Cyclic stretch induces vascular smooth muscle cell-derived connective tissue growth factor secretion and promotes endothelial progenitor cell differentiation and angiogenesis

Abstract

Endothelial progenitor cells (EPCs) play an important role in endothelial repair following vascular injury and maintaining the integrity of endothelium, but the underlying molecular mechanisms regulating the differentiation of EPCs remain to be studied. In this study, the effects of cyclic stretch and the neighboring vascular smooth muscle cells (VSMCs) on the differentiation of EPCs, as well as the underlying mechanism were investigated in a co-culture model. Bone marrow-derived EPCs were cocultured with VSMCs, which were exposed to cyclic stretch with a magnitude of 5% (which mimics physiological mechanical stress) at a constant frequency of 1.25 Hz. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cell (ECs), with upregulation of EC markers (CD31, vWF and KDR) and promoted angiogenesis. Meanwhile, cyclic stretch unregulated the mRNA level and secretion level of connective tissue growth factor (CTGF). After treated with recombinant CTGF (r-CTGF) at the concentration of 20 ng/ml, the mRNA expression of CD31, vWF and KDR in EPCs were increased, EPC tube formation and the protein level of FZD8 and β-catenin were promoted. Small interfering RNA (siRNA)-mediated knockdown of CTGF in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of FZD8/β-catenin signaling pathway in vitro. Consistently, in vivo matrigel plug assay demonstrated that r-CTGF treated EPCs enhanced angiogenesis. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which in turn activates FZD8 and β-catenin to promote co-cultured EPC differentiation into EC lineage and angiogenesis. Our findings provide insights into the mechanisms underlying the VSMC/EPC interactions and the pro-angiogenic function of cyclic stretch in modulating EPC differentiation. CTGF acts as a key intercellular mediator and may be as a potential therapeutic target for vascular injury.