PDMS Membrane Surface Micro-topography Regulated Smooth Muscle Cell Functuion By Endothelial Cell Exosomal MicroRNA

Atherosclerosis is a major cardiovascular disease which is prevalent throughout the world.The etiology of atherosclerosis is complicated with a large variety of causes involved.However the development of the disease finally leads to the disruption of endothelial cell(EC)and smooth muscle cell(SMC)communication signaling.And then SMCs differentiated into synthetic phenotype,secreting excess ECM and build up the plaque on the blood vessel wall.To treat this disease,vascular tissue engineering has become a promising therapeutic method.And researches are focusing on how to improve tissue engineering vascular material to provide a suitable microenvironment for vascular reconstruction.As far as the signaling between EC and SMC is concerned,previous researches largely lay emphasis on the cells participated in this process,such as inflammatory cells.Some research focused on the signaling pathway activated by injured ECs,such as PDGF pathway and so on.Recent research suggested that exosome is an important medium for cell-cell communications,as well as cells and their external environments.For example,exosome has been found to contribute to the dialogue between EC and SMC.On the other hand,it has been reported that biochemical factors can play an important role in regulating gene expression and exosome secretion in ECs.Biophysical cues,such as fluid shear stress,could also regulate exosome secretion in EC.Hence,theses findings provide a significant background for the hypothesis proposed in this project.In this study,we hypothesized that a simple external physical factor,such as PDMS material with grooved surface topology,might subsequently lead to a chain reaction,including the gene expression in EC,signal passing from EC to SMC by exosome and SMC function modification.To test these hypotheses,the thesis firstly tested the change of HUVEC morphology by parallel microgrooved PDMS,the gene and protein expression in SMC in a co-culture system.The morphology of HUVECs cultured on microgrooved PDMS showed much more elongated shape in compared to those cultured on flat PDMS materials.Meanwhile,in SMC,the expression of SMC-?-actin and MYH11 are downregulated.Accordingly,the expression of some microRNAs,especially miR-143 and miR-145 which targeting SM-?-actin and MYH11,were also been upregulated.This phenomenon was also confirmed in the conditioned medium system without direct communication of HUVEC and SMC,suggesting that the regulatory factors are in the supernatant of HUVECs.Then the thesis focused on confirming the regulatory factors are exosomes and their enclosed microRNAs secreted by morphology altered HUVECs.Comparing with flat surface,the grooved surface could induce HUVEC to secrete more exosomes,and the expreesion of miR-143 and miR-145 have also been upregulated in exosomes.The BODIPY staining result directly demonstrated that exosomes were transferred from HUVECs to SMCs.However,these results could be reversed by N-SMase Spiroepoxide inhibitor to inhibit the secretion of exosomes.Therefore,we confirmed that it was indeed exosomes and the exosomal miRNA,miR-143 and miR-145,exerted the important regulation effect on SMC.The last part of the thsis studied a broad range of microRNA expression profile in the morphology changed HUVECs.The results showed that lots of microRNAs and their target mRNAs have been affected.Apart from miR-143 and miR-145,miR-21 was upregulated and PTEN and Caspase-3 in HUVECs were downregulated accordingly,leading to the decrease of cell apoptosis in HUVECs.In conclusion,comparing to smooth PDMS material,the grooved PDMS material could change the morphology of HUVECs to elongated shape.However the expression of microRNAs in HUVECs also have been changed,especially miR-143 and miR-145 have been upregulated.HUVECs cultured on grooved PDMS membrane could secrete more exosomes,in this way the expreesion of miR-143,miR-145 and shrinkage phenotypic SMC markers have also been upregulated in SMCs.And it illustrated that HUVECs overexpreesing miR-21 caused by grooved PDMS material could decrease apoptosis in HUVECs.These results implied more possibilities in the future research on the regulatory effect on EC by micro-topography of the vascular tissue engineering material.