The effects of low density lipoproteins modified by incubation with chondroitin 6-sulfate on human aortic smooth muscle cells.

One of the first changes that take place within the artery intima at the inception of atherosclerosis is the accumulation of LDL-derived modified lipoproteins which appear as subendothelial lipid droplets and vesicles. With time, the LDL retention and interaction with intimal chondroitin sulfate-proteoglycans may induce further structural and functional modification of the lipoproteins. The aim of this study was to produce 'in vitro' modified lipoproteins by LDL incubation with chondroitin 6-sulfate (CS, at 37 degrees C, for 48 h, in the absence of antioxidants) and to test their effects on cultured human aortic smooth muscle cells (SMCs). CS induced LDL modification (CS-mLDL) consisted in formation of a mixture of fused particles (up to 150 nm diameter) and monomers with a small content of lipid peroxides and a partially degraded apo B-100, corresponding to a mild oxidation. Upon incubation with SMCs, CS-mLDL produced a concentration-dependent stimulation of 3H-thymidine incorporation, that, at low concentration (25 microg/ml), was 2-3-fold higher than that obtained when native LDL was used; this increase correlates well with the level of CS-mLDL uptake at the same concentration. Besides the mitogenic effect, CS-mLDL induced a significant stimulation of SMCs migration, comparable with that reported for oxidized LDL. Upon incubation with CS-mLDL, SMCs accumulated lipid droplets of various number and dimension, as revealed by Nile red staining and electron microscopy. Competition studies performed in the presence of 20-fold excess of native LDL and acetyl LDL showed that 125I-CS-mLDL were taken up both by LDL receptor and scavenger receptor. At high concentration (200 microg/ml), CS-mLDL had a cytotoxic effect that was not significantly different from that of native LDL. Together these results provide evidence of (i) the direct alteration produced by CS on LDL and (ii) the effect of CS-mLDL on SMCs migration, proliferation and transformation in lipid-laden cells, events that are crucial in the development of fibro-muscular atherosclerotic lesions.

Increased macrophage uptake of irreversibly glycated albumin modified-low density lipoproteins of normal and diabetic subjects is mediated by non-saturable mechanisms.

Diabetes mellitus is known as an independent risk factor in atherosclerosis. Among the prominent biochemical changes that occur in diabetic state, are the enhanced formation of advanced glycosylation end products (AGE) (especially linked to albumin and collagen) and the impaired oxidative-antioxidative balance. Previously, we have shown that AGE-albumin (AGE-Alb) significantly alters the physico-chemical characteristics of low density lipoproteins of normal (nLDL) and diabetic (dLDL) subjects. In this study we tried to establish if incubation of nLDL or dLDL, with AGE-Alb in autoxidative conditions, modifies the rate and/or the pathway of their uptake by macrophages. To this purpose, nLDL and dLDL were exposed to AGE-Alb, and after re-isolation and radiolabeling the lipoproteins were incubated with U937 or peritoneal macrophages (for various time and concentrations), in the absence or presence of different competitors (native LDL, acetylated LDL, AGE-Alb, mannan) or cytochalasin D. As controls, nLDL and dLDL, maintained in similar conditions, but without AGE-Alb, were used. The results showed that preincubation for 24 h and 72 h with AGE-Alb augmented the macrophage uptake for both nLDL and dLDL (1.7-fold). Either pre-incubated or not with AGE-Alb, dLDL was taken up at a constantly higher rate than nLDL; the difference appeared more prominent at 72 h (1.5 vs. 4 micrograms LDL protein/mg cell protein). The increased level of glycation of native dLDL as compared to native nLDL (266 +/- 35 vs. 160 +/- 24 mmol HMF/mol apoB) as well as of the lipid peroxides (1.34 +/- 0.47 vs. 0.3 +/- 0.09 nmol MDA/mg apoB) could account for the greater uptake of dLDL at any preincubation time. Competition experiments indicated that, generally, incubation with AGE-Alb diminished the apo B100,E receptor-mediated uptake in favour of 'scavenger' receptor pathway and phagocytosis. Macrophage uptake of AGE-Alb modified dLDL was reduced approximately 30% by native nLDL, approximately 70% by acetylated LDL and approximately 38% by cytochalasin D. Together, these data suggest that the consequence of the alterations induced by AGE-Albumin on LDL is the increased macrophage uptake, via non-saturable pathways, that ultimately may lead to accelerated formation of atherosclerotic plaques in diabetics.

Intimal thickenings of human aorta contain modified reassembled lipoproteins.

The aim of this study was to determine whether in human aortas early minute changes such as minimal intimal thickenings (MIT), developed in areas known to have a predilection to atherosclerosis, contain modified reassembled lipoproteins (MRLp) such as extracellular liposomes (EL) and lipid droplets (LD). These features have been previously detected in the aortic lesion-prone areas of rabbits and hamsters fed a fat-rich diet. Tissue samples of the aortic arch and thoracic aorta from 12 young subjects who died in accidents were selectively collected from grossly normal regions. By light microscopy, some of these regions were found to contain MIT. The normal areas and the MIT were separately examined by electron microscopy or subjected to fractionation and partial biochemical characterization. The MIT (approximately 25-100 microns thick) were constituted by a pronounced proliferation of extracellular matrix, especially elastin and microfibrils, with interspersed lipid deposits appearing as EL and LD. Commonly, MIT did not contain smooth muscle cells, macrophages, foam cells or cytolytic debris. Such components were only occasionally found in specimens excised from the vicinity of fatty streaks. Saline extracts of MIT or grossly normal aortic regions were subjected to a four-step purification procedure consisting of gel filtration, affinity chromatography on anti-apo B and anti-albumin Sepharose, followed by density gradient ultracentrifugation. The entire procedure was monitored by negative staining, lipid assays, SDS PAGE and immunoblotting. From the initial MRLp mixture, two fractions were obtained: fraction 1 containing multilamellar EL and LD, and fraction 2 composed mostly of unilamellar EL. As compared with serum LDL, the cholesteryl ester/unesterified cholesterol ratio was 4-6-fold lower in fraction 1 and 15-19-fold lower in fraction 2. On SDS-PAGE the fraction 2 displayed a single protein band of 66 kDa, immunochemically identified as albumin. The MRLp isolated from human aortas with minimal intimal thickenings appeared to be similar to those purified from the prelesional stage aorta of hyperlipidemic rabbits and hamsters.