Role Of NLRP3Inflammasome And Downstream Cytokines In Atherosclerosis

Background: Coronary artery disease (CAD) is the leading cause of mortality inmost developed countries. Atherosclerosis, which is a progressive disorder of the vesselwalls with the formation of plaques throughout the arterial system, is the essentialpathological process of CAD. It is general accepted that a complex endothelialdysfunction induced by free radicals, low density lipoproteins (LDL), infectiousmicroorganisms, shear stress, toxins, hypertension or a combination of these and otherfactors will lead to an chronic inflammatory response which leads to atherosclerosis.Chronic inflammation is recognized as a major driving force in atherogenesis. The sitesof atherosclerotic plaque development in the arterial wall are characterized bycholesterol accumulation and infiltration of peripheral blood monocytes, whichgradually differentiate into macrophages.Pattern recognition receptor (PRR)-mediated signaling pathways have recentlybeen elucidated to bridge innate immune system and atherosclerosis, they can detectpathogen-associated molecular patterns (PAMPs) and endogenous danger signals(DAMPs). Once activated, the monocytes and macrophages secrete a myriad ofcytokines that promote inflammation in the arterial wall. NOD-like receptor family,pyrin domain containing3(NLRP3) is a member of the NLR family. Upon activation,NLRP3recruits the adapter protein and forms NLRP3inflammasome. The NLRP3inflammasome assembles in response to a variety of exogenous and endogenousactivators as an initial priming step，and thereby initiates IL-1β and I-18processing, akey step in the innate immune response. As the role of NLRP3inflamasome in humanatherosclerosis is unclear yet, confirm potential role of NLRP3inflamasome in thedevelopment of atherosclerosis will provide a new theoretical basis for the innateimmune system participating in atherosclerosis development and highlight the potentialof NLRP3inflammasome as a target for prevention and treatment of atherosclerosis. Method: There are two parts in this study: the first part we observed theexpression of NLRP3and downstream cytokines in patients with CAD by ELISA,real-time PCR and Western Blot. Sixty-four CAD patients (patients with stable anginaand unstable angina) and twenty healthy controls were enrolled in this study. In addition,thirty patients with STEMI were recruited in this study. The expression levels ofNLRP3and downstream cytokines were determined at four different time points: within6hours after symptom onset (3.5±1.1h), acute phase at48~72hours (57.0±9.4h),convalescence stage at1week (7±1d) and stable period at4~6weeks (5.1±0.7w); thesecond part we study the role of NLRP3on the cell level, ox-LDL、HDL、ox-HDL wereused to stimulate human mononuclear cell line (Thp-1) and human umbilical veinendothelial cell line (ECV304), then expression of NLRP3and downstream cytokineswere observed. The following, we used NLRP3siRNA to down regulate the NLRP3mRNA expression, again gave ox-LDL、HDL and ox-HDL act on human mononuclearcell line Thp-1, observed the activation of NLRP3and downstream caspase-1、IL-1βandIL-18.Results:1. These results showed that the expression of NLRP3and downstreamcytokines gradually increased along with the increasing severity of CAD;2. NLRP3anddownstream cytokines had a dynamic variation in patients with STEMI;3. ox-LDL、ox-HDL acted on mononuclear cells can promote the NLRP3expression, and furtheractivated the downstream caspase-1, inducd the secretion of IL-1β and IL-18; theactivation effect was concentration dependent, NLRP3and downstream cytokinesdecreased with the treatment of HDL;4.The mRNA and protein expression of NLRP3and downstream cytokines were existed in human umbilical vein endothelial cells(ECV304), ox-LDL and ox-HDL can promote the expression of NLRP3and release ofdownstream IL-1β and IL-18;5. Intervention with NLRP3inflammasome couldsignificantly inhibit the release of downstream IL-1β and IL-18.Conclusion：1. the expression of NLRP3and downstream cytokines graduallyincreased along with the increasing severity of CAD, indicating that NLRP3anddownstream cytokines might be relevant with the severity of CAD；2. NLRP3anddownstream cytokines had a dynamic variation in STEMI, indicating that NLRP3anddownstream cytokines might be involved in the acute inflamatory response of STEMI；3.Lipoprotein acted on human mononuclear cells and endothelial cells can showdifferent degree of activation and inhibition, NLRP3expressed in a variety of cells,inducing the release of inflammatory mediators, fully participated in the occurrence and development of atherosclerosis.