People with diabetes suffer from an increased incidence of atherosclerosis, possibly due to the hyperglycaemia associated with this disease. Glucose may covalently modify proteins via glycation and glycoxidation reactions. Reactive aldehydes (e.g. methylglyoxal and glycolaldehyde) generated from these glycation and glycoxidation reactions, lipid peroxidation and other metabolic pathways may also modify proteins in glycation and glycoxidation reactions. These reactions can result in the formation of advanced glycation end-products, which are increased in diabetes and associated complications such as atherosclerosis. Low-density lipoproteins (LDLs) are the main source of lipid in atherosclerotic plaques, and the lipid-laden foam cells contained within. Modification of the single protein in LDL, apolipoprotein B-100 (apo B) by glucose and aldehydes may result in recognition of these altered LDL particles by macrophage scavenger receptors and cellular accumulation of cholesteryl esters; such accumulation is characteristic of atherosclerotic foam cells. The extent and nature of the modifications of LDLs that give rise to this behaviour have been poorly characterised, especially in regards to modification/oxidation of protein versus lipid components induced by glucose and low-molecular-mass aldehydes. Therefore the aims of this project were to: 1) characterise LDL modification by glucose, methylglyoxal and glycolaldehyde; 2) examine the effect of these modified LDLs on arterial cells by monitoring cellular viability, proliferation and cholesterol and cholesteryl ester levels; and 3) examine macrophage handling of apo B from these modified LDLs. Glycolaldehyde induced more rapid and more extensive changes to LDL than methylglyoxal, which was significantly more modified than LDL exposed to glucose, in the presence or absence of Cu2+. LDL was modified by glycolaldehyde and methylglyoxal in a time- and concentration-dependent manner. These aldehyde-modified LDLs were significantly more negatively charged relative (determined by changes in relative electrophoretic mobility), more aggregated (by SDS-PAGE) and lost more Arg, Lys and Trp residues (assessed by fluorescence-based assays) than glucose-modified and control LDLs. Glucose-modified LDL had more modest increases in net negative charge, aggregation and only significantly lost Arg residues. Under the conditions examined none of the modified LDLs contained significant levels of the protein oxidation products DOPA and o-tyrosine, the lipid oxidation products 7-ketocholesterol and cholesteryl ester hydro(pero)oxides, nor marked depletion of the major antioxidant α-tocopherol or significant radical formation (EPR spectroscopy). Therefore these LDLs were glycated, but not (glyc)oxidised, and so allowed the cellular uptake of glycated LDL, rather than glycoxidised LDL, to be examined. These glycated LDLs had no effect on the cellular viability (assessed by LDH release), cell protein (BCA assay), and cholesterol and cholesteryl ester levels (quantified by reverse-phase HPLC) of endothelial and smooth muscle cells. The glycated LDLs also had no effects on human and mouse macrophage viability, protein and free cholesterol levels. However, exposure of macrophages to some of the glycated LDLs resulted in significant accumulation of cholesteryl esters and apo B. The greatest cellular accumulation of cholesteryl esters was in cells exposed to glycolaldehyde-modified LDL, which occurred in a time- and concentration-dependent manner. Less cholesteryl ester accumulation was observed in cells exposed to methylglyoxal-modified LDL, but some conditions resulted in significantly more cellular cholesteryl esters as compared to control LDLs, unlike glucose-modified LDL. Macrophages endocytosed significantly more apo B from glycolaldehyde-modified LDL labelled with 125I on the apo B, than methylglyoxal-modified 125I-LDL. Apo B from methylglyoxal-modified 125I-LDL was also endocytosed and degraded in greater amounts than control 125I-LDLs, unlike glucose-modified 125I-LDLs. The glycation of LDL by some low-molecular-mass aldehydes have been shown to result in model foam cell formation as characterised by cholesteryl ester and apo B accumulation. This accumulation correlated with increases in net negative charge, aggregation and loss of Lys and Trp residues of the apo B in glycated LDL particles. However, the differences in cellular uptake of glycolaldehyde- versus methylglyoxal-modified LDL were not completely resolved and it is postulated that this may arise from the extent or type of products formed on key amino acid residues, resulting in differential uptake by macrophage scavenger receptors, rather than loss of particular amino acids per se. Therefore these studies provide a potential mechanism to explain the increased atherosclerosis in people with diabetes, and a suitable model to examine the potential inhibition of the effects of glycated LDLs. This could provide potential therapeutic interventions to reduce diabetes-induced atherosclerosis.
| Identifer | oai:union.ndltd.org:ADTP/220731 |
| Date | January 2005 |
| Creators | Brown, Bronnwyn Elizabeth |
| Publisher | University of Sydney. Central Clinical School |
| Source Sets | Australiasian Digital Theses Program |
| Language | English, en_AU |
| Detected Language | English |
| Rights | Copyright Brown, Bronnwyn Elizabeth;http://www.library.usyd.edu.au/copyright.html |
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