Atorvastatin Rescues Vascular Endothelial Injury In Hypertension By WWP2-mediated Ubiquitination And Degradation Of ATP5A

Objective:Changes in the structure and function of endothelial cells are the common pathological basis of various cardiovascular diseases,such as hypertension,coronary heart disease and aortic aneurysm.The structure and function of vascular endothelium in hypertensive patients are seriously damaged.The damage of target organs such as heart,brain and kidney is closely related to the damage of vascular endothelium in hypertensive patients.It is of great significance to actively explore the treatment methods and mechanisms of vascular endothelial injury.Atorvastatin,a hydroxymethylglutaryl coenzyme A（HMG-CoA）reductase inhibitor,is one of the most commonly used prescription drugs for lowering serum cholesterol levels and preventing cardiovascular diseases in clinical practice.Atorvastatin plays a beneficial role in cardiovascular disease by reducing lipid,anti-inflammatory,reducing cardiac hypertrophy and remodeling.However,the effect of atorvastatin on vascular endothelial injury and function in cardiovascular diseases is still unclear.Here,we explored the effect and mechanism of atorvastatin on vascular endothelial injury.Ubiquitination is a key process in regulating post-translational modifications of proteins（such as apoptosis,inflammation,transcriptional regulation and cell cycle）.E3 ligase is one of the most critical factors and is related to the development of various cardiovascular diseases.Previously,our research team has found that WWP2 is a member of NEDD4E3 ubiquitin ligase and plays a protective role in heart remodeling.Through mass spectrometry analysis,we found a new physiological substrate ATP5A of WWP2.ATP5A is a mitochondrial ATP synthase F1 complex subunitα。ATP synthase is the most important enzyme for mitochondrial ATP synthesis,and its content is extremely rich in mitochondria.Traditionally,ATP5A is often used as an indicator of mitochondrial function.In recent years,it has been found that ATP5A is involved in cell damage through its reverse activity,regulation of calcium ion and formation of mitochondrial permeability transition pore.And the neuroprotective drug J47 plays its role precisely with ATP5A as the target.This study explored the regulation of atorvastatin on WWP2-ATP5A and its mechanism in endothelial injury.Methods:1.To investigate the effect of atorvastatin on Ang Ⅱ-induced vascular endothelial injury in hypertension.We first divided C57BL mice into three groups and administered Na Cl,Ang Ⅱ（2mg/kg/day）and Ang Ⅱ combined with atorvastatin（15mg/kg/day）for 14 days.The thickness of blood vessel wall,the level of fibrosis,the level of oxidative stress,the degree of apoptosis and the level of eNOS protein expression were measured.HUVEC endothelial cells were divided into three groups,and were given control,Ang Ⅱ（10 nmol/L 24h）and Ang Ⅱ（10 nmol/L 24h）combined with atorvastatin（1μmol/L 24h）stimulation.The levels of eNOS and WWP2 protein were detected.2.Discuss the interaction between WWP2 and ATP5A.Firstly,the protein ATP5A interacting with WWP2 was identified by mass spectrometry.HUVEC endothelial cells were used to detect the endogenous binding of WWP2 and ATP5A.HUVEC endothelial cells were divided into two groups.One group overexpressed HA-WWP2,and the other group was used as control to detect the binding of WWP2 and ATP5A.HUVEC were divided into three groups:control group,Ang Ⅱ stimulation group,Ang Ⅱ combined with atorvastatin treatment group,and the binding of WWP2 and ATP5A was detected.HUVEC endothelial cells were divided into four groups:transfected with HA-WWP2 of 0ug,5ug,10ug and 15ug respectively,and the expression of ATP5A was detected.HUVEC endothelial cells were divided into two groups,one group was control,the other group was transfected with siRNAWP2,and the expression of ATP5A was detected.3.To explore the mechanism of action of WWP2 on ATP5A.We divided HUVEC endothelial cells into six groups:siRNA-control+CHX（0h）group,siRNA-control+CHX（4h）group,siRNA-control+CHX（8h）group,siRNA-WWP2+CHX（0h）group,siRNA-WWP2+CHX（4h）group,siRNA-WWP2+CHX（8h）group,and detected the level of ATP5A.We divided HUVEC endothelial cells into six groups:siRNA-control+MG132（0h）group,siRNA-control+MG132（4h）group,siRNA-control+MG132（8h）group,siRNA-WWP2+MG132（0h）group,siRNA-WWP2+MG132（4h）group,siRNA-WWP2+MG132（8h）group,and detected the level of ATP5A.We divided HUVEC endothelial cells into two groups:control group and MG132 group,and detected the binding of WWP2 and ATP5A.We divided HUVEC endothelial cells into two groups:HA-vector group and HA-WWP2 group,and detected the ubiquitination level of WWP2 to ATP5A.We divided HUVEC endothelial cells into two groups:siRNA-control group and siRNA-WWP2 group,and detected the ubiquitination level of WWP2 to ATP5A.4.To explore the mechanism of action of atorvastatin on WWP2-ATP5A in Ang Ⅱ-induced injury.We divided HUVEC endothelial cells into six groups:HA-vector group,HA-vector+Ang Ⅱ（1nmol/L 24h）group,HA-vector+Ang Ⅱ（1nmol/L24h）+atorvastatin（1μmol/L 24h）group,HA-WWP2 group,HA-WWP2+Ang Ⅱ（1nmol/L 24h）group,HA-WWP2+Ang Ⅱ（1nmol/L24h）+atorvastatin（1μmol/L 24h）group,the expression of HA,ATP5A,PARP1,Cleaved-capase3,Bax,Bcl-2 and eNOS were detected.We divided HUVEC endothelial cells into six groups:siRNA-control group,siRNA-control+Ang Ⅱ group,siRNA-control+Ang Ⅱ+atorvastatin group,siRNA-WWP2 group,siRNA-WWP2+Ang Ⅱ group,siRNA-WWP2+Ang Ⅱ+atorvastatin group,and detected the expression of HA,ATP5A,PARP1,Cleaved-caspase3,Bax,Bcl-2,and eNOS,as well as the level of oxidative stress and the degree of apoptosis.5.To explore the regulatory mechanism of atorvastatin in the treatment of vascular endothelial injury in hypertensive rats.First,we constructed vascular endothelial specific knockout WWP2 mice:Cdh5Cre-;WWP2^FL/FL mice and Cdh5Cre+;WWP2^FL/FL mice.We divided the mice into 6 groups:Cdh5Cre-;WWP2^FL/FL+Na Cl+distilled water,Cdh5Cre-;WWP2^FL/FL+Ang Ⅱ（2mg/kg/day）+distilled water,Cdh5Cre-;WWP2^FL/FL+Ang Ⅱ（2mg/kg/day）+atorvastatin（15mg/kg/day）,Cdh5Cre+;WWP2^FL/FL+Na Cl+distilled water,Cdh5Cre+;WWP2^FL/FL+Ang Ⅱ（2mg/kg/day）+distilled water,Cdh5Cre+;WWP2^FL/FL+Ang Ⅱ+atorvastatin（15mg/kg/day）.The diastolic and systolic blood pressure were measured after drug stimulation for 14 consecutive days.On the 15th day after administration,blood vessel tissue was taken and Septin4αExpression levels of SMA,PARP1,Cleaved-capase3,ATP5A,Bax,Bcl-2 and eNOSResults:1.After treatment with atorvastatin,vascular injury,remodeling and dysfunction caused by Ang Ⅱ were improved,but there was no salvage effect on vascular fibrosis caused by Ang Ⅱ stimulation.After Ang Ⅱ stimulation injury,the expression of WWP2 in HUVEC decreased,and the expression of WWP2recovered after atorvastatin treatment.2.There is an interaction between WWP2 and ATP5A.The binding ability of WWP2 and ATP5A increases after the HUVEC cells are injured by Ang Ⅱ stimulation.After treatment with atorvastatin,the binding ability of WWP2 and ATP5A decreases.With HA-WWP2 gradient overexpression,the expression of ATP5A decreased.After knockdown of WWP2,the expression of ATP5A increased.3.After stimulation with CHX,the level of ATP5A protein in the control siRNA group was significantly lower than that in the WWP2-siRNA group over time.After MG132 was used in the control siRNA group,the level of ATP5A protein increased with time;After administration of MG132 in WWP2-siRNA group,ATP5A protein remained at a high level.The binding between WWP2 and ATP5A was enhanced when MG132 was stimulated.After the overexpression of WWP2,the ubiquitination level of ATP5A increased significantly,and decreased significantly after the knockdown of WWP2.4.After HUVEC endothelial cell injury,the expression of ATP5A,PARP1and Cleaved-caspase3 was significantly increased,the expression level of eNOS and the ratio of Bcl-2/Bax were significantly decreased,and the overexpression of WWP2 significantly decreased the expression of ATP5A,PARP1 and Cleaved-caspase3 induced by Ang Ⅱ,and increased the level of eNOS expression and the ratio of Bcl-2/Bax.Compared with Control-siRNA group,WWP2-siRNA group further increased the expression of ATP5A,PARP1 and Cleaved-caspase3induced by Ang Ⅱ,and further decreased the expression level of eNOS and the ratio of Bcl-2/Bax.After atorvastatin treatment,the protein expression changes induced by the above injury basically recovered.Atorvastatin showed the effect on Ang Ⅱ-induced vascular endothelial injury.With the increase of WWP2overexpression,it became less obvious with the knockdown of WWP2.5.We successfully constructed vascular endothelial specific WWP2knockout mice:Cdh5Cre-;WWP2^FL/FL mice and Cdh5Cre+;WWP2^FL/FL mice.The systolic and diastolic blood pressure of mice in Ang Ⅱ stimulation group reached the level of hypertension,and the blood pressure of mice in Ang Ⅱ and atorvastatin combined application group recovered to a certain extent.After Ang Ⅱ stimulation,Septin-4 andα-SMA increased significantly;and comparison with Cdh5Cre-;WWP2^FL/FL mice,in Cdh5Cre+;WWP2^FL/FL mice,Septin-4 andα-SMA increased more significantly;after atorvastatin treatment,the content of Septin-4 was recovered in Cdh5Cre-;WWP2^FL/FL mice,but Cdh5Cre+;WWP2^FL/FL mice did not recover;after atorvastatin treatment,α-SMA content did not recover in Cdh5Cre-;WWP2^FL/FL mice and Cdh5Cre+;WWP2^FL/FL mice.After Ang Ⅱ stimulation,PARP1 and Cleaved-caspase3 increased significantly;compared with Cdh5Cre-;WWP2^FL/FL mice,the increase of PARP1 and Cleaved-caspase3 were found more significant in Cdh5Cre+;WWP2^FL/FL mice;after atorvastatin treatment,PARP1 and Cleaved-caspase-3 recovered in Cdh5Cre-;WWP2^FL/FL mice,but not in Cdh5Cre+;WWP2^FL/FL mice.After Ang Ⅱ stimulation,the expression of ATP5A increased,the expression level of eNOS and the ratio of Bcl-2/Bax decreased;compared with Cdh5Cre-;WWP2^FL/FL,increase of ATP5A in WWP2^FL/FL mice was more significant in Cdh5Cre+;WWP2^FL/FL mice,the expression level of eNOS and the ratio of Bcl-2/Bax decreased more significantly in Cdh5Cre+;WWP2^FL/FL mice;after atorvastatin treatment,the expression level of ATP5A,eNOS and the ratio of Bcl-2/Bax in the vascular tissues of Cdh5Cre-;WWP2^FL/FL mice recovered;but not in Cdh5Cre+;WWP2^FL/FL mice.Conclusion:1.Atorvastatin improves endothelial damage in hypertensive blood vessels by regulating WWP2-ATP5A.2.Atorvastatin upregulates WWP2,promotes the ubiquitination and degradation of ATP5A,stabilizes the apoptosis mechanism of mitochondrial Bcl-2/Bax pathway,thus playing a role in protecting vascular endothelial cells.