The Role And Mechanism Of E3 Ubiquitination Ligase WWP2 Modifing HO-1 In Atherosclerotic Vascular Disease Induced By AGEs

Objective:Coronary atherosclerotic heart disease（CHD）,also known as coronary heart disease,is an important cause threatening human life,health and quality of life in the world and the leading cause of death from chronic noncommunicable diseases in low-and middle-income countries.The main cause of CHD is atherosclerosis（AS）,which is driven by lipids.Endothelial cells,smooth muscle cells and leukocytes participate in the chronic pathological process of forming atherosclerotic plaque.Among them,vascular smooth muscle cells（VSMCs）play an important role in the synthesis of foam cells and extracellular matrix.Due to the thickening of arterial wall and the formation of atherosclerotic plaque,the blood flow rate slows down and the flow decreases after the stenosis of vascular lumen.Therefore,the nutrient supply of myocardial tissue in the area covered by blood flow is insufficient and leads to the change of cardiac function.There are many risk factors for CHD.Among them,diabetes mellitus（DM）is one of the increasing risk factors.Rencent data showing that the global DM population is increasing significantly,and the mortality rate is increasing year by year.The incidence of CHD in DM patients is characterized by high invisibility,high incidence rate,early onset,rapid development,and high disability.Advanced glycation end products（AGEs）are non-enzymatic glycosylation products that exist in all organisms and can be derived from in vivo production and in vitro dietary intake.When the accumulation of AGEs in the body exceeds the metabolic level,it will affect the insulin secretion function and cause oxidative stress,inflammation and endothelial damage,which may lead to diabetes mellitus and its complications,including the occurrence of diabetic atherosclerosis.HO-1 is an important antioxidant factor to maintain cell and body homeostasis.At the same time,it can play the role of anti-inflammatory,compression,anti apoptosis,anti-aging and so on.HO-1 can be stimulated by a variety of cytokines,physical or chemical factors,and directly or indirectly regulated by a variety of micro RNAs to participate in different signal pathways.Therefore,HO-1 plays an important role in a variety of growth and development and disease development.It is known that HO-1 can protect myocardial tissue,improve endothelial structure,restore cardiac function and blood perfusion,and resist the damage of external stimulation to cardiovascular system.WWP2 is a HECT type E3 ubiquitin ligase,which is expressed in many body apparatuses and participates in multiple cellular physiological and pathological processes.WWP2 mainly degrades substrate proteins through ubiquitin proteasome pathway to regulate the occurrence and progress of diseases.It can also reverse activate cytokines through single ubiquitination to regulate cell differentiation and protein post-translational modification.At present,WWP2 has been proved to play a variety of functions in tumor cells,bone and cartilage.In cardiovascular disease,WWP2 passes through TGFβ/Smad2 and ubiquitinated degradation PARP1 participate in the regulation of myocardial fibrosis remodeling,promote hypertensive vascular disease by regulating STAT3deacetylation,and play a protective role regulated by PPP1R3A in the early stage of arrhythmia.However,there is no correlation between the role of WWP2 and the role of HO-1 in the pathogenesis of diabetic atheromatous disease.Whether WWP2 can serve as a substrate protein in the regulation of cell function is also not supported by literature.In this study,animal models and cell models were used to investigate the effect of WWP2on the atherosclerosis induced by diabetes mellitus and the proliferation and migration of AGEs induced smooth muscle cells.Methods:1.To confirm the effects of AGEs at different concentrations on the expression of WWP2 and HO-1 in VSMCs.The cells were divided into four groups.Different volumes of AGEs solutions were added to make the concentrations 0,5,10 and15 ng/ml respectively.The expression levels of WWP2 and HO-1 were detected by western blot experiment.2.To confirm the effect of overexpression of WWP2 on the proliferation and migration of VSMCs.The cells were divided into four groups:HA-Control+BSA group,HA-Control+AGEs group,HA-WWP2+BSA group and HA-WWP2+AGEs group.The activity,proliferation and migration of VSMCs were detected by CCK-8 experiment,Transwell experiment,phalloidin staining experiment,and the related indexes of proliferation and migration and the expression level of HO-1 protein were detected by western blot experiment.3.To confirm the effect of WWP2 knockdown on the proliferation and migration of VSMCs.The cells were divided into four groups:Si-Control+BSA group,Si-Control+AGEs group,Si-WWP2+BSA group and Si-WWP2+AGEs group.The cell activity,proliferation and migration ability of VSMCs were detected by CCK-8 experiment,Transwell experiment,phalloidin staining experiment,and the related indexes of proliferation and migration and the expression level of HO-1 protein were detected by western blot experiment.4.To confirm the interaction between WWP2 and HO-1 in VSMCs and the effects of AGEs and MG132 on the interaction.The cells were divided into two groups:WWP2（HO-1）group and control group.The expression level of HO-1（WWP2）was detected by immunoprecipitation assay;After that,the cells were divided into three groups:blank control group,AGEs（MG132）group and Ig G control group.The expression level of WWP2 protein was detected by immunoprecipitation assay.5.To confirm the effect of WWP2 on HO-1 expression level.The cells were divided into4 groups and transfected with 0,2,4 and 6μg HA-WWP2 respectively.The expression level of HO-1 protein was detected by western blot experiment.6.To confirm the effects of MG132 and CHX on the expression level of HO-1 regulated by WWP2.Firstly,the cells were divided into six groups:HA-Control+MG132（CHX）0 hour group,HA-Control+MG132（CHX）3 hour group,HA-Control+MG132（CHX）6 hour group,HA-WWP2+MG132（CHX）0 hour group,HA-WWP2+MG132（CHX）3 hour group and HA-WWP2+MG132（CHX）6 hour group.The expression level of HO-1 was detected by Western blot;Then the cells were divided into six groups:Si-Control+MG132（CHX）0 hour group,Si-Control+MG132（CHX）3 hour group,Si-Control+MG132（CHX）6 hour group,Si-WWP2+MG132（CHX）0 hour group,Si-WWP2+MG132（CHX）3 hour group and Si-WWP2+MG132（CHX）6 hour group.The expression level of HO-1 was detected by western blot experiment.7.To confirm the effect of WWP2 on HO-1 ubiquitination.Firstly,the cells were divided into three groups:HA control group,HA-WWP2 group and Ig G control group.HA-Ub was co-transfected into HA control group and HA-WWP2 group.The expression levels of HO-1 and HA were detected by immunoprecipitation assay;Then the cells were divided into three groups:Si-Control group,Si-WWP2 group and Ig G control group.Among them,Si-Control group and Si-WWP2 group were co-transfected with HA-Ub.The expression levels of HO-1 and HA were detected by immunoprecipitation assay.8.Build SM22α-Cre;WWP2^FL/FL mice,diabetic mice were induced by intraperitoneal streptozotocin（STZ）,and blood glucose and body weight were continuously monitored.9.To confirm the effect of WWP2 on atherosclerosis in mice stimulated by hyperglycemia.Mice were divided into SM22α-Cre-;WWP2^FL/FL+SSC group,SM22α-Cre-;WWP2 ^FL/FL+STZ group,SM22α-Cre+;WWP2 ^FL/FL+SSC group,SM22α-Cre+;WWP2 ^FL/FL+STZ group was divided into 4 groups.The thickness of arterial wall was detected by abdominal aortic ultrasound and H&E staining of aortic tissue,the degree of arterial fibrosis was detected by Masson staining,and the proliferation and migration related indexes of aortic vascular tissue and the expression level of HO-1protein were detected by western blot experiment.Results:1.With the increase of AGEs concentration in VSMCs,the expression level of WWP2 increased gradually,while the expression level of HO-1 decreased gradually,and the change was most obvious when the concentration of AGEs was 15ng/ml.2.Compared with the control group,overexpression of WWP2 promoted the proliferation activity and migration ability of VSMCs,made the cytoskeleton more disordered,and the expression level of proliferation migration protein increased,while the expression level of HO-1 protein decreased.At the same time,overexpression of WWP2 expanded the effect of ages on the proliferation and migration of VSMCs.3.Compared with the control group,knockdown WWP2 inhibited the proliferation activity and migration ability of VSMCs,made the cytoskeleton more regular,and the expression level of proliferation migration protein decreased,while the expression level of HO-1 protein increased.At the same time,knockdown WWP2 reduced the effect of ages on the proliferation and migration of VSMCs.4.In VSMCs,WWP2 and HO-1 can bind to each other.Adding AGEs can enhance the binding ability between them.At the same time,MG132 can also enhance the binding ability of WWP2 and HO-1.5.After gradient overexpression of WWP2,the expression level of HO-1 decreased with the increase of WWP2 expression level.6.The expression level of HO-1 increased with the increase of MG132 dosing stimulation time and decreased with the increase of CHX dosing stimulation time,and overexpression or knockdown of WWP2 will affect the effect of MG132 and CHX on the expression level of HO-1.7.The ubiquitination level of HO-1 increased after overexpression of WWP2,and decreased after knockdown of WWP2.8.The mouse genotypes were SM22αCre-;WWP2 ^FL/FLand SM22αCre+;WWP2 ^FL/FL,and after intraperitoneal injection,the blood glucose level of mice continued to be higher than the normal level,and the body weight gradually decreased.9.Compared with the control group,the hyperglycemia group had higher vascular wall thickness and degree of fibrosis,higher expression level of proliferation and migration protein,and lower expression level of HO-1 protein,compared with SM22αCre+;WWP2 ^FL/FL group,SM22αCre-;WWP2 ^FL/FL significantly alleviated the stimulation of hyperglycemia on aortic vascular disease.Conclusion:1.WWP2 can promote the proliferation and migration of VSMCs induced by AGEs,and regulate the decrease of HO-1 expression through ubiquitin proteasome pathway.2.In animal models,WWP2 can promote aortic vascular lesions induced by hyperglycemia and reduce the expression of HO-1 in the blood vessels of mice.