Slit2/Robo-1 Signaling in Monocyte and Macrophage Function: A Role in Vascular Inflammation

Atherosclerosis is a maladaptive inflammatory response initiated by the retention of cholesterol-rich low-density lipoproteins. These are rendered pro-inflammatory upon oxidation, leading to activation of endothelial cells and recruitment of monocyte-derived macrophages. While the clearance of oxidized low-density lipoprotein (oxLDL) by macrophages is initially beneficial, excessive accumulation results in cholesterol-laden foam cells that drive disease progression. The uptake of oxLDL is largely mediated by the scavenger receptor CD36 and requires dynamic actin reorganization driven by Rac GTPases. The Slit family of secreted proteins, together with their transmembrane receptor Robo, inhibit the migration of a variety of cell types, in part by inactivating Rac and blocking dynamic actin rearrangement. We show that Slit2 inhibited adhesion of monocytic cells to activated human endothelial cells, and to immobilized ICAM-1 and VCAM-1. Microfluidic live cell imaging showed that Slit2 inhibited the ability of monocytes tethered to endothelial cells to stabilize their actin-associated anchors and to resist detachment in response to increasing shear forces. Transfection of plasmids encoding constitutively-active Rac1 revealed that Slit2 inhibited post-adhesion stabilization of monocytes on endothelial cells by preventing activation of Rac1. Slit2 also inhibited chemotaxis of monocytes towards the chemokines, CXCL12 and CCL2, and administration of Slit2 to atherosclerosis-prone LDL receptor-deficient mice inhibited monocyte recruitment to nascent atherosclerotic lesions. The role of Slit2 in foam cell formation was investigated using a neutral lipid probe, BODIPY 497/503, and quantified by immunofluorescence microscopy and flow cytometry, revealing a decrease in cholesterol ester accumulation in Slit2 treated cells. The binding and uptake of oxLDL was investigated using fluorescently (DiI)-labeled oxLDL, and quantified by microscopy and flow cytometry. Slit2 decreased oxLDL-mediated foam cell formation in human and murine macrophages, by blocking Rac-dependent uptake of oxLDL by CD36. Thus, Slit2 may represent a powerful new tool to inhibit pathologic monocyte recruitment in vascular inflammation, and block foam cell formation in progressive atherosclerosis.Ph.D.2018-07-08 00:00:0