Pleiotropic Effects Of Survivin In Vascular Endothelial Cells

Arteriosclerosis is a common disease in middle and old age.With the increasing incidence of hypertension,hyperglycemia and hyperlipidemia,there are more fatal diseases,such as myocardial infarction,cerebral infraction and limb ischemia necrosis caused by atherosclerosis,which were characterized by arteriostenosis,arterial embolization and intimal hyperplasia.As intimal hyperplasia always accompanied development,the current treatments including operation or drugs can not reverse this pathological change and result in the reocclusion after bypass surgery and interventional operation,which is most common in lower limb arteriosclerosis.In peripheral vascular disease(PAD),the establishment of collateral circulation can relieve the corresponding symptoms,reduce the progress of the disease and even achieve healing.The key that guarantee of the establishment of collateral circulation is the activation,proliferation,migration and tube formation of vascular endothelial cells(VECs)which finally form a new blood vessel network.The collateral circulation can increase the peripheral circulation,improve the microcirculation of the blood perfusion and solve the problem of arterial long-term patency rate after periphery revascularization.However,researches indicate that the defection of comparatively low activity,growth rate and update speed of mature VECs greatly restrict its application in clinical treatment.Therefore,how to ensure VECs with a powerful ability of anti-apoptosis,proliferation and migration is a difficult problem to be solved urgently at present in the treatment of arteriosclerosis.Survivin is the smallest member of the inhibitor of apoptosis(IAP)family of proteins,reacting with multiple cell cycle proteins and mitotic proteins to increase the survival rate of cells and regulate cell apoptosis through caspase dependent or non-dependent pathways.Reseachers speculate that SVV can bind to caspase 3 receptor or effector,thereby inhibiting cell apoptosis,and also can directly bind to caspase 9 that inhibit its apoptotic activity.Current study indicate SVV also can been found to be prominently expressed on both tumour cells and embryonic stem cells and even nontumorous cells such as esophageal epithellal cells,kidney tubules and gastric mucosal cells indicating its yet unexplored role in generation and maintenance.Therefore,this study verify the expression of SVV in endothelial cells and over-expressing it in rat aortic endothelial cells(RAECs)to exam the pleiotropic effects in proliferation,apoptosis,migration and angiogenesis,lay a theoretical foundation for the gene targeting therapy of peripheral vascular diseases.PART Ⅰ THE VALIDATION OF SURVIVIN GENE IN ENDOTHELIAL CELLSObjective:To observe the expression of survivn in endothelial cells.Methods:RAECs are harvested in the logarithmic growth phase and lysed by eukaryotic cell lysis solution and RNA extraction Kit to extract the protein and mRNA.The expression of Survivn in protein and nucleic acid levels are detected by western-blot and RT-PCR.Results:The outcomes of western-blot and RT-PCR reveal that mature RAECs have relatively light expression of Survivn in protein(grey level to 14.64±0.32)and nucleic acid levels(2-ΔΔCt to 1.165±0.17).Conclusion:The mature RAECs express Survivin gene at a low level.PART Ⅱ ADENOVIRUS TRANSFECTION AND OVER-EXPRESSION OF SURVIVIN IN ENDOTHELIAL CELLSObjective:Up-regulation the Survivin gene in RAECs to analysis the pleiotropic effects in vitro.Methods:RAECs are transfected by the recombinant adenovirus,which contain BIRC-5 and Green Fluorescent Protein(GFP)in MOI gradient(100 MOI,200MOI,300MOI)and confirmed the over-expression of Survivin after 24h and 48h through western-blot and RT-PCR.Results:At 300 MOI of 24h,Ad-GFP/SVV displayed the highest transfection efficiency(up to 95%).And the outcomes of western-blot and RT-PCR reveal that Ad-GFP/SVV group present a prominent upgrade of Survivin in protein and nucleic acid levels.Conclusion:Ad-GFP/SVW has a high transfection rate and significantly increase the expression of Survivin in RAECs.PART III The Pleiotropic Effects of Survivin on RAECs in vitro Objective:To confirm that Survivin over-expression can promote proliferation,migration and anti-apoptosis in RAECs and verify the regulation targets.Methods:RAECs are transfected by Ad-GFP/SVV at the 300 MOI.After 48h,we detect the effects of Survivin on proliferation,migration and apoptosis through CCK-8 Kit,transwell and flow cytometry in vitro and related factors such as INCENP,Aurora B,caspase-3 and bcl-2 are confirmed by western-blot and RT-PCR.The influence on the secretion of MMP-2,MMP-9 and Ang-2 is detected by ELISA.Results:Upregulation of Survivin in RAECs prominently promote proliferation and elevate the expression of cyclin D and some integral component of chromosomal passenger complex(CPC)such as INCENP,Aurora B and P-Aurora B which ensures proper segregation of chromosomes and cytokinesis during cell division.Apoptosis is significantly restrained by regulation the apoptotic factors such as up-regulate bcl-2 and down-regulate caspase-3.Survivin over-expressed increases the secretion of MMP-2,MMP-9 and Ang-2 result in a promotion of migration in vitro.Conclusion:Upregulation of Survivin in RAECs has a prominently effects on the proliferation,migration and anti-apoptosis and overcome the defects of low biological activity under the physiological conditions.PART IV The Effects of Survivin on Angiogenesis in vivoObjective:To confirm the promotion of Survivin for angiogenesis in nude mouse muscle tissue.Methods:The RAECs over-expressed Survivin are transplanted into hinder limbs muscle tissue of nude mouse.Paraffin section and frozen section are harvested 2 weeks later to analyze the quantity of regenerative capillary and heterotypic by immunohistochemistry(detect RAECs expressed CD31 positive),and HE staining(observation of nucleus morphology).We confirm the expression of Survivin in the regenerative capillary by double immunofluorescence histochemistry(signing CD 31 and Survivin).Results:Over-expression of Survivin in RAECs promote angiogenesis in the muscle tissue 2 weeks after intramuscular injection and do not induce tumorous lesions such as large nuclear hyperchromatism and karyoplasmic ratio increasing.The regenerative capillary express Survivin and CD31 positive at same time.Conclusion:Ad-GPF/SVV elevate Survivin expression in RAECs and promote its biological activity to obtain a stronger angiogenesis ability,and to a certain extent offset the allogeneic exclusion reponse.