The Relationship Between SERCA2 C674 Inactivation And Underlying Mechanisms In Atherosclerosis From The Perspective Of Macrophage

Cardiovascular diseases have the highest morbidity and mortality among diseases in the world.It is the major cause of death in China.Atherosclerosis is the major pathological basis that causes stroke and coronary heart disease.Atherosclerosis is a chronic progressive disease accompanied with plaque formation that mainly affects the large and middle arteries.Monocyte and macrophage are key players in the development of atherosclerotic plaques by ingesting lipids and secreting pro-inflammatory factors.Increased adherence of monocytes in the circulation to the vessel wall is an early manifestation of atherosclerotic lesions,and macrophage are the major components of atherosclerotic plaques.Sarco/endoplasmic reticulum calcium ATPase?SERCA?is a key enzyme in regulation of intracellular calcium cycle and calcium signaling and is essential for the maintenance of intracellular calcium homeostasis and endoplasmic reticulum homeostasis.SERCA2 is a major subtype of SERCA in the cardiovascular system and is extremely important for the maintenance of cardiovascular homeostasis.The sulfhydryl group located on the cytosolic side of SERCA2 at position 674?cysteine 674,C674?is its primary redox site.Under physiological conditions,glutathionylation of C674 can increase the activity of SERCA2 and thus relax the blood vessels;under pathological conditions,the irreversible oxidation of C674 thiol?C674-SO₃H?leads to dysfunction of SERCA2.Aortic plaques in patients with atherosclerosis have significant irreversible oxidation of C674,and its primary cellular source is macrophage.We speculate that irreversible oxidation of C674 in atherosclerosis-susceptible environment may induce macrophage dysfunction by interfering with calcium homeostasis to activate endoplasmic reticulum stress,thereby accelerating atherosclerosis.This project aims to study the relationship between the inactivation of C674 and atherosclerosis and the underlying regulatory mechanisms from the perspective of macrophage.In order to simulate the inactivation of the C674 under pathological conditions,we constructed a SERCA2 C674S knock-in mice?SKI?on the C57BL/6 background and transferred to atherosclerosis-susceptible low density lipoprotein receptor deficient?LDLR-/-?genetic background,which fed with a high-fat diet to accelerate atherosclerosis.We found that the aortic atherosclerotic plaques were significantly increased in SKI/LDLR-/-mice compared to control LDLR-/-mice,and the cells in the plaques were mainly macrophage,indicating that the inactivation of C674 accelerates the formation of atherosclerosis.To investigate the mechanism by which C674inactivation accelerates atherosclerosis,we performed mechanism studies in the cultured bone marrow-derived macrophages.In the basic condition,compared with macrophages from LDLR-/-mice,the ones from SKI/LDLR-/-mice had increased expression levels of the endoplasmic reticulum stress-related proteins p-PERK,p-JNK,and downstream pro-apoptotic factor CHOP,and had increased expression levels of inflammatory transcription factor p-p65NF?B and adhesion molecule ICAM-1,and increase adhesion to endothelial cells.In macrophage from both LDLR-/-mice and SKI/LDLR-/-mice,oxidized low-density lipoprotein?ox-LDL?and acetylated low-lipoprotein?ac-LDL?could up-regulate the expression levels of scavenger receptor CD36 and endoplasmic reticulum stress-related proteins p-PERK and p-JNK,respectively,and increased the expression levels of the inflammatory transcription factor p-p65NF?B and adhesion molecules?VCAM-1 and ICAM-1?,which were more pronounced in macrophages from SKI/LDLR-/-mice.In mice fed with a high-fat diet to accelerate atherosclerosis,we also isolated and cultured macrophages to investigate the effects of C674 inactivation on the expression of endoplasmic reticulum stress-related proteins and adhesion molecules.We found that the inactivation of C674 increased the expression levels of endoplasmic reticulum stress-related proteins?p-PERK,ATF6,IRE1?,BiP?and their downstream pro-apoptotic factors?CHOP and Fas?;increased the expression levels of the inflammatory cytokines IL-6 and adhesion molecules?ICAM-1and VCAM-1?.To investigate whether inactivation of C674 in macrophage increased the expression of inflammatory and adhesion molecules by inducing endoplasmic reticulum stress and whether stimulating SERCA2 activity could ameliorate endoplasmic reticulum stress and inflammatory responses,we have tested the endoplasmic reticulum stress inhibitor 4-PBA and SERCA2 activator CDN1163 in macrophages from SKI/LDLR-/-mice.We found that both 4-PBA and CDN1163 can down-regulate the expression of endoplasmic reticulum stress-related proteins?ATF6,BiP,p-JNK and CHOP?and inflammatory response-related proteins?p-p65NF?B,IL-6,VCAM-1?to some extent.Our preliminary study showed that the inactivation of C674accelerated the formation of atherosclerosis.In macrophage,inactivation of C674accelerates atherosclerosis by inducing endoplasmic reticulum stress,which stimulates the release of inflammatory factors and increases the expression of adhesion molecules,thus promoting macrophage adhesion to endothelial cells and accelerating atherosclerosis.