study was designed to examine biological behaviors of CMVECs following CVB3 infection and its possible effects on cardiac remodeling. Data demonstrated that CVB3 increased caspase-3 activities, Bax/Bcl-2 protein ratio and TGF-β1 levels in CMVECs, accompanying with elevated microvascular permeability. Double immunofluorescence revealed co-localization of endothelial markers (CD31 and VE-cadherin) and mesenchymal markers (FSP1 and αSMA) in infected CMVECs. Western blot demonstrated that CVB3 significantly decreased the expression of endothelial markers and increased the expression of mesenchymal markers, which were reversed by SB431542 (inhibitor of TGF-β1), indicating that endothelial-to-mesenchymal transition following CVB3 infection was probably induced by CMVECs-derived TGF-β1. Excess extracellular matrix was produced by myocardial cells incubated with supernatants of infected CMVECs. Our results displayed that CVB3 induced notable biological changes of CMVECs, which may contribute to cardiac fibrosis.