Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2

Oxidized low-density lipoprotein (oxLDL) is considered one of the principal effectors of atherogenesis. To explore mechanisms by which oxLDL affects human mononuclear phagocytes, we incubated these cells in medium containing oxLDL, acetylated LDL (acLDL), or native LDL, or on surfaces coated with these native and modified lipoproteins. The presence of soluble oxLDL, acLDL, or native LDL in the medium did not stimulate H2O2 secretion by macrophages. In contrast, macrophages adherent to surfaces coated with oxLDL secreted three- to fourfold more H2O2 than macrophages adherent to surfaces coated with acLDL or native LDL. Freshly isolated blood monocytes secreted little H2O2 regardless of the substrate on which they were plated. H2O2 secretion was maximal in cells maintained for 4–6 d in culture before plating on oxLDL-coated surfaces. Fucoidan, a known ligand of class A macrophage scavenger receptors (MSR-A), significantly reduced macrophage adhesion to surfaces coated with oxLDL or acLDL. Monoclonal antibody SMO, which blocks oxLDL binding to CD36, did not inhibit adhesion of macrophages to oxLDL-coated surfaces but markedly reduced H2O2 secretion by these cells. These studies show that MSR-A is primarily responsible for adhesion of macrophages to oxLDL-coated surfaces, that CD36 signals H2O2 secretion by macrophages adherent to these surfaces, and that substrate-bound, but not soluble, oxLDL stimulates H2O2 secretion by macrophages

shRNA-Mediated Decreases in c-Met Levels Affect the Differentiation Potential of Human Mesenchymal Stem Cells and Reduce Their Capacity for Tissue Repair

Mesenchymal stem cells/marrow stromal cells (MSC) are adult multipotent cells that can augment tissue repair. We previously demonstrated that culturing MSC in hypoxic conditions causes upregulation of the hepatocyte growth factor (HGF) receptor c-Met, allowing them to respond more robustly to HGF. MSC preconditioned in hypoxic environments contributed to restoration of blood flow after an ischemic injury more rapidly than MSC cultured in normoxic conditions. We now investigated the specific role of HGF/c-Met signaling in MSC function. An shRNA-mediated knockdown (KD) of c-Met in MSC did not alter their phenotypic profile, proliferation, or viability in vitro. However, we determined that while HGF/c-Met signaling does not play a role in the adipogenic differentiation of the cells, the disruption of this signaling pathway inhibited the ability of MSC to differentiate into the osteogenic and chondrogenic lineages. We next assessed the impact of c-Met KD on human MSC function in a xenogeneic hindlimb ischemia injury model. A 70% KD of c-Met in MSC resulted in a significant decrease in their capacity to regenerate blood flow to the ischemic limb, as compared to the MSC transduced with control shRNA. MSC with only a 60% KD of c-Met exhibited an intermediate capacity to restore blood flow, suggesting that MSC function is sensitive to the dosage of c-Met signaling. The current study highlights the significance of HGF/c-Met signaling in the capacity of MSC to restore blood flow after an ischemic injury and in their ability to differentiate into the osteogenic and chondrogenic lineages