Oxidized High Density Lipoprotein Accelerates Atherosclerosis Progression Via NLRPp3 Inflammasome Activation

Atherosclerosis is an inflammatory disease characterized by lipid deposition in the arterial wall.Several clinical and epidemiological researches have indicated the level of high density lipoprotein(HDL)is negatively correlated with atherosclerosis,the atheroprotective effects of HDL include: reverse cholesterol transport,antiapoptotic,antioxidative and anti-inflammatory effects.However,in chronic inflammatory diseases such as type 2 diabetes,metabolic syndrome,chronic kidney disease,and cardiovascular disease,HDL has been proposed to convert to a dysfunctional form.Dyfunctional HDL undergos pronounced structural and functional modification,of which oxidative modification is the most common type.Oxidized high-density lipoprotein(ox-HDL)loses its atheroprotective effects and exerts pro-oxidative and pro-inflammatory effects.Inflammasomes are a class of cytosolic multiprotein complexs involved in innate inflammatory responses that convert pro IL-1β and pro IL-18 to mature forms and initiate pyroptosis via cleaving procaspase-1.Among all inflammasomes,NLRP3 inflammasome has been more extensively be studied and characterized.NLRP3 inflammasome is considered to a link between lipid metabolism and inflammation,cholesterol crystal and oxidized low-density lipoprotein(ox-LDL)activate NLRP3 inflammasome,and oxidative stress,mitochondrial dysfunction,endoplasmic reticulum stress,and lysosome rupture are involved in the processes.Like ox-LDL,ox-HDL could be phagocytosed by macrophages causing lipid deposition,inducing macrophage apoptosis by oxidative stress,and can also up-regulate the expression of VCAM-1 in endothelial cells by NF-κB activation and increase the release of IL-1β,IL-6 and TNF-α in human monocytes and mouse macrophages.However,the underlying mechanisms between ox-HDL and NLRP3 inflammasome is poorly understood.In this study,we investigated the correlation between serum level of ox-HDL and NLRP3 inflammasome in patients with coronary heart disease,the interaction of ox-HDL with macrophages and then investigated the in vivo behavior of ox-HDL in the atherosclerotic-prone modle apo E-/-mice.Objective:1.To investigate serum level of ox-HDL,NLRP3,IL-1β and IL-18 in patients with coronary heart disease,and then to evaluate the correlation between above inflammatory factors and gensini score,and the correlation between serum level of ox-HDL and NLRP3.2.In vitro to detect the intracellular lipid content,the intracellular ROS level,pyroptosis percentage,the protein level of NLRP3 inflammasome,cytotoxicity LDH release and the content of IL-1β,IL-18 in supernatant after macrophage exposed to ox-HDL.3.In vivo ox-HDL was injected to atherosclerotic-prone model apo E-/-mice,total plaque area,lipid and macrophage content,ROS production and the expression of NLRP3 inflammasome in the plaque,and serum level of IL-1β,IL-18 were determined.Methods:1.25 healthy volunteers and 55 patients with CAD were enrolled and divided into three groups: healthy subjects(25 cases),patients with stable coronary artery disease(SCAD,28 cases)and patient with acute myocardial infarction(AMI,27cases)according to the symptoms,coronary angiography,ECG and myocardial infarction markers.Venous blood were drawn to measure serum levels of ox-HDL,NLRP3,IL-1β and IL-18,gensini score was used to evaluated the severity of CAD.Correlation between inflammatory factors and gensini score,and the correlation between serum level of ox-HDL and NLRP3 were studied by using the Pearson coefficient.2.Macrophages were isolated from mouse peritoneal cavity and cultured in 1640 medium,the cells were identified by F4/80 staining,and the cells were exposed to ox-HDL of different concentration gradients for 24 hours.The intracellular lipid content was determined by using oil red O,the intracellular ROS level was measured by DHE stainning,and the percentage of dead cells was measured by annexin V-FITC/PI double staining,protein levels of NLRP3,ASC and caspase-1 were detected by western blot,the cytotoxicity LDH release and the content of IL-1 β and IL-18 in cell supernatant were detected by ELISA kits.3.Apo E-/-mice of 8 weeks were fed with high-cholesterol diet(0.15% cholesterol,21% fat),ox-HDL were injected via tail vein from 12 to 18 weeks,14 mg of HDL protein per kg of body weight,twice a week.All mice were sacrificed at 18 weeks,plaque volume were detected by MOVAT staining,lipid content were observed by oil red O staining,ROS production were detected by DHE stainning,pyroptosis cells in the lesions of the aortic root were detected by TUNEL staining,expression of NLRP3 and caspase-1 were detected by immunohistochemistry,serum levels of IL-1β,IL-18 were detected by ELISA kits.Results:1.Serum levels of ox-HDL in patients with SCAD and AMI were increased compare to the healthy subjects(1052.7±459.1 vs 851.4±408.5pg/ml,p=0.042;1168.4±320.3 vs 851.4± 408.5pg/ml,p=0.006),whereas serum level of ox-HDL in patients with AMI was not significant with ox-HDL in patients with SCAD.Serum levels of NLRP3,IL-1β,IL-18 in patients with SCAD and AMI were increased compare to healthy subjects(p<0.05).Gensini score in patients with AMI was increased compare to patients with SCAD(37.2±27.1 vs 11.1±9.1,p< 0.005).Serum levels of NLRP3,IL-1β,IL-18 were shown to correlate with gensini score in patient with CAD,serum level of ox-HDL was correlate with serum levels of NLRP3,IL-1β,IL-18.2.F4/80 positive cells accounted for 95% of the total cells.Intracellular lipid content,intracellular ROS production,the percentage of Annexin V/PI double positive cells,protein level of NLRP3 inflammasome,the release of LDH and the levels of IL-1β,IL-18 in cell supernatant were increased by ox-HDL in a dose-dependent manner.3.The serum levels of IL-1β,IL-18 in apo E-/-mice with ox-HDL injection were increased,plaque volume,lipid deposition,ROS production,TUNEL positive cells and the expression of NLRP3,caspase-1 in aortic plaque were up-regulated,the number of smooth muscle cell and collagen content were down-regulated.Conclusion:1.The serum levels of ox-HDL,NLRP3,IL-1β,IL-18 in patients with CHD were increased;The serum levels of NLRP3,IL-1β,IL-18 were positively correlated with the gensini score.The serum level of ox-HDL was positively correlated with the serum levels of NLRP3,IL-1β and IL-18.2.ox-HDL increased intracellular lipid deposition and intracellular ROS production in macrophages,and activated NLRP3 inflammasome to promote the release of LDH,IL-1β,IL-18,and promote macrophage pryroptosis depend on caspase-1 activation.3.ox-HDL increased plaque lipid deposition and plaque volume in apo E-/-mice.ox-HDL activated NLRP3 inflammasome via ROS and increased inflammation in plaque, ox-HDL induced macrophage pyroptosis by caspase-1 dependent and accelerated plaque progression.