The Research Of Aldehyde Dehydrogenase 2 In Atherosclerotic Foam Cell

Atherosclerosis is the primary cause of cardiovascular diseases.It is characterized by the accumulation of lipids in the large artery wall.During atherosclerosis,circulating monocytes in the blood adhere,roll on the endothelial cells of the artery wall and transmigrate into the subintima to become macrophages under inflammatory response.These macrophages transform into foam cells at the onset of the disease,after exposure to modified lipoproteins such as the oxidized form of low-density lipoproteins(ox-LDL)and uptaking lipids through macrophage scavenger receptors.Foam cell formation plays a critical role in the initiation and progression of atherosclerosis as a component of plaques.Moreover,foam cell death participates in the necrotic core enlargement and aggravates the unstability of the atherosclerotic lesion.The formation of foam cell mainly depends on lipid influx and efflux in macrophage.Numerous scavenger receptors,including scavenger receptor A(SRA),lectin-type oxidized low-density lipoprotein receptor 1(LOX-1),and CD36 are responsible for modified lipids uptake.As a respond to excessive lipids in foam cell,ATP-Binding cassette transporter A family member 1(ABCA1),A-TP-Binding cassette transporter G family member 1(ABCG1)etc.facilitate the efflux of lipids.Moreover,the free cholesterol in foam cell undergoes re-esterification by Acyl-CoA cholesterol ester transferase(ACAT),which can influence cholesterol trafficking and lipid efflux.Aldehyde dehydrogenase 2(ALDH2)is a tetrameric enzyme located in mitochondrial and is present abundantly in liver,kidney and heart.ALDH2 is best known for its metabolism capability of acetaldehyde and it is also a key metabolic enzyme of other reactive aldehydes such as 4-hydroxy-2-nonenal(4-HNE)and malondialdehyde(MDA).People with mutant ALDH2 allele were significantly more frequent in coronary atherosclerosis disease.ALDH2 plays a positive role in endothelial function and revascularization.Moreover,many studies indicated that ALDH2 protects cardiomyocytes from ischemia/reperfusion injury.In our previous study,we found that mice whose ALDH2 was knocked out with lentivirus-ALDH2-RNAi presented more atherosclerotic lesions of mouse aortas and exacerbated unstability of plaques of carotid arteries with increased lipid accumulation.In the present study,we confirmed that ALDH2 knockout with lentivirus-ALDH2-RNAi increased lipid content in the plaques on aortic roots.As foam cell formation is very important to lipid accumulation within atherosclerotic plaque,we hypothesized that ALDH2 plays a role on foam cell formation.To validate this hypothesis and examine the underlying signaling pathways,a series of in vivo and ex vivo experiments were designed and performed.Objectives:1.To study the effect of ALDH2 in the lipid deposition of atherosclerosis.2.To clarify the mechanism of ALDH2 regulating the lipid in foam cell formation and to provide further theoretical support of atherosclerosis pathophysiology.Methods:1.The effect of lipid deposition in ALDH2 deficient mice1.1 The lipid in the atherosclerotic plaque of ALDH2 knockout miceApoE-/-mice were use to establish the atherosclerotic animal model,after lentiviral suspension of GFP,ALDH2 siRNA were injected,the mice were fed a western diet.Aortic roots collection were used for Oil Red staining to test the lipid content.1.2 The effect of ALDH2 in foam cell formation.1.2.1 The extraction and identification of peritoneal macrophage from ALDH2 deficient miceALDH2-/-and ALDH2+/+ mice were intraperitoneally injection of thioglycollate,mouse peritoneal macrophages were harvested.Immunofluorescence assay were used for identify the macrophage and the expression of ALDH2.1.2.2 The effect of ALDH2 deficiency in foam cell formation.After ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Oil Red staining was used for lipid staining.Observation by the microscope and gather the counts of formation of foam cells.1.2.3 The effect of ALDH2 deficiency in foam cell lipid depositionAfter ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,using a TC assay kit to measure the cholesterol OD value for the content of lipid deposition.2.The effect of ALDH2 in foam cell lipid metabolism2.1 The expression of lipid intake related proteinAfter ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Western Blot was used to measure the protein expression of CD36,LOX-1 and SRA.2.1 The expression of lipid efflux related proteinAfter ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Western Blot was used to measure the protein expression of ABCA1,ABCG 1 and AC AT-1.3.The effect of ALDH2 in regulating CD363.1 The role of CD36 in foam cell formationCD36 inhibitor SSO were used before ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Oil Red staining was used for lipid staining.Observation by the microscope and gather the counts of formation of foam cells.3.2 The effect of CD36 inhibition in foam cell lipid depositionCD36 inhibitor SSO were used before ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+.TC assay kit was used to measure the cholesterol OD value for the content of lipid deposition.3.3 The effect of ALDH2 on CD36 mRNAAfter ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Trizol assay was used to extract the total RNA and RT-PCR was used to test the mRNA level of CD36.3.4 The effect of ALDH2 on CD36 transcriptional regulation factor PPAR?After ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Western Blot was used to measure the protein expression of PPAR?.4.The role of ALDH2 on apoptosis of foam cell4.1 The effect of ALDH2 on apoptosis of foam cellAfter ox-LDL stimuli the macrophage of ALDH2-/-and ALDH2+/+,Tunel assay was used to detect the apoptosis of foam cell and Western Blot was used to measure the expression of pro-apoptosis protein Activated Caspase-3.4.2 The effect of ALDH2 on toxic aldehydesMDA detection kit was used for measure the content of MDA in foam cell.Western Blot was used for detecting the 4-HNE addution of total proteins in foam cell.4.3 The effect of ALDH2 on foam cell apoptosis related signal pathwayWestern Blot was used for detecting the apoptosis related signal pathway p-AKT/AKT and p-P38/P38.Results:1.The effect of lipid deposition in ALDH2 deficient mice1.1 The lipid in the atherosclerotic plaque of ALDH2 knockout miceThe apoE-/-mice with ALDH2 siRNA showed more lipid deposition in atherosclerotic of aortic roots.1.2 The effect of ALDH2 in foam cell formation.1.2.1 The extraction and identification of peritoneal macrophage from ALDH2 deficient miceImmunofluorescence assay showed the peritoneal cells from ALDH2-/-mice were ALDH2 deficient macrophages and the peritoneal cells from ALDH2+/+ mice were ALDH2 normal macrophages.1.2.2 The effect of ALDH2 deficiency in foam cell formation.After ox-LDL stimuli,both ALDH2-/-and ALDH2+/+ macrophage form foam cell and the ALDH2-/-foam cells formation were less than ALDH2-/-.1.2.3 The effect of ALDH2 deficiency in foam cell lipid deposition Cholesterol content in ALDH2-/-was less than ALDH2+/+.2.The effect of ALDH2 in foam cell lipid metabolism2.1 The expression of lipid intake related proteinAfter ox-LDL stimuli,the CD36 protein expression of ALDH2-/-were less than ALDH2+/+,while the protein expression of LOX-1 and SRA were same in two group.2.1 The expression of lipid efflux related proteinAfter ox-LDL stimuli,the protein expression of ABCA 1,ABCG 1 and ACAT-1 were same between ALDH2-/-and ALDH2+/+.3.The effect of ALDH2 in regulating CD363.1 The role of CD36 in foam cell formationAfter CD36 inhibitor SSO stimuli,there were no differences between ALDH2-/-and ALDH2+/+ foam cell formation.3.2 The effect of CD36 inhibition in foam cell lipid depositionAfter CD36 inhibitor SSO stimuli there were no differences between ALDH2-/-and ALDH2+/+ cholesterol content in foam cell.3.3 The effect of ALDH2 on CD36 mRNAAfter ox-LDL stimuli,the ALDH2-/-CD36 mRNA expression was decreased compared with ALDH2+/+.3.4 The effect of ALDH2 on CD36 transcriptional regulation factor PPARyAfter ox-LDL stimuli,the protein expression of CD36 transcriptional regulation factor PPARyof ALDH2-/-was decreased compared with ALDH2+/+.4.The role of ALDH2 on apoptosis of foam cell4.1 The effect of ALDH2 on apoptosis of foam cellAfter higher level ox-LDL stimuli,the macrophage of ALDH2-/-showed more apoptosis than ALDH2+/+ and the expression of pro-apoptosis protein Activated Caspase-3 of ALDH2-/-was more than ALDH2+/+.4.2 The effect of ALDH2 on toxic aldehydesAfter higher level ox-LDL stimuli,the macrophage of ALDH2-/-showed more MDA and 4-HNE addution than ALDH2+/+.4.3 The effect of ALDH2 on foam cell apoptosis related signal pathwayAfter higher level ox-LDL stimuli,the p-AKT/AKT signal was activated and the p-P38/P38 signal pathway was inhibited in ALDH2-/-foam cell compared with ALDH2+/+.Conclusions and Significances:1.This study firstly discover the deficiency of ALDH2 decrease foam cell formation by promoting toxic aldehydes which could decrease CD36 transcriptional regulation factor PPARy,thereby reducing lipid intake through the down regulating the CD36.2.This study firstly reveals the lack of ALDH2 activates the apoptosis signal pathway p-P38/P38 and inhibits the proliferation signal pathway p-P38/P38.