Role Of P53 SUMOylation-mediated Endothelial Senescence And Apoptosis Evasion In The Development Of Atherosclerosis

Atherosclerosis(AS)is the formation of fibrous fatty lesions in the artery wall,which is an inflammatory disease of blood vessels.The progression of AS is slow,and the duration of disease is long before obvious clinical symptoms appear.If not effectively controlled,AS can further develop into cardiovascular diseases such as coronary heart disease,stroke,and peripheral arterial disease,which causes extremely high morbidity and mortality rates worldwide.As often coexists with vascular senescence,and cellular senescence is one of the major reasons of vascular senescence.Many studies have demonstrated that endothelial senescence occurs in atherosclerotic lesion sites.Endothelial dysfunction caused by endothelial senescence is the pathophysiological basis for the development of atherosclerosis.Advanced oxidative protein products(AOPPs)are the products of oxidative stress.AOPPs are accumulated gradually with age and increased in serum of patients with atherosclerosis.We have confirmed that AOPPs induced premature senescence of endothelial cells.Therefore,We used endothelial cells stimulated by AOPPs as the senescence cell model in this study to explore the internal molecular mechanism of endothelial senescence.This model has not been reported yet,and it is feasible and innovative.Do senescent cells undergo apoptosis or escape from apoptosis?What mechanism determines the fate of senescent cells?This issue has aroused academic controversy.At present,most studies on apoptosis evasion focus on cancers,while studies on endothelial evasion of apoptosis are scarce.Due to the low replacement rate of endothelial cells in the body,it is challenging to proliferate and repair after injury.We assume that the senescent endothelial cells are likely to escape from apoptosis rather than apoptosis,surviving in an senescent state for a long time and accumulate in the body,thus causing vascular senescence and dysfunction.Therefore,it is of great significance to clarify whether senescent endothelial cells escape from apoptosis and elucidate its mechanism for the prevention and treatment of aging-related diseases such as AS.Previous studies on cellular senescence were mostly related to telomerase and SIRT-mediated acetylation.Our previous study found that p53 SUMOylation increased in senescent endothelial cells,suggesting that p53 SUMOylation might mediate endothelial senescence.Studies have shown that the level of p53 and the post-translational modification of p53 play an important and decisive role in the fate of senescent cells.p53 SUMOylation is a novel post-translational modification,which is rarely seen in studies on endothelial senescence.Our study aims to clarify the role p53 SUMOylation in mediating endothelial senescence and apoptosis evasion.Objective:Based on the model of AOPPs-induced endothelial premature senescence,our study aimed to explore the role of p53 SUMOylation in AOPPs-induced endothelial senescence and apoptosis evasion.Methods:Human umbilical vein endothelial cells(HUVECs)were used in our study.AOPPs-induced endothelial premature senescence were used as the cellular senescence model.Transendothelial resistance(TER)and FITC-dextran transendothelial flux were used to detect endothelial monolayer permeability.The proliferation of endothelial cells were detected through CCK-8.Transwell assay was used to detect the endothelial migration.Matrigel assay was used to evaluate tube formation.Immunofluorescence and western blot were used to detect the autophagy level.Senescence-associated beta-galactosidase(SA-β-gal)assay was used to identify the senescent cells.Annexin V/propidium iodide apoptosis assay and flow cytometry were used to detect the apoptotic cells.RAGE inhibitors,autophagy agonists,autophagy inhibitors,RAGE siRNA transfected,and p53 K386 SUMO-deficient virus were used to investigate whether p53 SUMOylation is involved in AOPP/RAGE-induced endothelial senescence and apoptosis evasion.Mouse models of atherosclerosis were established by high-fat diet feeding combined with drugs intraperitoneal injection.The aortas of atherosclerotic mice were stained with Oil Red O for plaque evaluation.Immunohistochemistry,western blot,and immunoprecipitation were used to detect the expression and distribution of vascular proteins associated with senescence and autophagy.Results:Part Ⅰ:AOPP-BSA mediated endothelial barrier dysfunction and impaired angiogenesis1.HUVECs were treated with AOPP-BSA at different concentrations for different time.With the increase of the concentration and time of AOPP-BSA stimulation,the transendothelial resistance value of HUVECs gradually decreased and the leakage amount of FITC-Dextran gradually increased.2.With the increase of the concentration and time of AOPP-BSA stimulation,the proliferation ability of HUVECs decreased gradually,and there was a statistical difference when the concentration reached 200 μg/mL and the stimulation time reached 24 h.3.With the increase of the concentration and time of AOPP-BSA stimulation,the migration of HUVECs decreased gradually.There was statistical difference when AOPP-BS A concentration reached 200_μg/mL and the stimulation time reached 12h.4.When the AOPP-BSA concentration reached 50 μg/mL,the tube formation ability of HUVECs was significantly inhibited.It was difficult for HUVECs to form a complete tubular structure at any time point under the treatment of 50 μg/mL AOPP-BSA.Part Ⅱ:AOPP-BSA mediated endothelial senescence and apoptosis evasion through RAGE1.HUVECs were treated with 200 μg/mL AOPP-BSA for 12 h.Compared with the control group,AOPP-BSA increased the proportion of senescent cells and the expression of senescence-related proteins.2.Cells were treated with a water bath at 55℃ for 10 min to induce apoptosis as positive control.Cell apoptosis rates of all groups were detected by Annexin V-FITC/PI staining.The results showed that AOPP-BSA unchanged apoptosis of endothelial cells,suggesting that AOPP-BSA induced endothelial senescence and apoptosis evasion.3.The proportion of senescent endothelial cells induced by AOPP-BSA decreased significantly after RAGE was knocked down by RAGE siRNA transfection.Part Ⅲ:AOPP-BSA mediated endothelial senescence through autophagy inhibition1.HUVECs were stimulated by AOPP-BSA at different concentrations for different time.AOPP-BSA decreased the expression of autophagy-related proteins like LC3 Ⅱ and Beclin 1 and increased the expression of p62 in HUVECs.Immunofluorescence results showed that AOPP-BSA reduced the autophagosome formation of HUVECs in a time and concentration-dependent manner.2.After the expression of RAGE was knocked down or function of RAGE was inhibited,AOPP-BSA-induced autophagy inhibition was prevented,characterized by the autophagosome formation increased,the expression of LC3 Ⅱ significantly increased and the expression of p62 declined.3.The activation of autophagy with Rapa attenuated AOPP-BSA-induced expression of p21 and p16 in HUVECs.It also significantly decreased the p roportion of β-galactosidase-positive endothelial cells.The inhibition of autoph agy with 3-MA increased AOPP-BSA-induced expression of p21 and p16 in HUVECs.It also significantly increased the proportion of β-galactosidase-positi ve endothelial cells.Part Ⅱ:AOPP-BSA mediated endothelial senescence through p53 SUMOylation1.AOPP-BSA increased the p53 SUMOylation in HUVECs,but unchanged the expression of p53.2.AOPP-BSA-mediated p53 SUMOylation decreased after pretreated by Rapa in HUVECs,while 3-MA pretreated HUVECs increased AOPP-BSA-mediated p53 SUMOylation.3.After pre-transfected with p53 K386-deficient virus,the expression of p21 and p16 was decreased in HUVECs treated with AOPP-BSA,and the number ofβ-galactosidase-positive endothelial cells also decreased significantly.But the level of p53 acetylation at K386 unchanged.Part V:AOPP-BSA mediated vascular senescence through vascular autophagy inhibition1.The aortas of atherosclerotic mice were stained with Oil Red O.Compared with control group and BSA group,the plaque area of the AOPP-BSA group was significantly increased,while that of the AOPP-BSA+Rapa group was decreased.2.Compared with the control group and the BSA group,the expression of LC3 in the aorta of mice in the AOPP-BSA group was decreased,but the expression of p16,p21,and p62 was significantly increased.Compared with the AOPP-BSA group,the expression of LC3 in the aorta of mice in the AOPP-BSA+Rapa group was increased,while the levels of p16,p21,and p62 were decreased.3.AOPP-BSA induced vascular p53 SUMOylation.Besides,autophagy activation could effectively attenuate the AOPP-BSA-induced p53 SUMOylation.4.The results of immunohistochemistry about p16,LC3 and SUMO 1 were consistent with the results of western blot.In addition,the expression of RAGE was significantly increased in both AOPP-BSA group and AOPP-BSA+Rapa group.Conclusions:1.AOPP-BSA induced endothelial hyperpermeability in a dose-dependent and time-dependent manner and inhibited the abilities of proliferation,migration,and tube formation of endothelial cells.2.AOPP-BSA induced endothelial senescence and evasion of apoptosis through RAGE.3.AOPP-BSA inhibited endothelial autophagy and mediated endothelial senescence through autophagy inhibition.4.AOPP-BSA inhibited endothelial autophagy,leading to p53 SUMOylation.p53 K386 SUMOylation mediated AOPP-BSA-induced endothelial senescence.5.AOPP-BSA mediated vascular senescence via autophagy inihibition,thus leading to the acceleration of atherosclerosis.