Potential Role Of Cardiac Microvascular Endothelial Cells On Cardiac Fibrosis In Viral Cardiomyopathy And Its Underlying Mechanism

Viral myocarditis (VMC) is the inflammation of cardiac muscles due to viral infection. Coxsackievirus B3(CVB3) infection presents to be a significant cause of myocarditis. Most of patients could completely recovery from VMC, whereas there are still small portion (10%-15%) of patients have residual myocardial injury and develop to dilated cardiomyopathy (DCM). Among idiopathic DCM, evidence demonstrated that virus RNA could be detected in60%of DCM patients, indicating that virus infection is crucial in viral cardiomyopathy pathogenesis.Cardiac fibrosis is associated with an adverse prognosis and impaired response to therapeutic interventions in patients with VMC and DCM. Activated cardiac fibroblasts are at the heart of cardiac fibrosis and play a key role in regulating normal cardiac function and adverse myocardial remodeling. For many years, a common notion considered that activated fibroblasts generated primary from resident fibroblasts when injuries attack. However, recent findings have demonstrated that bone morrow stem cells are found to contribute to the pool of activated fibroblasts and endothelial cells via endothelial-to-mesenchymal transition (Endo-MT) can also be in the recruitment of activated fibroblasts and plays an important role of organ fibrosis. Endo-MT is a process by which endothelial cells disaggregate, change shapes and migrate into the surrounding tissue, accompanied by loss of endothelial cell markers like CD31and vascular endothelial cadherin (VE-cadherin), and up-regulation of mesenchymal markers like smooth muscle actin (aSMA) and fibroblast specific protein-1(FSP1).Coxsackievirus B3(CVB3) spreads mostly by respiratory and digestive channels, which invades into the circulation by primary viral replication at the portal of entry and viral release into bloodstream, leading to viremia. Then viruses in the circulation must either circumvent or infect cardiac microvascular endothelial cells (CMVECs) in order to reach target myocardial cells. Combined with emerging data showing that endothelial cells (ECs) appear to be crucial in modulating virus tropism and tissue access in murine studies, CMVECs are regarded as an important and last protective barrier for cardiomyocytes to fight against foreign triggers intrusion. Moreover, CMVECs are also regarded as one of important sources of the cytokines to maintain balance within cardiac microenvironment under dangerous condition.Previous studies were mostly focused on the changes of cardiac myocytes, whereas evidence on the CMVECs was scarce. Moreover, the role of infected CMVECs in the progression of viral cardiomyopathy remains unclear. Given the facts that fibrotic disorder is a critical process in viral cardiomyopathy pathogenesis and Endo-MT is one of the possible causative mechanisms of excessive collagen disposition, the purpose of this study was to clarify the potential role of infected CMVECs on viral cardiomyopathy and its underlying mechanism.Part1:Contribution of endothelial-to-mesenchymal transition to cardiac remodeling in human idiopathic dilated cardiomyopathy[Background] Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation and cardiac fibrosis. Emerging evidence has indicated that endothelial-to-mesenchymal transition (Endo-MT) is a crucial event during murine organ fibrosis. This study was performed to clarify whether human cardiac endothelial cells undergo Endo-MT and its underlying possible mechanism in human DCM heart samples.[Methods] The presence of endothelial markers (CD31and VE-cadherin) and mesenchymal markers (a-SMA and FSP1) was performed using immunohistochemistry in surgical myocardial specimen of40patients with DCM. Data were analyzed to evaluate the correlation between these markers and clinical features of patients. Coexpression of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Protein levels of Wnt, β-catenin and snail were evaluated using western blot.[Results] Compared to normal cardiac sections, all patients showed decrease of endothelial markers (CD31, P<0.01; VE-cadherin, P<0.01) and increase of mesenchymal markers (α-SMA, P<0.01; VE-cadherin, P<0.01). The labeling of endothelial markers demonstrated overt negative correlation with patients’LVEDD (CD31, r=-0.82, P<0.01; VE-cadherin, r=-0.73, P<0.01); while mesenchymal markers labeling demonstrated a positive relationship with LVEDD (α-SMA, r=0.65, P<0.01; FSP1, r=0.53, P<0.01). Western blot analysis showed that proteins levels of Wnt signaling were increased in DCM samples. [Conclusion] These results provided new insights in human level that endothelial cells contributed to the pool of myofibroblasts and Endo-MT was associated with human ventricular enlargement. Targeting endothelial derived mesenchymal cells from its underlying mechanism might be beneficial in the prevention of DCM progression. Part2:Impaired cardiac microvascular endothelial cells function induced by Coxsackievirus B3infection and its potential role in cardiac fibrosis[Background] Coxsackievirus B3(CVB3) infection presents to be a significant cause of myocarditis. Virus tropism and tissue access are modulated by cardiac microvascular endothelial cells (CMVECs) in the context of microvasculature. Data on the permission of CMVECs to CVB3infection was scanty. This study was designed to examine the potential of CVB3to infect CMVECs, the following pathophysiological changes, and the possible consequences of such infection in relation to chronic myocardial inflammation and cardiac remodeling.[Methods and Results] In the beginning, increased caspase3activities (P<0.01) and elevated Bax/Bcl-2protein ratio (P<0.05) were revealed in infected CMVECs, accompanied with decreased CMVECs growth rate, indicating that damaged biological events in CMVECs were induced by CVB3. Higher microvascular permeability was then verified by indirect evidence of In vitro vascular permeability assay and direct evidence of transmission electron microscopy, suggesting impaired endothelial cell line integrity after infection. Moreover, results of double immunofluorescence in vivo and in vitro indicated that the TGF-β1derived from infected CMVECs may induce endothelial-to-mesenchymal transition. Additionally, excess extracellular matrix was produced by myocardial cells incubated with supernatants of infected CMVECs.[Conclusions] These results demonstrated that endothelial cells of cardiac microvascular vessels can harbor a persistent CVB3infection, resulting in quantitative modification of biological characteristics themselves and cytokine production. These factors, may in turn contribute to the pathophysiological response and cardiac fibrosis during acute and chronic viral myocarditis pathogenesis. Part3:Contribution of endothelial-to-mesenchymal transition to viral myocarditis and its possible regulating factor[Background] Cardiac fibrosis is associated with an adverse prognosis and impaired response to therapeutic interventions in patients with VMC. Activated cardiac fibroblasts are at the heart of cardiac fibrosis and play a key role in regulating normal cardiac function and adverse myocardial remodeling. Evidence demonstrated that endothelial cells could contribute to the pool of cardiac myofiblasts via Endo-MT. This study was designed to evaluate the contribution of Endo-MT to viral myocarditis using murine model and try to find the possible regulating factor.[Methods] Viral myocarditis mice model was made and fourteen days after CVB3infection, echocardiography, Sirius Red staining and hematoxylin-eosin (HE) were performed to describe the cardiac function and histological characteristics. Colocation of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Western blot and double immunofluorescence were performed to quantify the expression of endothelial and mesenchymal markers in infected CMVECs. qPCR was used to detect the translating factors of Endo-MT in CMVECs.[Results] After fourteen days infection, CVB3-infected mice showed ventricular dilation and declined cardiac ejection fraction. HE and SR staining demonstrated inflammatory infiltrates, myofibrils disarrangement and replacement of fibrosis. Data depicted that endothelial and mesenchymal markers were co-localized exclusive in myocarditis specimen. Moreover, results of double immunofluorescence and western blot in vitro indicated that the TGF-β1derived from infected CMVECs may induce endothelial-to-mesenchymal transition.[Conclusions] These results discover a new underlying mechanism during myocardial fibrosis of viral cardiomyopathy that Endo-MT contributed to myofibroblasts recruitment in response to viral infection. Furthermore, TGF-β1derived from infected CMVECs was found to take responsibility of this process. Part4:BMP7counteracts TGF-β1induced Endo-MT in viral cardiomyopathy and its potential mechanism[Background] Cardiac fibrosis is the most important histological characteristics of viral cardiomyopathy and is associated with poor prognosis and therapeutic interventions. BMP7is a member of TGF-β superfamily and was reported to counteract the effects of TGF-β1. This study was designed to evaluate whether BMP7administration could reduce fibrosis induced by CVB3infection and its potential mechanism.[Methods] Viral myocarditis mice model was made and BMP7was administrated to infected mice. Fourteen days after CVB3infection, echocardiography, Sirius Red staining and hematoxylin-eosin (HE) were performed to describe the cardiac function and histological characteristics. Colocation of endothelial markers and mesenchymal markers were identified using confocal immunofluorescence staining. Western blot was performed to evaluate the TGF-β1/smad and Wnt/β-catenin signaling pathway.[Results] A mice model of CVB3myocarditis was made and BMP7was administrated to CVB3-infected mice. Histological data demonstrated that BMP7administration reduced inflammatory cells accumulation and cardiac fibrosis in response to CVB3challenge. Echo data described cardiac dysfunction was recovered after BMP7intervention. Double labeling of endothelial and mesenchymal markers showed BMP7-treated mice had significantly reduced the double-positive cells. Western blot described that TGF-β1/smad and Wnt/β-catenin signaling pathway was involved in this pathogenesis.[Conclusions] BMP7counteracts TGF-β1induced Endo-MT in viral cardiomyopathy through both TGF-β1/smad and Wnt/β-catenin signaling pathway.