Study On The Relation Between Resveratrol Targeting Protein And Proliferation Of Vascular Smooth Muscle Cell

Background:Coronary heart disease(CHD)is a leading cause of morbidity and mortality in both developed and developing countries.Percutaneous coronary intervention(PCI)has been used worldwide for the treatment of stenotic lesions as a result of atherosclerosis.However,the incidence of restenosis after PCI is still rather higher(15～20%).So it's important and urgent to prevent atherosclerosis and restenosis.It has been shown that the migration of vascular smooth muscle cells(VSMCs)from the media to the intima and their subsequent proliferation,secretion of extracelluler matrix play major roles in the complex process of atherosclerosis and post-angioplasty restenosis.This suggests that the VSMCs may be a valid target for the development of new anti-atherosclerotic and anti-restenotic therapies.So it is of vital importance to develop new antiatherosclerotic and antirestenotic drugs and elucidate the mechanism of these effects.Resveratrol is a polyphenol compound possessing cardiac protective effects. One of the important effects is that resveratrol reduces the incidence of atherosclerosis and restenosis through inhibiting the proliferation of vascular smooth muscle cell(VSMC).Resveratrol affinity column(RAC)was designed according to the construction feature of resveratrol.Resveratrol targeting protein (RTP)was purified using RAC.RTP was confirmed to specific binding with resveratrol by radio-ligand autograph.The amino acid sequence of resveratrol was identified to have homology(80%)with quinone oxidoreductase 2(NQO2) by mass spectrography analysis.Is the inhibitory effect on the proliferation of VSMC and endangium hyperplasty due to the direct acting of resveratrol on RTP? How to regulate the proliferation of VSMC by RTP? There are no answers for these questions.More studies need to be done.Objective:In this study,we will investigate these aspects:(1)To purify RTP from rabbit vascular smooth muscle tissue and identify the relation between RTP and NQO2;(2)To investigate the effects of resveratrol on the incidence of restenosis in experimental atherosclerosis model rabbit.To observe the effects of resveratrol on the level of RTP mRNA and the expression of RTP in vessel wall; (3)To observe the effects of resveratrol on the proliferation of VSMC;To investigate the effects of resveratrol on the level of RTP mRNA and the expression of RTP in VSMC;(4)To observe the change of the proliferation of VSMC,in which RTP is over-expression or knock-down.We wish to clarify the effects of resveratrol on RTP and the mechanisms of RTP's regulation on the proliferation of VSMC from three levels:global level,cellular level and molecular level.The theoretical and experimental proofs were provided by this study that resveratrol would be a new targeted-therapy for the restenosis of CHD.Methods:1.To prepare resveratrol affinity column and purify RTP from rabbit arteriae aorta smooth muscle tissue.To identify RTP using protein electrophoresis and western blot.2.48 New Zealand male rabbits,divided into 3 groups randomly,were fed with forages as follow respectively:(1)normal forage,(2)high-cholesterol forage (containing cholesterol 1.5%),(3)high-cholesterol forage with resveratrol. Restenosis model was established by denudating endothelium using PTCA balloon in right arteria iliaca after 12 weeks feeding.Continue feeding for another 4 weeks,all experimental rabbits were sacrificed.(1)Evaluation of the restenosis:immediately upon excision,cross sections of iliac arteria were stained using hematoxylin and eosin.All slides of specimen were examined using Leica Qwin image analysis system.Rate of restenosis and tunica initima/media area ratio were measured.(2)The levels of RTP mRNA in both arteria iliaca were measured using real-time PCR.(3)The expression of RTP in both arteria iliaca was measured using western blot.3.Cultured rabbit vascular smooth muscle cells were incubated with resveratrol (1,10 and 50μmol/L)for different time(24,48 and 72 hours).(1)The proliferation of VSMC was measured by cell counting.(2)The levels of RTP mRNA in VSMC were measured using real-time PCR.(3)The expression of RTP in VSMC was measured using western blot.4.RTP-26 over-expression plasmid was constructed and transduced to rat vascular smooth muscle cells.(1)The expression of RTP was analyzed using half-quantitative RT-PCR and western blot.(2)The proliferation of transduced VSMC was measured using BrdU Cell Proliferation Assay Kit.5.Lentiviral vector against RTP-26 was constructed and transduced to rat vascular smooth muscle cells.(1)The expression of RTP was analyzed using real-time PCR and western blot.(2)The proliferation of transduced VSMC was measured using BrdU Cell Proliferation Assay Kit.Results:1.A protein fragment,which molecular weight is 26kD,was purified from rabbit vascular smooth muscle tissue homogenate using resveratrol affinity column.This protein is denoted as resveratrol targeting protein 26(RTP-26). The molecular weight of this protein is identical to that of NQO2 in the previous literature.This fraction was analyzed for the presence of NQO2 using polyclonal antibody in western blot analysis.2.Resveratrol reduced the incidence of restenosis in atherosclerotic rabbit models.The rates of restenosis of control,model and resveratrol group are 63.04±3.33%,73.92±7.10%and 28.10±5.83%,respectively.(ANOVA test,P＜0.01).Resveratrol inhibited the expression of RTP-26 either in the mRNA level or in the protein level.Compared with control and model,the level of RTP-26 mRNA in resveratrol group was depressed by 39%and 44%, respectively(P＜0.01).Furthermore,the expression of RTP-26 in resveratrol group was decreased 35%and 32%,respectively,compared with control and model(P＜0.01).3.Rabbit VSMC proliferation was inhibited by resveratrol in a concentrationand time-dependent manner.Meanwhile,the expression of RTP-26 was inhibited by resveratrol in VSMC.RTP-26 mRNA decreased 56%,62%and 95%after treatment with 1,10,or 50μmol/L resveratrol,respectively (P＜0.01 compared with control).The expression of RTP-26 was inhibited 15%and 47%with 10 and 50μmol/L resveratrol,respectively(P＜0.05 and P＜0.01,respectively,compared with control).After exposure to 50μmol/L resveratrol,RTP-26 mRNA levels was reduced by 53%and 95%at 48 and 72 h,respectively(P＜0.01 compared with control).Furthermore,the expression of RTP-26 was significantly reduced by 36%and 58%at 48 h and 72 h,respectively(P＜0.01,compared with control).These results suggested that resveratrol inhibited RTP-26 mRNA and protein expression in dose- and time-dependent manners.4.RTP-26 over-expression plasmid was constructed successfully and was denoted as pEGFP-N1-NQO2.The mRNA level of RTP-26 was doubled in VSMC transduced pEGFP-N1-NQO2 for 48 hours than the wild type cell.A fusion protein including green fluorascent protein(GFP)and RTP-26 could be detected in transduced VSMC.However,the proliferation of transduced VSMC is similar with the wild type cell.5.Lentiviral vectors containing RTP-26 shRNA was constructed and denoted as Psc-NQO2.RTP-26 expression in lentiviral vector-transduced cells was evaluated by real-time quantitative PCR and western blotting.RTP-26 mRNA levels decreased by 73%in the Psc-NQO2 transduced rat VSMC after 72 h culture.Western blot analysis of 14 days post-transfection showed that the RNAi treatment significantly suppressed the expression of NQO2 protein.The proliferation was analyzed using BrdU Cell Proliferation Assay Kit.RTP-26 knockdown had a similar inhibitory effect on rat VSMC proliferation as 25μmol/L resveratrol.Conclusions:1.A protein fragment,which molecular weight is 26kD,was purified from rabbit vascular smooth muscle tissue homogenate using resveratrol affinity column.This protein is denoted as resveratrol targeting protein 26(RTP-26). RTP-26 was identified as quinone oxidoreductase 2(NQO2)using western blot according to literatures.2.Atherosclerotic rabbit model was successfully developed by feeding with 1.5%cholesterol diet for 12 weeks.The restenosis model was prepared by denudating endothelium using PTCA balloon.Resveratrol diminished restenosis in atherosclerotic iliac artery.Resveratrol also inhibited the expression of RTP-26,either at mRNA level or at protein level,in vascular smooth muscle tissue.3.Resveratrol inhibited the proliferation of cultured vascular smooth muscle cells.Resveratrol down-regulated the level of RTP-26 mRNA of smooth muscle cell in a concetration- and time-dependent manner.Furthermore, resveratrol decreased the expression of RTP-26 protein of smooth muscle cell in a concetration- and time-dependent manner.4.The proliferation ability was similar between native smooth muscle cells and cells in which RTP-26 was over-expressed induced by plasmid.5.The proliferation was inhibited by the down-expression of RTP-26 in vascular smooth muscle cells using RNA interfere method. RTP-26-knockdown smooth muscle cells showed a similar inhibitory effect of proliferation with 25μmol/L resveratrol-treated cells.