Structure, function and atherogenicity of apolipoprotein B containing lipoproteins

[摘要] Focal deposition of lipids in the form of lipoproteins is an important step in the pathogenesis of the early, fatty streak and the later advanced atherosclerotic plaque. In this thesis, two mechanisms potentially leading to lipid accumulation are examined, namely the interaction of arterial wall proteoglycans with lipoproteins and LDL oxidative modification. In study 1, chondroitin sulphate proteoglycan (CS-PG) was dissociatively extracted and purified from human aorta in guanidine-HCl. This was incubated with LDL and the extent of complex formation was determined by measuring turbidity at 600nm. The interaction of total LDL (d 1.019-1.063g/ml) was examined in 59 angiographically positive coronary heart disease patients. The extent of APG-LDL complex formation was positively correlated with the percentage of small, dense LDL-III within total LDL and with plasma triglyceride level, while it was inversely related to the percentage of the large buoyant LDL-I within total LDL and plasma HDL cholesterol. In the second study the reactivity of apoB containing lipoproteins towards APG was examined in three groups of subjects whose LDL subfraction profile consisted of mainly LDL-I, LDL-II or LDL-III. Two fractions from each lipoprotein class were isolated by cumulative density gradient centrifugation (VLDLl Sf 60-400, VLDL2 Sf 20-60, IDLl Sf 16-20, IDL2 Sf 12-16, LDLA Sf 8-12 and LDLB Sf 0-8. A similar pattern of reactivity was found in all subjects in which IDL2 and LDLA were the most reactive species towards APG followed by LDLB and IDLl and the least reactivity was found in the VLDL fractions. The magnitude of the reactivity of the lipoproteins was highest in the CHD group of patients with a predominance of small, dense LDL, intermediate in the group who had an LDL-II dominated subfraction profile and lowest in the group with the mainly high level of LDL-I. Lipid lowering treatment with ciprofibrate (100mg/day for 8 weeks) decreased the reactivity of all apoB containing lipoproteins towards APG, this was attributed to the triglyceride lowering effect of the drug. Neutral carbohydrate and sialic acid concentration were determined in all the apoB containing lipoproteins in study 4. A higher concentration of neutral carbohydrate, as measured by the phenol sulphuric acid method, was found in LDL-I compared with the small, dense LDL-III subfraction. Sialic acid concentration was highest in VLDLl and lowest in LDLB in all subjects. Neither the neutral carbohydrate nor sialic acid correlated significantly with the extent of APG-Lipoprotein complex formation. This was confirmed by subfractionation of LDL into 3 subfractions by affinity chromatography on Con- A on the basis of the high mannose structure of apoB. An unbound, a weakly bound and a strongly bound fraction were obtained. Each had similar binding reactivity towards APG. Finally a series of LDL modifications were performed to explore the mechanism of APG-LDL interaction. Neuraminidase treatment of LDL increased its reactivity towards APG by 76% and addition of GM3 ganglioside from plasma decreased it by 12%. Charge modification by cyclohexandione treatment and carbamylation blocked the interaction as did reductive methylation which alters the apoB protein structure. LDL incubation with apoE2 and apoE3 also blocked its binding with APG while apoCIII addition had no effect. These findings suggested that although APG-LDL interaction is charge related, conformation of apoB is critical in controlling the interaction process. The second major study was determination of the effect of Evening Primrose Oil (EPO) on plasma lipid levels and LDL subfraction profile and LDL susceptibility to oxidative modification in a group of non-insulin dependent diabetics (NIDDM). At a dose of 500mg/day no significant effect was found on lipid parameters. However the extent of in-vitro copper catalysed LDL oxidation measured by fluorescence at 430 was significantly increased after EPO treatment and was positively related to the percentage of small dense LDL within total LDL and plasma triglyceride concentration. In conclusion the main findings from the studies provide evidence that the heterogeneity of apoB containing lipoproteins as seen in an atherogenic lipoprotein phenotype (ALP) is an important determinant of the atherogenic properties of the lipoprotein. The potential atherogenicity of small dense LDL is linked to its enhanced oxidative modification and its preferential association with APG. These can be modulated pharmacologically.

[发布机构] University:University of Glasgow

[关键词] Molecular biology