Rock Inhibitor Fasudil Reduces Leukocyte-Endothelium Interactions in the Microvasculature of a Sickle Cell Mouse Model of Allergic Inflammation

Introduction: Sickle cell disease (SCD) vaso-occlusion process involves different cell types, such as red blood cells,activated endothelial cells, platelets and leukocytes. Endothelialdysfunction contributes to the vaso-occlusion process and leadsto inflammation. Data suggest that Rho-kinase signaling may regulate numerous aspects of the inflammatory process. Alterations in the Rho-A/Rho-kinase signaling pathway modulate pathophysiological aspects of the sickle cell disease such as priapism, and these enzymes are involved in increased reactive oxygen species generation and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced allergic inflammation in the lungs of SCD mice. However, the involvement of Rho/Rho-kinase signaling pathways in the mechanism underlying systemic vascular occlusion in SCD remains unclear. This study aimed to determine whether Rho/Rho-kinase pathways are involved in the mechanism of microvascular SCD vaso-occlusion. We investigated the effect of fasudil, a specific inhibitor of Rho-kinase, in the initial steps of the leukocyte transmigration process in a model of allergic inflammation using intravital microscopy of the cremaster muscle in SCD mice. Methods: Experimental groups consisted of male homozygous Tim Townes transgenic sickle cell mice and C57BL/6JuniB control mice. SCD and control mice were actively sensitized with a subcutaneous injection of 100 μg of ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 in 0.9% NaCl (Day zero). On day 7, mice received a second injection of 100 μg of OVA. On day 14, mice were subcutaneously challenged with eotaxin (100 ng/per dose) with or without pre-treatment with intraperitoneal fasudil (10 mg/kg) 1 hour before eotaxin. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results: Intravital microscopy showed that eotaxin challenge in OVA-sensitized animals increased rolling and adhesion of leukocytes to the endothelium, and accumulation of leukocytes outside the vessels was observed. However, this increase is significantly higher in SCD mice compared to control animals (Rolling: 23±1.9 and 15±1.2 leukocyte min-1; Adhesion: 16.3±0.9 and 11.7±0.5 leukocyte adhered 100µm-1; Extravasation: 3.88±0.3 and 2.4±0.2 leukocyte perx100x50 µm2, p<0.05, respectively). The leukocyte rolling flow was similarly inhibited by fasudil treatment in control animals by 50% and in SCD mice by 42%. Eotaxin-induced firm adhesion after fasudil was also reduced by 57% in control animals and 63% in SCD mice. Notably, pre-treatment with fasudil caused a greater decrease in leukocyte extravasation in SCD mice (44%) than in control animals (16%), p<0.0001. Conclusion: Our data show that inhibition of Rho-kinase decreased endothelial-leukocyte interaction. These findings suggest that Rho-kinase inhibitors may have therapeutic benefits in the vaso-occlusive process in SCD, limiting the extravasation of leukocytes, and reducing vascular inflammation. Disclosures Conran: Bayer AG: Research Funding. Fertrin: Alexion Pharmaceuticals: Consultancy.