Mechanism Of Vascular Smooth Muscle Nox4 And SERCA2 Involved In Vascular Remodeling

Cardiovascular disease is the type of disease that causes the most deaths in the world,including aortic aneurysm,atherosclerosis,pulmonary hypertension,etc.Vascular remodeling is the common pathological basis of these diseases,especially the remodeling of vascular smooth muscle layer.Massive generation of reactive oxygen species(ROS)and the inactivation of some downstream enzymes are the key to vascular remodeling.NADPH oxidase is the major enzyme to generate ROS.NADPH oxidase 4(Nox4)is most abundantly expressed in the vascular system and is the main source of hydrogen peroxide.Sarcoplasmic/endoplasimic reticulum calcium ATPase(SERCA)is a vital enzyme for maintaining calcium homeostasis in cells.SERCA2 is the main subtype of SERCA in blood vessels.Cysteine at position 674(cysteine 674,C674)is the crucial redox site that regulates the activity of SERCA2.Under physiological conditions,the sulfhydryl group on C674 is modified by glutathione under the action of nitric oxide to increase the activity of SERCA2.Under the pathological conditions where a large amount of ROS is generated,C674 is irreversibly oxidized(C674-SO₃H),which hinders the glutathione modification of this site and interferes with the function of SERCA2.In vascular smooth muscle,Nox4 is the major source of ROS that leads to the irreversible oxidation of C674 in SERCA2.This topic explored the mechanism of Nox4 involved in aortic aneurysm and atherosclerosis from the perspective of vascular smooth muscle,and the mechanism of irreversible oxidative inactivation of C674 in SERCA2 involved in pulmonary vascular remodeling.PART 1:the mechanism study of smooth muscle Nox4 in angiotensin ?(angiotensin ?,Ang ?)-induced aortic aneurysm and atherosclerosis.Osteopontin(OPN),collagen ?&?(Col ?&?),matrix metalloproteinases(MMP)2,vascular cell adhesion molecule 1(VCAM1)are the important proteins involved in vascular remodeling.In aortic smooth muscle cells(SMC),Ang ? significantly up-regulates the expression of Nox4,OPN,MMP2,Col ?&? and VCAM1.It is speculated that Ang ? promotes aortic aneurysm and atherosclerosis formation by up-regulating the expression these vascular remodeling-related proteins.We found that downregulating Nox4 decreased the expression of OPN,Col ?&?,and MMP2,while overexpressing Nox4 increased the expression of these proteins in aortic SMC.These results indicate that Nox4 directly regulates the expression of these vascular remodeling-related proteins in the aortic SMC.In addition,treatment with exogenous OPN increases the expression of MMP2,Col ?&? and VCAM1 in aortic SMC.It is speculated that OPN mediates the regulation of smooth muscle Nox4 on vascular remodeling-related proteins.In human aortic aneurysm and Ang ?-induced mouse aortic aneurysm and atherosclerosis samples,the expression of Nox4 and OPN increased in layer of smooth muscle.It is speculated that smooth muscle Nox4 may participate in the process of aortic aneurysm and atherosclerosis formation by regulating OPN.To clarify whether smooth muscle Nox4 is involved in the process of aortic aneurysm and atherosclerosis formation and its regulatory mechanism.In the FVB/N genetic background,we construct a smooth muscle specific overexpression of human Nox4 dominant negative(SDN)mouse to antagonize the expression of Nox4 raised in some pathological cases.In the Ang ?-induced aortic aneurysm and atherosclerosis model,compared with non-transgenic mice(NTg)littermate control mice,the incidence of aortic aneurysm,the number and area of aortic aneurysms,the collagen deposition and degradation of elastin were significantly decreased in SDN mice.In addition,the Oil red O positive staining atherosclerotic lesions also decreased.In the aorta and aortic SMC,compared with NTg mice,the expression of OPN,MMP2 and Col ?&? decreased in SDN mice.In SDN aortic SMC,treatment with exogenous OPN increased the expression of MMP2,Col ?&? and VCAM1 and promoted cell proliferation,migration and macrophage adhesion to SMC,which further proved that OPN mediates the regulation of smooth muscle Nox4 on vascular remodeling-related proteins.These results indicate that the up-regulated smooth muscle Nox4 in pathological conditions is directly involved in aortic aneurysms and atherosclerosis formation.One of the important mechanisms is to increase the expression of vascular remodeling-related proteins by up-regulating OPN,leading to vascular remodeling.PART 2:the mechanism study of pulmonary vascular remodeling caused by the irreversible oxidative inactivation of C674 in SERCA2.Pulmonary hypertension(PH)is a common cardiopulmonary vascular remodeling disease,which threatens human health.ROS is closely related to pulmonary vascular remodeling and PH,but the regulatory mechanism is still unclear.We found that C674-SO₃H in SERCA2 significantly increased in hypoxia-induced PH.It is speculated that the ROS generated by hypoxia can aggravate the irreversible oxidation of C674,causing the inactivation of the redox status of C674,which impaired the function of SERCA2,thereby promote pulmonary vascular remodeling and PH.In order to explore the relationship between the irreversible oxidative inactivation of C674 in SERCA2 and pulmonary vascular remodeling and PH,we mutated the cysteine at position 674 to serine(C674S)and constructed the SERCA2 C674S knock-in(SKI)mice on the background of C57BL/6J,to simulate the inactivation of C674 under pathological conditions.Compared with wild type(WT)mice,the right ventricular systolic pressure(RVSP),right ventricular end diastolic pressure(RVEDP),and right ventricular hypertrophy index increased with age in SKI mice.SKI mice had obvious pulmonary vascular remodeling,such as the tunica media thickness increased,muscularization of distal pulmonary arterioles,the percentage of cell proliferation index Ki67 positive cells increased,neointimal lesions,plexiform lesions,thrombosis,and pulmonary venous hypertrophy,all increased with age.Pulmonary artery smooth muscle cells(PASMC)is involved in most pulmonary vascular remodeling.Therefore,we explored the mechanism by which C674 inactivation in SERCA2 regulates pulmonary vascular remodeling from the perspective of PASMC.In PASMC,C674 inactivation does not affect the m RNA and protein expression levels of SERCA2,of which SERCA2b is the main genotype and increases the intracellular Ca²⁺,which indicates that C674inactivation impaired with the function of SERCA2.C674 inactivation increased the expression of cell cycle-related proteins,including cyclin(A1,A2,B1)and cyclin-dependent kinase(CDK)(CDK 1 and CDK 2),accelerated cells cycle and promoted cell proliferation.It was speculated that C674 inactivation accelerated the cell cycle and promoted the proliferation of PASMC by increasing the expression of these cell cycle-related proteins.C674 inactivation induces endoplasmic reticulum stress,by activating inositol-requiring enzyme-1?-(IRE1?)-spliced X-box-binding protein-1(XBP1s)pathway.In WT PASMC,overexpression of XBP1s increases the expression of cell cycle-related proteins and promotes cell proliferation.In SKI PASMC,all of the endoplasmic reticulum stress inhibitor 4-PBA,IRE1?-XBP1s pathway inhibitor 4?8C,and SERCA2 activator CDN1163 can decreased the expression of XBP1s and cell cycle-related proteins and inhibits the process of cell cycle and proliferation.In WT PASMC,overexpression of the C674 mutant(S674)of SERCA2a or SERCA2b can activate the IRE1?-XBP1s pathway and promotes cell proliferation,indicating that the inactivation of the C674 site in SERCA2a or SERCA2b is associated with the regulation of PASMC proliferation.In SKI PASMC,overexpression of SERCA2b inhibits the IRE1?-XBP1s pathway and cell proliferation,indicating that SERCA2b is very important in regulating the proliferation of PASMC.In vivo,we found that 4?8C and CDN1163 significantly improved pulmonary vascular remodeling in SKI mice and inhibited the expression of XBP1s and cycle-related proteins in lung tissues.It was further demonstrated that C674 inactivation in SERCA2 increases the expression of cell cycle-related proteins,promotes cell proliferation,and causes pulmonary vascular remodeling by activating the IRE1?-XBP1s pathway.In summary,we studied the mechanism of Nox4-derived ROS and irreversible oxidative inactivation of C674 in vascular smooth muscle in vascular remodeling.In the aorta,OPN mediates the regulation of smooth muscle Nox4 on vascular remodeling related proteins and the process of Ang ?-induced aortic aneurysm and atherosclerosis formation.In pulmonary vessels,inactivation of C674 in SERCA2 promotes cell proliferation and pulmonary vascular remodeling by activating the IRE1?-XBP1s pathway.Intervention for vascular smooth muscle Nox4 and SERCA2 and their downstream pathways can improve vascular remodeling under pathological conditions.