Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

Background-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. Methods and Results-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. Conclusions-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. (Circulation. 2012;126:1385-1395.)Key Words: blood cells Ⅲ blood platelets Ⅲ thrombosis C ardiovascular disease leading to heart attack and stroke remains the leading cause of mortality and morbidity in the Western world. 1,2 Atherosclerosis is a progressive disease characterized by accumulation of inflammatory cells and vascular smooth muscle cells (VSMCs) within the intima of injured blood vessels. The atherosclerotic plaque is made up of immune cells, including monocytes, macrophages, neutrophils, T lymphocytes, and VSMCs, that migrate from the media to the intima where they proliferate and secrete extracellular matrix proteins. Platelets have also been implicated in the initiation of atherosclerotic lesion formation. 3,4 These events result in progressive narrowing of the vessel, allowing platelet aggregation and activation to ultimately form vascular occlusive thrombi, precipitating acute coronary syndromes and ischemic stroke. 5 Clinical Perspective on p 1395Strategies that block recruitment to the intima of immune cells and VSMCs are partially protective against vascular injury in both animal models and human patients. Inhibiting monocyte and VSMC recruitment to selected chemoattractants partially prevents atherosclerosis and its clinical manifestations, and simultaneous blockade of 2 chemotactic pathways confers additional, but not complete, benefit...