Effect Of Platelet Microparticles On The Progression Of Atherosclerotic Lesions In ApoE^-/- Mice And Its Effect On Vascular Endothelial Cell Function

BackgroundAcute cardiovascular and cerebrovascular events,due to rupture or erosion of an atherosclerotic plaque,are one of the leading causes of high morbidity and mortality worldwide.Atherosclerosis is a slowly progressive disease of the vascular wall involving a local accumulation of cholesterol and non-resolving inflammation.The pathogenic events of atherogenesis are characterized by lipid deposition,local oxidative stress,endothelial dysfunction,monocyte/macrophage activation,smooth muscle cell?SMC?migration and proliferation,extracellular matrix?ECM?synthesis.Advanced atherosclerotic lesions associate large necrotic cores with thin fibrous caps,cholesterol deposits,increased local inflammation,intralesional hemorrhage and neovascularization,all of which are closely related with plaque growth and instability.Vulnerable plaque rupture occurs frequently during the evolution of coronary atherosclerotic lesions accompanied by naked vascular tissue and the erosion of necrotic lipid core,which may finally resulting in the release of a large amount of procoagulant substances and activation of the coagulation system and platelets.The recruited platelets accumulate at the site of plaque rupture,leading to formation of thrombus and obliteration of vessels lumen.Numerous studies have reported that platelets are procoagulant mediators as well as important inflammatory and immune cells,contributing to in the formation and progression of atherosclerotic lesion.A large number of platelet microparticles?PMPs?,with a diameter of 0.1¹.0?m,are released into circulation when platelets experience activation or apoptosis.PMPs account for about70%⁹0%of circulating extracellular vesicles,containing glycoproteins,p-selectin,bioactive lipids and miRNA et.al derived from platelet activation,are thought to play a more lasting and extensive pathophysiological role in the progression process of AS.In our previous studies,we have found that staphylococcal superantigen-like protein5?SSL5?-activated PMPs increased the expression of inflammatory cytokines such as IL-1?,TNF?,MCP-1,MMP-9 in monocytes,thereby aggravating the inflammatory response of atherosclerotic plaque.Recently,we also demonstrate that PMPs promote angiogenesis by up-regulating MMPs expression?especially MMP-2?in ECs via mechanism extending beyond the direct delivery of angiogenic factors.Therefore,we hypothesized that PMPs in vivo and vitro could be promote the development of the AS plaque lesions and further result in atherosclerotic plaque destabilization implicated in aggravated inflammation,intralesional neovascularization and endothelial dysfunction,thus laying a foundation for further understanding of the important functions of PMPs in the body and the role of PMPs in cardiovascular diseases.Methods1.Forty male apolipoprotein E knockout(ApoE^-/-)mice,aged 8 weeks and fed for 4weeks with high-fat diet,were randomly divided into four groups?n=10 in each group?according to the table of random digit.The mice were respectively injected with PBS?PBS group?,PMPs-free supernatants?SUP group?,low-dose PMPs?LDP group?and high-dose PMPs?HDP group?once a week through tail vein injection for 8 weeks.At the end of the experiment,the mice were euthanized and the blood samples were collected to detect blood routine,blood lipids,liver and kidney function and inflammatory factors including CRP,IL-1?,TNF-?.;The aorta and brachiocephalic trunk were sampled to detect the atherosclerotic plaque formation.Oil Red O staining,H&E staining,Masson staining and immunohistochemistry?CD68,MMP-9,?-SMA?were used to examine the stability of the atherosclerotic plaque in each group.2.Culture human microvascular endothelial cells?HMECs?,Tyrode's buffer as blank control?Con?,PMPS-free supernatant isolated from PMPs preparation as solvent control?Sup?,detect PMPS pairs by ELISA The expressions of endothelial function-related factors PGI2 and TXA2 in HMECs were detected by immunofluorescence probe assay.The expression of phosphorylated eNOS and ET-1 was detected by Western Blot.The effects of PMPs on the function of endothelial cells of HMECs were studied.Results1.Effect of PMPs on the formation and stability of atherosclerotic plaques in ApoE^-/-mice.?1?PMPs had no effect on body mass,blood routine?WBC,RBC,PLT,HGB?,blood lipid?TG,TCH,LDL,HDL?,liver function?ALT,AST?,renal function?UN,Cr?and other indicators in mice.?2?Compared with the blank control group and SUP group,PMPs group can promote the formation of AS plaque in mice,increase the proportion of lipid core in the total plaque area,improve the infiltration of macrophages in the plaque,increase the expression of mmp-9 in the plaque,and reduce the collagen content and smooth muscle cell content in the plaque area.?3?Compared with the blank control group and SUP group,the expression levels of serum CRP,IL-1?and TNF-?were significantly up-regulated in the PMPs group.?4?PMPs elevated the expression of TXA2 and ET-1 in HMECs,accompanied by lower NO production and decreased expression level of phosphorylated eNOS?p-eNOS?and PGI2.ConclusionsPMPs can promote the formation and instability of AS plaques in high-fat ApoE^-/-mice manifested by local macrophage infiltration,enhanced inflammatory reactions,reduced smooth muscle cells and collagen content.PMPs increased the expression of TXA2 and ET-1 in HMECs,inhibited the phosphorylation of eNOS,and decreased the expression of NO and PGI2,leading to deteriorated endothelial function.This study found that PMPs can promote the development of atherosclerotic lesions and reduce plaque stability in mice.In vitro,PMPs increased endothelial cells dysfunction.Our studies clarified the role of PMPs in cardiovascular diseases,which may contribute to provide novel therapeutics for prevention and treatment of AS.