P185	LDL from diabetic and hypercholesterolemic patients induce an increase in scavenger receptors CD36 and LOX-1 gene expression in human endothelial cells.
1C.Stancu, 1G.Botez, 1D.Alexandru, 2M.Vladica, 1A.Sima
1Institute of Cellular Biology and Pathology “Nicolae Simionescu”, Bucharest, RO; 2Institute of Diabetes, Nutrition and Metabolic Diseases “N.C. Paulescu", Bucharest, RO.

Background. Dyslipidemia is an important component of the constellation of abnormalities characteristic for the metabolic syndrome, the latter being a major risk factor for both type 2 diabetes mellitus and cardiovascular disease. Aim. Our objective was to determine biochemical modifications in circulating lipoproteins (Lp) from patients with dyslipidemia and to evaluate their biological effect on human endothelial cells. Methods. Three groups of subjects were involved: (i) 5 diabetic patients (D), (ii) 5 hyperlipemic patients (H), and (iii) 4 control healthy subjects (N), the first 10 subjects being obese, while controls were normal-weight. Serum total cholesterol (C), triglycerides (TG), and glucose levels were measured with commercial kits. Lp were separated by FPLC (Pharmacia) on a Superose 6 column and characterized by C, TG and protein content. The biological effect of D, H and N sera was assessed on human endothelial cells (EAhy926); intracellular accumulation of esterified cholesterol (EC) was measured with a Wako kit and the gene expression for scavenger receptors CD36 and LOX-1 was evaluated by Real-time PCR (MJ Research), using the appropriate primers. In addition, in vitro oxidized and non-enzymatically glycated (in the presence of EDTA and BHT) low density lipoproteins (LDL) isolated from normal human sera were incubated with EAhy926 for 24h, followed by measurement of EC content and of gene expression for CD36 and LOX-1. Results. C and TG content of Lp from H and D sera differed from N sera: an increase in the percentage of C in VLDL and LDL, and a decrease in high density lipoproteins (HDL) C were determined; TG levels increased in VLDL and decreased in LDL and HDL. FPLC profiles suggested a modification of LDL and HDL from H and D sera, compared to N sera, the characteristic peaks being shifted towards smaller particles. Sera from D and H patients induced a 2-3 fold increase in the EC content of EAhy926, and a 3 fold increase in CD36 gene expression, compared with N sera. Glycated LDL incubated with EAhy926 induced increases in: EC content (x2), CD36 mRNA (x12) and LOX-1 mRNA (x5). Oxidized LDL incubated with EAhy926 induced increases in: EC content (x3), CD36 mRNA (x5) and LOX-1 mRNA (x10). Conclusions. The accumulation of EC in EAhy926 by incubation with H and D sera might be due to the increase in the gene expression of CD36 and LOX-1, as was demonstrated by incubating EAhy926 with in vitro glycated or oxidized LDL.

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