The Effects Of CD151 On Migration And Proliferation Of Vascular Smooth Muscle Cells And Antisense CD151 Gene's Inhibition To Experimental Restenosis Of Common Carotid Artery After Balloon Injury In Rats

Part IThe Effect of Foreign CD151 Gene's Transfection on the Migration and Proliferation of Vascular Smooth Muscle Cells in RatsObjective To explore the effects of sense or antisense sequence of foreign CD151 gene on migration and proliferation of cultured vascular smooth muscle cells (VSMCs) in rats . Methods Functional fragment of CD151 gene was amplified by RT-PCR and inserted into the vector pcDNA3.1 in the sense direction and antisense direction, respectively. Recombinant vectors pcDNA3.1-anti-CD151 and pcDNA3.1-CD151 were transfected into cultured VSMCs in vitro by means of Iiposome(lipofectamine2000), their effects on mRNA and protein expression of CD151 were measured by RT-PCR and Western blot, the effects on migration of cells were evaluated by using Boyden chamber and wound healing test, and their effects on cell's proliferation were evaluated by employing MTT assay and flow cytometry.All results were compared with those of vector control [transfected with pcDNA3.1(+)], liposome control (transfected only with lipofectamine -2000), and PBS control (given with similar volume of PBS). Results In comparison with the controls, the expression of CD151 in VSMCs transfected with pcDNA 3. l-anti-CD151 was decreased significantly by 58% in mRNA and by 52% in protein , and the migrated cells count per field (× 200) and healing area of wouned cells were less or smaller significantly; while in VSMCs transfected with pcDNA 3.1 -CD 151, the expression of CD151 was increased significantly by 171 % in mRNA and by 133% in protein, and migrated cells count per field (×200) and healing area of wouned cells were more or larger significantly.There were no signifficantal differences among VSMCs transfected with CD151 gene ,VSMCs transfected with antisense CD151 gene,and controls in cell's optical density ( tested by MTT assay ) and S+G₂M % (evaluated by flow cytometry).Conclusion CD151 has no effecs on proliferation of VSMCs of rat in vitro. Whereas CD151 plays an important role in regulating migration of cultured VSMCs. Sense CD151 gene upregulates the expression of VSMCs and promotes the cell's migration; antisense CD151 gene could attenuate the expression of CD151 and inhabite the cell migration, which may provides a new gene target for anti-sense gene therapy in preventing and treating restenosis after angioplasty.Part IIThe Local Transfer of Foreign Antisense CD151 Gene InhibitedExperimental Restenosis of Common Carotid Artery afterBalloon Injury in RatsObjective To determine whether direct transfer of the gene encoding antisense CD151 into artery could inhibit restenosis of vessel after balloon injury, in order to explore an effective target gene for gene therapy of arterial restenosis Methods 45 SD adult male rats were randomly divided into gene therapeutic group, vector group and NS (normal saline) group, respectively. Experimental restenosis in common carotid artery were built up in all animals by means of balloon catheter-induced injury. In gene therapeutic group, the antisense CD151 cDNA was transfected into the segments of the injured carotid arteries either by perfusing pcDNA3.1-anti-CD151 plasmid plus liposomes in lumen, or by coating the surface of adventitia. As controls, pcDNA3.1(+) plasmid or NS also were delivered into injured segments by same procedure in vector group or NS group, respectively . RT-PCR were employed to measure the mRNA expression of CD151 in transfected segments, and the protein expression of CD151 and its morphological distribution in the arteries were also evaluated by means of Western blot analysis and Immunohistochemical Assays. The effects of anti-sense CD151 cDNA on intimal hyperplasia and restenosis extent of injured arteries were observed in pathological sections. Results There was lower expression of CD151 mRNA and protein in gene therapeutic group than in both vector group and NS group. Immunohistochemical assay in transfected tissues showed faint expression of CD151 in subintima in gene therapeutic group, whereas in both control groups, there were intensive expression of CD151 protein. The intima area, and the ratio of intima area to midia area (IA/MA) were both smaller significantly in gene therapeutic group than in vector group and NS group respectively. Gene therapeutic group also showed less extent of restenosis of injured anteries in comparision with the two control groups significantly. Conclusion These results demonstrated that local direct transfer of gene encoding antisense CD151 could attenuate the expression of CD151 mRNA and protein in the injured segments of common carotid arteries in rats, and therefore reduced the intima hyperplasia and inhibited the restenosis of injured arteries. We also provided evidence that CD151 plays an important role in mechanism of restenosis after balloon catheter-injured arteries. Part IIIExpression of CD151 in Human Atherosclerotic Artery and ItsImplicationObjective To investigate the expression of CD151 in human atherosclerotic arteryand explore it's clinical implication.Methods Western blot and immunohistochemical techniques were used todetermine the protein expression of CD151 in artery tissues with atherosclerosistaken from 36 patients, including 26 cases who received bypass operation forperipheral atherosclerotic desease and 6 cases who died from coronary heart disease.The expression of CD151 in normal arterial tissues from 15 healthy organ donatorswere also mearsured to serve as control. In the patients, the effect of risk factors foratherosclerosis on the protein expression of CD151 was also evaluatedsimultaneously.Results The results showed that the expression of CD151 protein in atheroscleroticarteries was significantly higher than that in normal artery. In atherosclerotic arteries,CD151 expressed in vascular smooth muscle cells (VSMCs) in all tunica media and inpartial subintima , while in normal artery , sparse expression was found in tunicamedia near adventitia. There was no significant difference in protein expression ofCD151 between the patients with and without same risk factor.Conclusion It is concluded that high CD151 protein expression in artery isassociated with atherosclerosis and CD151 might play an important role in themechanism of atherosclerosis related to VSMC. The expression of CD151 in humanatherosclerotic artery is dependent on the extent of atherosclerotic damage, and isindependent of risk factors.