To Investigate The Role Of Sarco (Endo) Plasmic Reticulum Calcium ATPases 2 (SERCA2) Cysteine 674 Inactivation In Atherosclerosis And The Underlying Mechanisms Involved From The Perspective Of Endothelial Cells

Atherosclerosis is a kind of chronic disease with great social harmfulness,which characterized by deposition of lipids under the endothelium,infiltration of inflammatory cells and neointimal formation.Due to its high morbidity and mortality,atherosclerosis becomes the leading cause of death.So far,there are many theories about the pathogenesis of atherosclerosis,and the most recognized of which is the theory of the injury response of vascular endothelial cells and the derivative inflammation theory.Endothelial dysfunction leads to increased endothelial permeability,for what low-density lipoprotein（LDL）enters the endothelium.Then activated vascular endothelial cells produce a variety of adhesion molecules to recruit monocyte and promote its adhesion to endothelial cells and infiltration.Afterwards monocytes differentiate into macrophages under the stimulation of macrophage colony-stimulating factor.At the same time,various pro-inflammatory factors and growth factors secreted by macrophages stimulate the proliferation and migration of vascular smooth muscle cells to form neointima.Macrophages together with the vascular smooth muscle cells in the neointima further uptake amounts of lipid and transform into foam cells,eventually forming atherosclerosis plaque.The sarco/endoplasmic reticulum calcium ATPase（SERCA）is a key enzyme in regulation of intracellular calcium.Its main function is to pump calcium ions(Ca²⁺)from the cytoplasm to the sarcoplasmic reticulum or endoplasmic reticulum（ER）.ER plays a significant role in maintaining intracellular Ca²⁺homeostasis and ER homeostasis.In patients with left ventricular hypertrophy,heart failure,atherosclerosis and other cardiovascular diseases,the activity or expression of SERCA was significantly downregulated.SERCA2 is a major isoform of SERCA in the vasculature,of which cysteine 674（C674）is the most reactive cysteine redox site to regulate the function of SERCA.C674 is a key glutathionylation site regulated by nitric oxide,a vasodilator,that increases SERCA2 activity and thus relaxes blood vessels;pathological conditions that cause a dramatic increase of reactive oxygen/nitrogen species can cause irreversible oxidation of C674,leading to SERCA2 inactivation and disorders of ER Ca²⁺reabsorption,and participating in the progression of cardiovascular diseases.The irreversible oxidation of C674 was significantly increased in human atherosclerosis vessels.It remains unclear that the contribution of SERCA2 and its C674 irreversible oxidation to atherosclerosis.To test these,we mutated C674 to serine（C674→S674）to mimic the inhibition of glutathionylation at the site by the irreversible oxidation of the C674 locus and built C674S mutated knock-in mice under the control of the SERCA2 promoter（SERCA2 C674S knock-in mice,SKI）.Our previous studies showed that homozygous SKI died at a critical period of blood vessel development.Heterozygous SKI（hereinafter referred to as SKI）have slowed recovery of blood flow after ischemia of the hind limb,suggesting that C674 inactivation is related to pathological angiogenesis disorders,of which SKI endothelial cells dysfunction is involved in the ruined formation of new blood vessels.Since endothelial dysfunction is the initiating factor of atherosclerosis,and it is speculated that the irreversible oxidation of C674 in endothelial cells is directly involved in the progression of atherosclerosis.This study intends to explore the relationship between inactivation of SERCA2 C674and atherosclerosis and its underlying regulatory mechanisms from the perspective of endothelial cells,providing a new theoretical basis and intervention targets for clinical prevention and treatment of atherosclerosis,which is of great theoretical and clinical impotance.Materials and Methods:In order to investigate the relationship between inactivation of SERCA2 C674 and atherosclerosis and its underlying regulatory mechanisms,we backcrossed SKI into the atherosclerosis-susceptible low-density lipoprotein receptor deficient（LDLR-/-）genetic background and fed a high-fat diet to accelerate the progression of atherosclerosis.In the isolated mouse endothelial cells from cononary arteries,the expression of endothelial function and the expression of endoplasmic reticulum stress-related proteins and inflammatory factors were analyzed.SERCA2b is the major gene type of SERCA2 in blood vessels.The effects of overexpression of the defective adenovirus SERCA2b C674S on endoplasmic reticulum stress-related proteins and inflammatory factors were analyzed compared to the defective adenoviral empty vector（Adv）that served as control.Results:（1）There was no difference in the body weight and serum concentrations of cholesterol and triglyceride between SKI/LDLR-/-mice and their littermate controls LDLR-/-mice,but lipid deposition in the aortic arch and aortic root was significantly increased in SKI/LDLR-/-mice,indicating that SERCA2 C674 dysfunction accelerates atherosclerosis.（2）In the isolated and cultured mouse endothelial cells from coronary arteries,compared with endothelial cells from wild type mice（WT）,the ones from SKI:1)the expression levels of endoplasmic reticulum stress-related protein p-eIF2αand CHOP were significantly increased,while the expression level of ERO1αwas decreased,indicating that inactivation of C674 in endothelial cells induces endoplasmic reticulum stress;2)Both the expression of the inflammatory factor VCAM1 and Cav-1,which regulates the expression of inflammatory cytokines,were increased,and macrophages adhesion to endothelial cells was increased;3)The expression of autophagy-related protein LC3 was significantly increased.（3）In the isolated and cultured mouse coronary endothelial cells from WT,compared with overexpression of Adv,overexpression of SERCA2b C674S:1)Significantly increased the endoplasmic reticulum stress related proteins Bip,ATF6,CHOP and LOX-1 that mediates endoplasmic reticulum stress,while the expression of ERO1αwas decreased;2)Significantly increased the expression of inflammatory factor and adhesion molecules such as VCAM1 and ICAM1,and increased the expression of chemokine MCP-1 and inflammatory cytokines TNF-αand IL-6;3)Upregulated the expression of OPN and MMP2 that regulate vascular remodeling and downregulated the expression of TIMP-1 that inhibited MMP activities;4)Upregulated the expression of Cav-1 and downregulated anti-inflammatory factor PPARγ,indicating that inactivation of SERCA2b C674 promotes inflammatory responses in mouse coronary endothelial cells.5)Upregulated autophagy-related protein LC3 and apoptosis-related protein cleaved-caspase 3.Conclusions:The irreversible oxidation of SERCA2 C674 under pathological conditions accelerates the progression of atherosclerosis.In endothelial cells,partial inactivation of SERCA2 C674 induces endoplasmic reticulum stress,activates endothelial cells,and promotes macrophage adhesion to endothelial cells.In endothelial cells,overexpression of SERCA2b C674S induces endoplasmic reticulum stress and increases the expression of inflammatory and apoptosis-related proteins.We hypothesize that the irreversible oxidation of SERCA2 C674 in pathological conditions,causes a decrease in the ER Ca²⁺content that leads to endoplasmic reticulum stress,which induces endothelial cell inflammation and apoptosis,and accelerates the progression of atherosclerosis.