The cell motility modulator Slit2 is a potent inhibitor of platelet function.

Vascular injury and atherothrombosis involve vessel infiltration by inflammatory leukocytes, migration of medial vascular smooth muscle cells to the intimal layer, and ultimately acute thrombosis. A strategy to simultaneously target these pathological processes has yet to be identified. The secreted protein, Slit2, and its transmembrane receptor, Robo-1, repel neuronal migration in the developing central nervous system. More recently, it has been appreciated that Slit2 impairs chemotaxis of leukocytes and vascular smooth muscle cells toward diverse inflammatory attractants. The effects of Slit2 on platelet function and thrombus formation have never been explored. We detected Robo-1 expression in human and murine platelets and megakaryocytes and confirmed its presence via immunofluorescence microscopy and flow cytometry. In both static and shear microfluidic assays, Slit2 impaired platelet adhesion and spreading on diverse extracellular matrix substrates by suppressing activation of Akt. Slit2 also prevented platelet activation on exposure to ADP. In in vivo studies, Slit2 prolonged bleeding times in murine tail bleeding assays. Using intravital microscopy, we found that after mesenteric arteriolar and carotid artery injury, Slit2 delayed vessel occlusion time and prevented the stable formation of occlusive arteriolar thrombi. These data demonstrate that Slit2 is a powerful negative regulator of platelet function and thrombus formation. The ability to simultaneously block multiple events in vascular injury may allow Slit2 to effectively prevent and treat thrombotic disorders such as myocardial infarction and stroke

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

Chemokine Signaling Enhances CD36 Responsiveness toward Oxidized Low-Density Lipoproteins and Accelerates Foam Cell Formation

Excessive uptake of oxidized low-density lipoproteins (oxLDL) by macrophages is a fundamental characteristic of atherosclerosis. However, signals regulating the engagement of these ligands remain elusive. Using single-molecule imaging, we discovered a mechanism whereby chemokine signaling enhanced binding of oxLDL to the scavenger receptor, CD36. By activating the Rap1-GTPase, chemokines promoted integrin-mediated adhesion of macrophages to the substratum. As a result, cells exhibited pronounced remodeling of the cortical actin cytoskeleton that increased CD36 clustering. Remarkably, CD36 clusters formed predominantly within actin-poor regions of the cortex, and these regions were primed to engage oxLDL. In accordance with enhanced ligand engagement, prolonged exposure of macrophages to chemokines amplified the accumulation of esterified cholesterol, thereby accentuating the foam cell phenotype. These findings imply that the activation of integrins by chemokine signaling exerts feedforward control over receptor clustering and effectively alters the threshold for cells to engage ligands

The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils

In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP-2. In all instances, Slit2 reduced neutrophil recruitment effectively (P<0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.Peer reviewed: YesNRC publication: Ye

The cell motility modulator Slit2 is a potent inhibitor of platelet function

Background\u2014Vascular injury and atherothrombosis involve vessel infiltration by inflammatory leukocytes, migration of medial vascular smooth muscle cells to the intimal layer, and ultimately acute thrombosis. A strategy to simultaneously target these pathological processes has yet to be identified. The secreted protein, Slit2, and its transmembrane receptor, Robo-1, repel neuronal migration in the developing central nervous system. More recently, it has been appreciated that Slit2 impairs chemotaxis of leukocytes and vascular smooth muscle cells toward diverse inflammatory attractants. The effects of Slit2 on platelet function and thrombus formation have never been explored.Peer reviewed: YesNRC publication: Ye