TWEAK/Fn14 Promotes The Proliferation And Collagen Synthesis Of Rat Cardiac Fibroblasts

BACKGROUND:Hypertension is a common disease, which seriously impairs public health. Hypertension exerts adverse effects on the target organs such as the heart, the brain,the kidney and so on. Myocardial fibrosis is one of the important pathological bases in hypertension heart disease (HHD). Myocardial fibrosis is a process of the proliferation of cardiac fibroblasts (CFs)and increased collagen deposition increased, which gradually leads to cardiac diastolic and systolic dysfunction, and finally induces many severe complications including congestive heart failure, arrhythmia and sudden cardiac death, et al. However, It is not known that the role of the pathogenesis of hypertension myocardial fibrosis yet. Nowadays, the study on the pathogenesis and prevention and cure of myocardial fibrosis has become a worldwide hot topic.Recent studies have indicated the inflammatory reaction could greatly influence the development of myocardial fibrosis. Inflammation forms a complex regulation network. The fibrotic myocardium releases more inflammatory cytokines with the stimulation of other inflammatory cytokines, and finally aggravates to myocardial fibrosis. Inflammatory cytokines and their receptors play an important role in cardiac remodelling, and they may facilitate both acute heart failure and progression to chronic cardiac dysfunction. Several experiments revealed that there were inflammatory cell infiltrate and myocardial fibrosis in the myocardial infarction area or not after myocardial infarction, and the fibrosis was positive correlated with the level of the inflammatory cytokines.Recently, several reports have suggested that the tumor necrosis factor (TNF) played a crucial role in myocardial fibrosis, which promoted myocardial cell apoptosis and changed the expression of related cytokines. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a new member of the TNF superfamily of ligands. TWEAK is a typeⅡtransmembrane, and it can be released into a soluble factor with biological activity. This multifunctional cytokine regulates cellular activities including proliferation, differentiation, inflammation, apoptosis and angiogenesis by binding to its receptor named fibroblast growth factor-inducible 14 (Fn14). Fn14 is a type I transmembrane. Fn14 cytoplasmic tail contains a single TNF receptor-associated factor (TRAF) consensus binding site, and TRAF are able to bind this site. Moreover, TWEAK links Fnl4 through this site to induce a variety of signaling cascades. Futhermore,several studies have revealed that TWEAK/Fn14 axis affected pathogenesis of cardiac remodelling and heart failure,and the heart failure be closely related with myocardial fibrosis. However, little is known about TWEAK/Fn14 potential role in myocardial fibrosis.OBJECTIVE:To explore the effect of TWEAK/Fn14 on the proliferation and collagen synthesis of rat cardiac fibroblasts (CFs) and the relationship between TWEAK/Fn14 and myocardial fibrosis.METHODS:1. CFs were isolated by trypsin digestion method from Wistar rats and identified by invert microscope and vimentin immunofluorescence.2. CFs in all groups were incubated for 24h and 48h.Then CFs proliferation was examined by MTT, and qRT-PCR and ELISA methods were used to detect collagen type I mRNA and protein respectively. CFs were divided into three groups:①control group:CFs were cultured without any stimulus;②100μg/L group:CFs were incubated with 100μg/L Recombinant human TWEAK (rhTWEAK);③500μg/L group:CFs were incubated with 500μg/L rhTWEAK.RESULTS:1. This isolated CFs were observed with invert microscope, and primary CFs appear fibroblast morphology, fusiform or triangular with light cytoplasm and big oval nucleus.No spontaneous beat could be seen. When identified by immunofluore- scence,the cells showed positive for vimentin.2. Cell proliferation was detected by MTT assay(A492 Value). The A492 values of the 100μg/L group and 500μg/L group were higher than that of the control group (P<0.01), and the difference between 500μg/L and 100μg/L TWEAK group was statistically significant (P<0.01) on 24h or 48h. The A492 values of the 100μg/L and 500μg/L group on 48h were higher than on 24h. TWEAK increased the number of CFs in a time and does dependentl manner.3. The expression levels of mRNA and protein of collagen type I in the 100μg/L and 500μg/L group were higher than in the control group (P<0.05), and the 500ng/mL TWEAK group was greater than 100ng/mL TWEAK group (P<0.05) on 24h or 48h. The 100μg/L and 500μg/L group on 48h were higher than on 24h. TWEAK increased the type I collagen mRNA and protein synthesis in a time and does dependentl manner.CONCLUSIONS:1. TWEAK is a novel regulator of CFs proliferation and collagen synthesis.2. Our results indicate that TWEAK-induced these effects in CFs are may mainly dependent on Fn14, and this suggests that TWEAK/Fn14 may play an important role on myocardial fibrosis.