The Effect And Mechanism Of Adenovirus-mediated Angiotensin Ⅱ Type 1 Receptor-associated Protein (ATRAP) On Proliferation And Apoptosis Of Vascular Smooth Muscle Cells

Background and ObjectiveCardiovascular and cerebrovascular disease is currently a common cause of death in developed and developing countries.Atherosclerosis(AS)is a multifactorial chronic disease,so far its risk factors have been found in a variety,and is stillincreasing.There are three stages into AS development pathology process: the first stage is the formation of lipid,the second is the formation of fibrous plaques,and the third stage is formation of atherosclerotic plaque.At present,two mainly treatment methods of atherosclerosis caused by vascular lesions are percutaneous angioplasty and arterial bypass surgery,although the treatment of the above technology has been very mature,the occurrence of vascular anastomosis endometrial hyperplasia,stent restenosis and other complicationsaftersurgery can not benefit to patients in long-terms,resulting in higher mortality.The Rennin-angiotensin-aldosterones system(RAAS)is an important humoral regulation system that regulates water and salt balance,angiotasis and sympathetic nervous system activity.Angiotensin Ⅱ(Ang Ⅱ)is an important effector molecule involved in the formation of atherosclerosis,which requires a biological effect through its receptor,and Ang Ⅱ Type I receptor(AT1R)mediates theoverwhelming majority of the biological effects of Ang Ⅱ.Activation of receptor can promote vascular endothelial cell proliferation,stimulate cells to produce a large number of growth factors,chemokines and cytokines,so that inflammatory cell infiltration,induced strong vascular inflammation.In addition,activation of AT1 R can also lead to NF-κB transfer to the nucleus,increase the transcription and translation activities in cells,promote vascular smooth muscle cell(VSMC)migration and proliferation.Therefore,AT1 R gene naturally become an important candidate gene for atherosclerosis.In recent years,Dzau,V.J have cloned the Angiotensin Type 1 Ⅱ Receptor-Associated Protein(ATRAP).At present,it has been confirmed that ATRAP gene is involved in the regulation of RAAS and strengthens the internalization of AT1 R on VSMC,so it antagonizes the biological effect of Ang Ⅱ-mediated by AT1 R,which can significantly inhibit the proliferation of VSMC.Nonetheless,the exact molecular mechanism of ATRAP as a new target gene in the treatment of vascular disease methods in Ang Ⅱ-mediated vascular disease remains unclear.With the progress of science and technology,gene therapy has been paid more and more attention.There are many types of gene therapy reported,but the results are unsatisfactory.Therefore,we intend to use the gene delivery system to target gene in the direct exposure of the surgical site into the cells,that can upregulate the related genes to inhibit atherosclerosis,and it may inhibit vascular inflammation and vascular intimal hyperplasia,which properbly become the most reliable method of treatment of atherosclerosis.Commonly used gene vector system are retrovirus,liposome,etc.,and adenovirus vector is selected for the infection efficiency of this experiment.The aim of this study was to construct the recombinant adenovirus pAD-ATRAP-HIS * 6,with a mouse ATRAP,which was used to in vitro culture of VSMC and rat carotid artery injury model.The proliferation and apoptosis of VSMC,the expression of apoptotic family genes bax and bcl-2 and the changes of vascular intimal hyperplasia were observed to evaluate the efficiency of adenovirus-mediated ATRAP-infected VSMC.We will demonstrate the effect of ATRAP gene on the proliferation and apoptosis of VSMC and the apoptotic Mechanism.MethodsIn this study,we use the adenovirus vector loaded with ATRAP,infecte in vitro culture VSMCs and rat injury of the common carotid artery.(1)to construct adenovirus pAD-ATRAP-HIS * 6,to detect its titer and infection ability.(2)in vitro experiments: Adenovirus-mediated ATRAP infection VSMC,ATRAP observed in vitro culture of VSMC proliferation and apoptosis.(3)in vivo experiments: The model of intimal injury hyperplasia was established by carotid artery injury in rats.Adenovirus infection was used to observe the inhibitory effect of ATRAP on proliferative endometrium.Results 1.The successful construction of adenovirus pAD-ATRAP-HIS * 6,and the titer is 1.34×109LFU/mL.2.High levels of ATRAP mRNA and protein expression were observed in VSMC infected with adenovirus in vitro.3.In Ang Ⅱ-stimulated VSMC infected with adenovirus,we observed that ATRAP can inhibit the proliferation of VSMC.4.In Ang Ⅱ-stimulated VSMC infectedwith adenovirus,we observed ATRAP can promote the apoptosis of VSMC,and observed the high expression of bax mRNA levels and protein levels and the low levels of bcl-2 mRNA and protein.5.Infection of ATRAP at the injured arteries was observed to reduce intimal hyperplasia,whereas intimal hyperplasia in the control group was severe.ConclusionATRAP can significantly inhibit the proliferation of VSMCmediated by AT1 R,promote the apoptosis of VSMC,and inhibit the intimal hyperplasia after injury,thus inhibiting the development of atherosclerosis.