Low Molecular Weight Hyaluronic Acid Regulates The Nuclear Translocation Of S100A4 To Promote Myocardial Fibrosis Via CD44

BackgroundMyocardial fibrosis(MF)is an important pathological feature of cardiovascular diseases and cardiac aging,which can eventually lead to a series of serious pathological damage such as cardiac insufficiency,and is also one of the main causes of death of cardiovascular diseases.Therefore,it is urgent to find a new target to inhibit myocardial fibrosis and reverse ventricular remodeling.Low molecular weight hyaluronic acid(LMW-HA)is not only the outcome of fibrosis,but also can be used as a driver to stimulate fibrosis.However,the specific mechanism of action of LMW-HA leading to myocardial fibrosis remains unclear.Objective This study aimed to investigate the promotion effect of low molecular weight hyaluronic acid(LMW-HA)on phenotypic transformation of mice cardiac fibroblasts and the role of CD44 and S100A4 in this process.Methods1.Cardiac fibroblasts(CFs)and myocardial cells(MCs)were isolated from1-3-day-old SPF grade neonatal ICR mice,and then cultured to the third generation for next experiments.2.With LMW-HA stimulation,the proliferation of CFs was measured by CCK-8 and Ed U to determine the optimal concentration of LMW-HA.3.WB,q RT-PCR and IF were used to detect the phenotypic transformation(α-SMA,Collagen III)and the levels of CD44 and S100A4 in CFs after LMW-HA stimulation.4.We extracted nuclear and cytoplasmic proteins of CFs simultaneously,and detected the expression changes of CD44 and S100A4 proteins separately to specified the nuclear translocation by WB and IF.Co IP was carried out to define the interaction of CD44 and S100A4 in CFs.5.BRIC-235,a specific inhibitor of CD44,was used to block the binding of LMW-HA and CD44.WB,q RT-PCR and IF were performed after BRIC-235 and LMW-HA stimulation.6.The recombinant mouse adenovirus S100A4-sh RNA was synthesized and transfected into CFs before LMW-HA stimulation.The levels of S100A4 and myocardial fibrosis markers were detected by WB,q RT-PCR,IF and Co IP.Results1.The m RNA and protein expressions of CD44 and S100A4 were detected in CFs and MCs of neonatal mice,and more CD44 and S100A4 existed in CFs than in MCs.2.LMW-HA can stimulate CFs proliferation at the concentration of 0.2mg/ml,and the optimal concentration of LMW-HA stimulation is 0.8mg/ml.3.After LMW-HA stimulation,CFs transformed into myofibroblasts,and the expression of myocardial fibrosis markers(α-SMA and Collagen III)and S100A4 increased,while the level of CD44 remained unchanged.4.CD44 and S100A4 expressions were decreased in the cytoplasm and increased gradually in the nucleus.At the same time,Co IP showed that these two proteins combined in CFs and interacted with each other.5.After CD44 was inhibited by BRIC-235,the expressions of myocardial fibrosis markers(α-SMA and Collagen III)were significantly reduced and the nuclear translocation of CD44 and S100A4 was unhappened.6.Downregulation of S100A4 reduced the expression of myocardial fibrosis markers(α-SMA and Collagen III).The protein of CD44 transferred to the nucleus,but did not bind to S100A4 protein intracellularly.ConclusionsLMW-HA can promote the differentiation of mice cardiac fibroblasts,which is regulated by the combination of LMW-HA and CD44 on the cell membrane surface,the nuclear translocation of CD44 and S100A4 and the activation of downstream signaling pathway.Blocking the binding of LMW-HA with CD44 or down-regulating S100A4 can inhibit the phenotypic transformation of cardiac fibroblasts and alleviate cardiac fibrosis.This may be a new therapeutic target of myocardial fibrosis in the future.