Study On The Role Of CD73 In Artherosclerosis

Ecto-5’-nucleotidase(CD73) is a glycosylphosphatidylinositol (GPI)-anchored protein, which can metabolize adenosine 5’-monophosphate (AMP) to adenosine. Adenosine released can activate receptors(A1R, A2aR, A2bR, A3R) to control inflammation reaction, dilate blood vessels, enhance endothelial barrier function, stimulate angiogenesis, inhibit platelet aggregation and reduce production of free radicals. CD73 has been found associated with biosynthesis of lipid droplets (LDs) and adiposomes. Hyperlipidemia is a risk factor for arteriosclerosis. Based on this, it suggests an important role of CD73 in the progress of AS. However, how CD73 influence the progress of AS remains unknown. In this study, CD73-/-ApoE-/-m ice were developed by crossbred, backcross and intercross of the CD73-/- and ApoE-/- mice. By feeding with high-fat diet, atherosclerotic mouse models were developped to study the role of CD73 on arteriosclerosis in vivo. Our research laid the basis for further study of effect and mechanism of CD73 on AS and put forward a potential target for treatment of AS.This study includes three parts:1.Cultivation of CD73-/-ApoE-/- mice. We got the first generation F1 by crossing CD73-/- mice with ApoE-/- mice of the same genetic background. The F1 mice hybridize backcross ApoE-/- mice to generate the second generation (F2). CD73+/-ApoE-/- mice of F2 hybridize intercross to generate the third generation (F3). We screened CD73-/-ApoE-/- mice from F3. Genetypes of mice were detected by PCR. The band of 280bp or 245bp is the product respectively for CD73-/-or ApoE-/- mice by gel electrophoresis.2.Atherosclerotic mice model construction. Atherosclerotic models of 4-week-old CD73+/+, CD73-/-, ApoE-/-, CD73-/-ApoE-/- mice were setup by high-fat diet for 8 weeks and 16 weeks. Bodyweight, blood lipid and plaques were tested. It showed no statistical difference between CD73+/+ and CD73-/- mice in bodyweight and blood lipid. Alternatively, bodyweight, total cholesterol, total triglyceride and low density lipoprotein in ApoE-/- mice are much higher than those in CD73-/-ApoE-/- mice, while high density lipoprotein and lipoprotein(a) showed no statistical difference. The aortas from iliac artery branch to the 2cm inferior- left subclavian artery was stained with Sudan IV. Ratios(area of plaque/area of aorta) were much higher in ApoE-/- mice than that in CD73-/-ApoE-/- mice after 16-week-western diet. In summary, these studies suggest that CD73 can increase blood lipid level and maybe involved in the development of atherosclerosis.3.The potential role and mechanism of CD73 in lipid metabolism. Total RNAs were extracted from the livers of ApoE-/- mice and CD73-/-ApoE-/-mice. The mRNA expressions of key enzymes and correlated genes in lipid metabolism were measured by real-time PCR, including FAS, LCAT, HL, ABCA1, SRBI, GPAT, HSL, PPARα, PPARγ, CYP7A1, FDPS. The mRNA levels of LCAT and CYP7A1 were much higher in CD73-/-ApoE-/- mice, while those of HL, PPARa, PPARy and FDPS were much higher in ApoE-/- mice. Rest of the genes diden’t change.Conclusions:1.CD73 affect the formation of atherosclerotic plaque and it is closely related to lipid metabolism.2.CD73 increases blood lipid level and mainly affects total cholesterol, triglyceride and low density lipoprotein while has no effect on high density lipoprotein and lipoprotein(a).3.CD73 affects lipid metabilsm and plaque formation potentially by transcriptional regulation of LCAT, HL, PPARα, PPARγ, CYP7A1 and FDPS.