| Rheumatic fever and rheumatic heart disease (RHD) were serious diseases burden worldwide and its pathogenesis was somewhat unclear. In traditional theory, droup A streptococcal infection and patiets'autoimmune reaction were taken as the pathogenesis of rheumatic heart disease and rheumatic valvulitis because of the shared antigen epitopes between group A streptococcal M peptides and valves components. Pathological locations were mainly distributed among heart valve, myocardium and joints whose extracellusr matrix (ECM) endure highest and most frequent mechanical stress. And the proceeding of rheumatic heart valvular disease (RHVD) was closely related with mechanical strain. Tenascin-C (TN-C), a hexameric extracellular matrix glycoprotein, is a major component of extracellular matrix (ECM). TN-C was reported present during and played important role in ECM remodeling, which was also identified to be among one of the important molecules directly regulated by mechanical stress. Therefore, our assay was focused on TN-C expression and regulation of TN-C among RHVD pathogenesis.Aim: To evaluate concentration of TN-C within sere of patients with RHVD. To culture valves interstitial cells in vitro. To study TN-C expression with designed strain-producing instrument under mechanical stress and judge pathogenesis of RHVD with new direction. Our research would supply a new idea on prevention of RHVD and understanding interaction between valves ECM and surrounding cells, which may help us to construct tissue-engineered biological heart valves with self-repair function.Methods: Sera from the normal subjects, patients with RHVD or congenital valvular malformation and IFN-γ,TNF-αand TN-C concentrations were measured. Aortic valves were obtained in sterile conditions after operation and their endothelial layer was deprived carefully. The valves interstitial cells were isolated by collagenase I and then cultured in vitro. Cellularα-smooth muscle actin was detected by confocal staining technique. The expressed IFN-γand TNF-αreceptors were analyzed by flow cytometry. Equi-biaxial cyclic strain production device was developed. TN-C mRNA was measured by quantitative real-time RT-PCR after cytokines and mechanical application. Signal transduction pathways induced by cytokines and mechanical stress were assessed after usage with different protein kinase inhibitors.Results: Concentrations of IFN-γ, TNF-αand TN-C of patients with RHVD were significantly higher than those of healthy volunteers or with congenital valve malformation. Valves interstitial cells culture system were established successfully and proved to be myofibroblast. More IFN-γreceptors and TNF receptors were expressed on rheumatic aortic valves interstitial cells than non-rheumatic ones and their expression were patients'sera dependent. Equi-biaxial cyclic strain production device was developed successfully. Antibodies neutralizing IFN-γor TNF-αcould attenuate patients'sera induced TN-C transcription by isolated rheumatic aortic valves interstitial cells. Combined with cyclic strain, TNF-αand IFN-γinduced TN-C transcription through RhoA/ROCK signaling pathway. At the same time, p38 mitogen-activated protein kinase was involved in TNF-αand IFN-γinduced TN-C transcription. TNF-αalso increased TN-C mRNA level by additional PKC and ERK 1/2 activation.Conclusion: 1. TN-C could be taken as sera active biomarker of RHVD. 2. Cellular phenotype was altered under rheumatic conditions. 3. TN-C was constitutively expressed by non-rheumatic valves interstitial cells. Th1-like cytokines IFN-γ, TNF-αand mechanical stress had synergistic effects on TN-C transcription and expression through the common and distinct signal transduction pathways in RHVD pathogenesis. Our finding revealed a new insight into ECM remodeling during RHVD pathogenesis and new mechanisms involved in the clinical anti-IFN-γand anti-TNF-αtherapy. |
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