Mechanobiological Roles Of MicroRNA-33 In Neointimal Hyperplasia Induced By The Vein Graft

Coronary artery bypass graft(CABG)is an economical clinical revascularization strategy for patients with coronary artery disease.Autologous veins,such as saphenous veins,are important,convenient,and frequently used conduit for surgical revascularization.However,the neointimal hyperplasia of the vein after transplantation can lead to the failure of bypass graft.The mechanical environment caused by arterial blood flow and blood pressure is significantly changed after vein graft,which is characterized by the abnormal increase of fluid shear stress,cyclic stretch and transmural pressure.Recent studies have shown that the changed mechanical environment is one of the key pathogenesis factors of intimal hyperplasia and lumen occlusion.However,the molecular mechanism in this process is still unknown.The present research focused on the role of arterialized cyclic stretch in the proliferation of venous smooth muscle cells(SMCs),and explored the mechanobiological mechanism of micro RNA(mi R)in the neointimal hyperplasia of vein graft.The mechanobiological mechanism on the intimal hyperplasia of vein grafts will provide new insight into understanding the pathology of cardiovascular disease,and reveal potential therapeutic targets.Vein grafts were performed using the ?cuff? technique,and the contralateral vein was used as control.To address the progress of venous remodeling after graft surgery,Elastin-Van Gieson staining was used to detect neointimal hyperplasia after 7-,14-,and 28-day vein graft.The neointimal hyperplasia in the grafted vein was significantly thickened after 7-,14-,and 28-day graft surgery.In situ Brd U proliferation assay was used to detect cell proliferation in vivo,and the result revealed that cell proliferation was significantly increased in 7-,14-,and 28-day grafted veins.mi R sequencing was used to detect the differentially expressed mi Rs between 14-d grafted veins and the contralateral control.The results showed that the expression of virious mi Rs including mi R-33,mi R-21 and mi R-143 were significantly changed in grafts.RT-PCR confirmed that mi R-33 was significantly decreased in grafted veins.To investigate the function of the mi R-33 in the progress of neointimal hyperplasia,mi R-33 MI or IN transfection was used respectively and the proliferation of venous SMCs was detected in vitro.mi R-33 MI inhibited while mi R-33 IN promoted venous SMC proliferation.To identify possible mi R-33 targets,three web-accessible databases,i.e.Pic Tar(http://www.pictar.org/),Target Scan(http://www.targetscan.org/),and mi Randa(http://www.microrna.org)were searched,and BMP3 was found to be a potential target for mi R-33.A dual luciferase reporter assay verified the results of the algorithm prediction.mi R-33 IN and MI were used to further validate the effect of mi R-33 on BMP3 expression.mi R-33 IN upregulated while MI downregulated the expression of BMP3 in venous SMCs.The expressions of BMP3 downstream molecules,i.e.p-smad5 and p-smad2 were also negatively regulated by mi R-33.The expression of BMP3,p-smad5 and p-smad2 was also significant increased in the 7-,14-,and 28-day grafts.To demonstrate the effect of BMP3 on the proliferation of venous SMCs,recombinant BMP3 and BMP3 specific si RNA were used.BMP3 recombinant protein promoted while BMP3 si RNA inhibited the proliferation of venous SMCs,and the expression of p-smad5 and p-smad2.In order to explore whether the effect of mi R-33 in venous SMCs is BMP3 dependent or not,BMP3 recombinant protein was used in venous SMCs under mi R-33 MI transfection,and BMP3 si RNA was used under mi R-33 IN transfection.The results showed that BMP3 recombinant protein promoted the proliferation of venous SMCs and the expression of p-smad5 and p-smad2 under mi R-33 MI transfection;while BMP3 si RNA inhibited the proliferation of venous SMCs as well as the expression of p-smad5 and p-smad2 under mi R-33 IN transfection.10%-1.25Hz-cyclic stretch was used in vitro to simulate the mechanical environment of venous SMCs in vivo,and the proliferation of venous SMCs,the expression of mi R-33,as well as the expression of BMP3,p-smad5 and p-smad2 were then detected.10%-1.25 Hz cyclic stretch promoted the proliferation of venous SMCs,reduced the expression of mi R-33,and induced the expression of BMP3,p-smad5 and p-smad2 in venous SMCs.mi R-33 MI transfection under cyclic stretch was used to illustrate whether mechanical stretch modulates venous SMC proliferation and BMP3/smads signaling pathway via mi R-33.The results indicated that mi R-33 decreased venous SMC proliferation and expression of BMP3,p-smad5 and p-smad2 under cyclic stretch.To confirm whether the effect of cyclic stretch on venous SMC proliferation was dependent on BMP3,venous SMCs were transfected with BMP3 specific si RNA and then applied to 10%-1.25Hz-cyclic stretch.The results showed that BMP3 promoted venous SMC proliferation and the expression of p-smad5 and p-smad2 under cyclic stretch.Perivascular multi-point injection of agomi R-33 or BMP3 overexpressive lentivirus was performed in vivo to identify the potential therapeutic role of mi R-33 and BMP3 in neointimal formation.Agomi R-33 reduced the expression of BMP3 and its downstream signaling molecules,decreased cell proliferation as well as alleviated intimal hyperplasia in vivo.BMP3 lentivirus not only activated p-smad5 and p-smad2,but also promoted cell proliferation,neointimal hyperplasia and even blockage of grafted vein.In summary,cyclic stretch plays an important role in the neointimal hyperplasia of vein graft.Abnormally increased cyclic stretch participates in cell proliferation via down-regulation of mi R-33 and up-regulation of BMP3,which susequently activates p-smad5 and p-smad2.In vivo experiments revealed that the perivascular multi-point injection of agomi R-33 alleviated intimal hyperplasia of the grafted vein.The present study suggests that mi R-33 and BMP3 may be potential therapeutic targets for neointimal hyperplasia in vein grafts.