Macrophage released proteoglycans are involved in cell-mediated aggregation of LDL

Aggregated low density lipoprotein (LDL) is taken up by macrophages at enhanced rate, leading to macrophage cholesterol accumulation and foam cell formation. Since macrophages were shown to mediate self aggregation of modified forms of LDL, we sought to study the effect of macrophages on the susceptibility of native LDL to aggregation. Incubation of LDL (100 microg of protein/ml) with J-774A.1 macrophage-like cell line for 18 h at 37 degrees C, led to a 114 and 56% enhanced susceptibility of LDL to aggregation by vortexing and by Bacillus cereus SMase respectively. Macrophage conditioned media (MCMs) that were obtained from J-774A.1 cells also enhanced the susceptibility of LDL to aggregation by vortexing and SMase by 134 and 75% respectively, suggesting the involvement of macrophage secretory products in the enhanced aggregation of LDL. As proteoglycans were shown to be involved in lipoprotein aggregation, we analyzed the possible involvement of macrophage-released proteoglycans in LDL aggregation. Incubation of LDL (100 microg protein/ml) with 25 microg of proteoglycans that were isolated from MCM led to a dose-dependent enhanced susceptibility of LDL to aggregation by vortexing or by SMase by up to 62 and 77% respectively. The stimulatory effect of the MCMs on LDL aggregation was markedly reduced upon MCMs treatment with the glycosaminoglycan hydrolyzing enzyme chondroitinase ABC, chondroitinase AC, but not heparinase. On the contrary, incubation of LDL (100 microg of protein/ml) with increasing concentrations (up to 50 microg/ml) of chondroitin sulfate, or heparan sulfate enhanced the susceptibility of LDL to aggregation by up to 98 or by only 18% respectively, in comparison with non-treated LDL. Since macrophages under atherogenic conditions (cholesterol-loading, cellular lipid peroxidation and activation) demonstrate enhanced secretion of proteoglycans, we finally studied the effect of J-774A.1 macrophages on the susceptibility of native LDL to aggregation under the above atherogenic conditions. Incubation of LDL with cholesterol-loaded macrophages led to a 62% enhanced susceptibility of LDL to undergo aggregation by vortexing, in comparison with LDL that was incubated with non-loaded cells. Macrophage activation with phorbol myristate acetate (5 microM of PMA) also significantly increased cell-mediated aggregation of LDL by 50%, in comparison with non-activated cells. Lipid peroxidized macrophages obtained by cell treatment with either FeSO4 (50 microM), or angiotensin II (10(-7) M) enhanced the susceptibility of LDL to aggregation by 22 or by 39% respectively. These results suggest that under atherogenic conditions, macrophages release proteoglycans, and mainly chondroitin sulfate, which can contribute to cell-mediated formation of aggregated LDL, a potent inducer of macrophage foam cells which are the hallmark of early atherogenesis.