Tumor Necrosis Factor-like Weak Inducer Of Apoptosis Promotes Expression Of Matrix Metalloproteinase 2 And Collagen Ⅰ In Rat Cardiac Fibroblasts Via The ERK1/2 Pathway

BACKGROUND:With the improvement of living standard and the economic development, the incidence of cardiovascular disease shows an uptrend year by year. Data suggest that cardiovascular disease is the main cause of globally chronic disease deaths and hypertension is closely related to the occurrence of stroke and myocardial infarction. As one of the hypertensive target organic injury, left ventricular hypertrophy is an independent risk factor of several cardiovascular diseases, which may cause poor prognosis including arrhythmia, heart failure, sudden death. Myocardial fibrosis plays a significant role during the process of left ventricular hypertrophy from compensation to decompensation or even heart failure.One most significant characteristic of the process of myocardial fibrosis is the imbalance in activity between synthesis and degradation of myocardial interstitial collagen. The tiny disorder of all kinds of collagen’s Proportion and structure can negatively affect the stiffness of left ventricular, which will further influence the left ventricular diastolic function. The MMPs in myocardial tissue are mainly secreted by myocardial fibroblasts, which mediate the degradation of myocardial extracellular matrix, especially the collagen protein. The increasing activity and expression of MMPs, as well as the imbalance between MMPs and the inhibitor TIMPs, play a crucial role in the process of myocardial fibrosis. There is evidence that MMP2 can accelerate the development of myocardial fibrosis by degrading collagen protein, disassembling the cell to cell junctions, hydrolyzing and activating regulating factors.Besides mechanical stress and neurohumoral regulation, inflammation plays an important role in the process of myocardial fibrosis. Inflammatory factors (TNF) superfamily has effects of anti-tumor and inflammation promotion, and also gets involved in the occurrence and development of myocardial fibrosis. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of TNF superfamily and expressed in various cells, tissues and organs. TWEAK participates in the occurrence and development of cardiovascular diseases, such as myocardial infarction, dilated cardiomyopathy, hypertension by promoting cardiomyocyte hypertrophy, inducing endothelial cell injury, accelerating vascular smooth muscle cell migration, altering myocardial extracellular matrix components. TWEAK is able to facilitate cardiac fibroblasts proliferation and collagen synthesis, which is mediated by P38MAPK signaling pathway. P38MAPK, as well as ERK1/2 pathway, JNK pathway, P38 pathway and ERK5 pathway, exists in the eukaryotic cells widely. In addition, it is verified that ERK1/2 is associated to cardiomyocyte hypertrophy and myocardial fibrosis as well. Thus, this study aims to explore whether TWEAK controls the expression of type Ⅰ collagen in rats’ CFs and MMP2 by EKR1/2 pathway, and elucidate the mechanism of TWEAK promoting myocardial fibrosis further.Objective:To elucidate the mechanism of tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-mediated expression of MMP2 and collagen Ⅰ in cultured neonatal rat cardiac fibroblasts (CFs)Methods:1. CFs were isolated and cultured using trypsin enzyme digestion technique, and identified using cellular immunofluorescence technique.2. The interventional concentration and duration of rhTWEAK and inhibitor PD98059 of ERK1/2 pathway in CFs was optimized according to the expression level of phosphorylated ERK1/2 (p-ERK1/2) using Western blot.3. The expression level of mRNA and protein of MMP2 and collagen Ⅰ was investigated subsequently by real-time PCR and western blot, respectively.4. Methyl Thiazolyl Tetrazolium (MMT) was applied to determine cell proliferation with different treatments.Results:1. CFs were in fusiform shap, or irregularly polygonal shape with reansparent cytoplasm and ellipsoidal nucleus. Cell immunofluorescence staining showed strongly positive vimentin.2. The expression level of ERK protein in CFs stimulated with TWEAK and PD98059:100μg/L TWEAK significantly upregulated the expression of p-ERK1/2 protein, and showed a time-dependent as well as a concentration-dependent effect. PD98059 inhibited the expression of p-ERK1/2.3. TWEAK significantly improved the expression of MMP2 at transcriptional and translational level. PD98059 inhibited the expression of MMP2 by blocking ERK1/2 signaling pathway.4. Collagen I were significantly increased with TWEAK stimulation at transcriptional and translational level, which could be suppressed by inhibitor PD98059.5. TWEAK significantly improved the proliferation of CFs. PD98059 could restrain this effect by blocking ERK1/2 signaling pathway..Conclusions:TWEAK promotes the expression of MMP2 and collagen I in CF through ERK 1/2 signaling pathway.