Binding and Uptake of Oxidized Low Density Lipoprotein (LDL) by Macrophage Scavenger Receptors Are Enhanced by Substrate-Bound Fibronectin

Abstract

The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages and the resulting accumulation of low density lipoprotein (LDL) lipids within the cells has been implicated in the pathogenesis of atherosclerosis. The effect of fibronectin (FN) on the binding and uptake of ox-LDL by macrophages was investigated using thioglycollate-induced mouse peritoneal macrophages. The ability of the macrophages to bind ox-LDL was assessed by the binding of mouse red blood cells (RBC) pre-coated with ox-LDL (ox-LDL-RBC) prepared in vitro to macrophages at 37°C. The binding of ox-LDL-RBC to macrophages was significantly enhanced when the macrophages were plated on a FN-coated substrate. Similar enhancement was observed when the macrophages were plated on a substrate pre-coated with Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide, an adhesive sequence of FN involved in binding to the cells, but not with the control Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) peptide. The effect of FN was inhibited when GRGDSP, but not GRGESP, was present during the macrophage attachment to the FN-coated substrate, suggesting that the specific interaction of this sequence and the FN-receptor is responsible for the effect of FN. The addition of FN or GRGDSP in solution to the macrophage layers on an uncoated substrate was ineffective. Thus, attachment to a substrate is necessary for FN to be effective on the macrophages. The FN-coated substrate also enhanced the uptake of ¹²⁵I-labeled ox-LDL by the macrophages, and this enhancement was found to be due both to an increased binding of ¹²⁵I-labeled ox-LDL to the cells and to increased internalization of the surface-bound ¹²⁵I-labeled ox-LDL into the cells. Consistently, enhanced accumulation of lipid droplets and cholesterol within the cells was observed when the macrophages were plated on the FN-coated substrate. These results indicate that substrate-bound FN (solid-phase FN) enhances the activity of the macrophage receptors for ox-LDL (scavenger receptors) to bind and take up ox-LDL, and thus causes enhanced accumulation of ox-LDL lipids. It is, therefore, possible that insoluble FN present in an extracellular matrix and on other cell surfaces in the artery wall plays a promoting role in atherogenesis.