Role Of Vascular Smooth Muscle Cell Derived-CTGF In Endothelial Progenitor Cell Differentiation Under Cyclic Stretch

Cardiovascular diseases are the one of the leading causes of death globally.Percutaneous coronary intervention(PCI)is an important therapeutic option in the treatment of coronary heart disease and atherosclerosis.However,PCI can cause endothelial damage and result in dysfunction of endothelium.Endothelial repair following vascular injury plays a crucial role in maintaining the integrity of endothelium and preventing the development of cardiovascular diseases.Once the tissue is damaged,endothelial progenitor cells(EPCs)are mobilized from bone marrow,enter blood stream and localize to the injured vessel at the sites of injury.As EPCs home to the endothelial injury sites,they may communicate with exposed vascular smooth muscle cells(VSMCs)in the media.It has been reported that VSMCs and shear stress exert effects on EPC differentiation towards to mature endothelial cells in the process of endothelium repair.However,the effects of neighboring VSMCs and mechanical stretch generated from the pulsatile nature of blood flow on EPC function still need to be studied.Therefore,we focused on examining the role of cyclic stretch and VSMCs in the differentiation of EPCs and further provided first insights into the mechanisms.In this study,EPCs were isolated from bone marrow of Sprague Dawley(SD)rats by density-gradient centrifugation.In an improved coculture model,EPCs were seeded on the inner side of membrane in a cell culture insert,and VSMCs were plated on flexible silicone bottom plates.Application of cyclic stretch(5% magnitude at 1.25 Hz)to VSMCs cocultured with bone marrow-derived EPCs modulated EPC differentiation to mature endothelial cells after 12 h,with upregulation of EC markers(CD31,vWF and KDR),which revealed that EPCs differentiated towards to mature endothelial cells.Matrigel angiogenesis assays revealed that EPC tube formation capacity in vitro was dramatically enhanced after cocultured with VSMCs under 5% cyclic stretch.Connective tissue growth factor(CTGF),a secretion protein rich in cysteine,plays important roles in cardiovascular diseases,such as atherosclerosis,hypertension and vascular injury.We found that CTGF secretion level was increased significantly in VSMCs under 5% cyclic stretch.Ingenuity Pathway Analysis indicated that the target molecules of CTGF were highly related to cellular development;in addition,FZD8 protein,a key regulator in Wnt/?-catenin signaling pathway,may involve in this process.Recombinant protein(r-CTGF)induced EPC differentiation,as well as tube formation by increasing the protein expression of FZD8 and ?-catenin.VSMCs transfected with CTGF specific small interfering RNA inhibited cyclic stretch-induced expression of FZD8 and ?-catenin,and reversed EPC differentiation,as well as tube formation capacity.In vivo Matrigel plug assay was performed by injecting subcutaneously Matrigel mixed with r-CTGF treated EPCs into nude mice.7 days after injection,the Matrigel plugs were retrieved.There were more blood vessels in Matrigel plugs with r-CTGF stimulated EPCs,indicating increased angiogenesis.Tissue sections results revealed that EPCs treated with r-CTGF increased evidently CD31 expression by immunofluorescence staining.In summary,cyclic stretch and VSMCs play important roles in the process of EPC differentiation towards to mature endothelial cells.Physiological cyclic stretch(5%)induces CTGF secretion from VSMCs,then paracrine CTGF modulates EPC differentiation towards to mature endothelial cells with enhanced angiogenic ability by activating FZD8 and ?-catenin.The current study emphasizes the importance of synergy effect of VSMCs and mechanical stretch on EPC function during vascular repair.CTGF acts as a key intercellular mediator and may be as a potential therapeutic target for endothelial repair following vascular injury.