| Objective:Vascular smooth muscle cells?VSMCs?proliferation and migration play a fundamental role during the process of hypertensive angiopathy.Angiotensin-??Ang-??is one of the robust phenotype-modulating agents,which changes VSMCs to efficiently proliferate and migrate.The mechanism of the proliferation and migration is not well understood yet.Autophagy is the major intracellular process for lysosome-mediated dynamic recycling system.The purpose of autophagy is to eliminate the damaged cytoplasmic material and produce new building blocks and energy for cellular renovation and homeostasis.Recent studies have approved that autophagy is a critical determinant in the changes of VSMC proliferative and migratory properties.Septin4,as a member of GTP binding protein family,is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes.But it is still nounknown yet that if Septin4 plays a role in the cardiovascular diseases.We designed experiments to explore if Septin4 was involved in VSMCs proliferation and migration induced by Ang-?.Furthermore,we discussed the mechanism whether it was related to autopahgy activity.Methods:1.We used mice as animal model.Osmotic pumps with Ang-??1.5mg/kg/day?or 0.9%NaCl as control were implanted subcutaneously.The aortas of the mice were harvested after 14 days,which were further stained by hematoxylin-eosin?HE?and Masson protocols.2.After aorta tissue was lysed,Septin4 protein amount was evaluated by Western blot assay.3.Human aortic smooth muscle cells?HASMCs?were cultured and Ang-? at concentration gradient as0M,10-8M,10-7M,10-6M,10-5M were added and incubated for48 hours.CCK-8 and transwell tests were conducted in order to evaluate the proliferative and migratory activity.Septin4 protein amount was evaluated by western blot assay.At the same time,the most appropriate treating concentration of Ang-? was screened.4.Overexpressing Septin4 was performed by plasmid transfection.Ang-? at concentration gradient as0M,10-8M,10-7M,10-6M,10-5M were added and incubated for48 hours.CCK-8 assay was conducted to evaluate the proliferative activity.We used10-6 M Ang-? as the best treating concentration to incubate and perform transwell test to examine the migratory activity.5.We set 4 groups as follows:Flag-control+0 M Ang-?,Flag-control+10-6 M Ang-?,Flag-Septin4+Ang-?+0 M Ang-?,Flag-Septin4+10-6 M Ang-?.PCNA,col-I,MMP2,p62,LC3 protein amount was evaluated by Western blot assay.6.We designed six groups as follows,Flag-control,Flag-control+Ang-?,Flag-control+Ang-?+chloroquine?CQ?,Flag-Septin4,Flag-Septin4+Ang-?,Flag-Septin4+Ang-?+CQ.PCNA,col-I,MMP2,p62,LC3 protein was calculated through western blot assay.Besides,CCK-8 and transwell tests were performed to evaluate the proliferative and migratory activity.7.Mass spectrometry?MS?was conducted to test protein binding to Septin4.Results:1.Ang-? increased Septin4 expression,the intima-media thickness and collagen content in mice aortas.2.With the increase of Ang-? concentration,Septin4 expression was increased,as well as the capacity of VSMCs proliferation and migration.3.Stimulation effects of Ang-? on proliferation and migration were alleviated by Septin4overexpression.4.Stimulation effects of Ang-? on autophagy were inhibited by Septin4 overexpression.5.Ang-? stimulated proliferation and migration in Septin4 overexpressing VSMCs were further inhibited by autophagy inhibitor.6.Mass spectrometry?MS?showed 14 protein binding to Septin4.By literature analysis,we speculated that binding to CCT2 may be the mechanism by which Septin4 modulated autophagy.Conclusion:Septin4 was involved in the VSMCs switching to a proliferative and migratory state stimulated by Ang-? through modulating autophagic activity and the mechanism may be binding to CCT2. |
PDF Full Text Request