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°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 μg/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 μg/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.