This study identified a novel endogenous modification of LDL, converting it to the proatherogenic form, increasing binding to arterial PGs, and thereby producing increased retention of LDL in the arterial wall.
The decrease in particle size of MGmin
-LDL with retention of mass of the major components is interpreted as an increase in density, and hence, MGmin
-LDL is a new type of sdLDL. Measurements of LDL subfraction diameters by electron microscopy established that the mean particle diameter decreases with increasing density (32
). Recent studies suggest that the LDL particle has a flattened discoidal shape, with the wide circumference surface a high-density region containing apoB100. Part of apoB100 protrudes into the solvent, producing a lower-density region (33
). We speculate that MG modification has the effect of decreasing the protrusion and increasing particle density. A loss of cholesterol esters and a decreased cholesterol/apoB100 ratio is often found in sdLDL in vivo. This is partly linked to the triglyceride/cholesterol ester exchange by the cholesterol ester transfer protein (5
). This is not available in the conversion of LDL to sdMGmin
-LDL in vitro, and so the cholesterol/apoB100 ratio remains unchanged.
Modification of LDL by MG was directed to arginine residues of the protein component—mainly apoB100—forming hydroimidazolone MG-H1 residues. This modification is nonoxidative and, hence, does not form oxidized LDL, as judged by the lack of increase in TBARS content; nor was there an increase in methionine sulfoxide, dityrosine, and 3-nitrotyrosine, which are markers of protein oxidation (16
). MG may modify basic phospholipids, phosphatidylethanolamine, and phosphatidylserine (34
). There appeared to be little modification of nonprotein sites, however, because the estimation of total MG adducts in MGmin
-LDL by preparation with radiolabeled [14
C]MG was similar to the total increase in MG-derived AGEs. By facilitating trapping of LDL by arterial PG, however, MG modification may promote LDL oxidation indirectly (35
). LDL was decreased in size by 11% on modification by MG, suggesting that MG modification of LDL causes particle remodelling to an atherogenic form. The sdLDL particles gain entry and are retained in the arterial wall, especially at sites of atherosclerotic plaque lesion development (36
The initial stages of atherosclerosis in the arterial wall involve the accumulation of aggregates of small lipid droplets and vesicles of up to 400 nm in diameter in the extracellular matrix and the binding of these to PGs (20
). Characteristic of atherogenic sdLDL, MG-modified LDL had an increased tendency to form aggregates and increased binding affinity for arterial PGs and cell surface heparan sulfate (10
). PG binding has been implicated in subendothelial retention of LDL in animal models of atherogenicity (37
Chemical modification of LDL by arginine-specific modifying agents has been investigated previously. A nonphysiologic arginine-modifying agent, 1,2-cyclohexandione, was used, and LDL with extremely high supraphysiologic extents of modification was prepared: ~66 mol adducts per mol of LDL (44% of total arginine residues). This modified LDL at sites other than the hotspot site discovered in this study and thereby produced a markedly abnormal LDL with impaired binding to the LDL receptor and PGs (9
). This masked the effects we found. LDL in vivo has one MG-H1 modification on 2–12% of total LDL (16
), and hence, the minimal modification of MGmin
-LDL is appropriate to model MG modification in vivo.
Hotspot modification by MG was located at R18 in apoB100 of LDL. This is the first example of post-translation modification at site B-Ia enhancing LDL binding to PG. Molecular graphics analysis suggested that R18 is a critical stabilizing residue in this domain, and loss of charge after MG-H1 formation led to loss of β-strand secondary structure, with increased protrusion of the site binding PG on the protein surface. Increased modification of LDL by MG in diabetic apoE−/−
mice is also expected because murine apoB48 residues 1–2152 of apoB100, the major protein of LDL in apoE−/−
), has high sequence homology with the same domain of human apoB100 (40
), and in situ concentrations of LDL and MG were increased. Although MG modification of LDL is not an oxidative modification, diabetic apoE−/−
mice suffer oxidative stress, as indicated by a threefold increase of urinary isoprostanes (41
). Increased retention of LDL in the arterial wall by MG modification may synergize with increased oxidative stress to produce increased oxidized LDL in the arterial wall and escalation of atherosclerosis.
MG modification of LDL likely contributes to the increased atherogenicity of LDL in diabetes (16
). Agents that scavenge MG, such as metformin, aminoguanidine, and thiazolium compounds, prevented the development of atherosclerosis in diabetic apoE−/−
). Irbesartan, an angiotensin II receptor blocker, decreased the formation of MG-derived MG-H1 in clinical diabetes (44
) and decreased the development of atherosclerotic plaques in diabetic apoE−/−
). Treatment with high-dose thiamine supplements might be expected to prevent diabetic atherosclerosis because this treatment corrected increased MG and dyslipidemia in experimental diabetes (46
), but this remains to be evaluated. MG is increased in the plasma of patients with diabetes (12
) but less in patients treated with metformin (17
), with concomitant lowered risk of CVD (48
Metformin has an antiatherogenic effect that has hitherto been unexplained, but results from this study show it is likely due to a decrease of MG-modified LDL (16
). Metformin treatment of patients with type 2 diabetes also decreased sdLDL (49
). The nonoxidative nature of the MG modification may partly explain why antioxidant intervention to prevent CVD has been less effective than expected (50
). Quantitation of MG-modified LDL may improve epidemiologic CVD risk models, and therapeutics to decrease plasma MG may improve current treatment to decrease CVD in diabetes, renal failure, and in healthy people.