Fasudil, a ROCK inhibitor, attenuates endotelial‐leukocyte interaction in sickle cell transgenic mice

Sickle cell disease (SCD) is one of the most common genetic disorder worldwide, caused by point mutations in the gene encoding the hemoglobin β chain. The most common form of SCD is homozygous sickle cell anemia (SCA; HbSS), where the presence of two βs globin alleles results in an almost exclusive production of hemoglobin S (HbS) instead of HbA in erythrocytes. In SCD, leukocyte plays an important role in the pathophysiology of disease, and leukocyte adhesion may be, in fact, the initiating event in vaso‐occlusive episodes. The neutrophil appears to be the leukocyte that most participates in the vaso‐occlusive process. Changes in the Rho‐A/Rho‐kinase signaling pathway can modulate some pathophysiological aspects of the sickle cell disease, such as priapism, increased ROS production 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 the allergic lung inflammation in SCA mice. Objective The aim of this study was to investigate the effect of acute treatment with Fasudil, a specific inhibitor of Rho‐kinase, in the initial steps of the leukocytes transmigration processes in an in vivo model of inflammation‐induced vaso‐occlusion in SCA mice model. Methods Inflammation was induced by the administration of TNF‐α (0.5 μg, i.p .) in male HbSS knock‐in mouse model of SCA (Townes; Wu et al., Blood 2006) and C57BL/6JuniB control mice. Fasudil (10 mg/kg) was intraperitoneally administrated in the animals 1 h before TNF‐α. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results Intravital microscopy demonstrated that TNF‐α induced an increase in the number of rolling and adhesion leukocytes to endothelium and accumulation of leukocytes outside the vessels. However, this augment is significantly higher in SCA mice compared to control animals (Rolling: 15.8±2.3 and 4.5±0.7 leukocyte min −1 ; Adhesion: 15.3±0.7 and 10.1±0.6 leukocyte adhered 100μm −1 ; Extravasated: 5.7±0.5 and 3.5±0.5 leukocyte ×100×50 μm 2 , p<0.05, respectively). Pre‐treatment with Fasudil had no effect on leukocyte rolling (10.5±1.8 leukocyte min −1 ), however, significantly reduced firm adhesion by 30% (11.1±0.8 leukocyte adhered 100μm −1 ) and decreased leukocyte extravasation by 46% (3.1±0.4 leukocyte ×100×50 μm 2 ) in SCA mice. Conclusion In summary, our data demonstrated that Rho‐kinase signaling regulates inflammation‐induced vaso‐occlusion in SCA mice model in vivo and that Rho‐kinase inhibition may be useful in the treatment of vaso‐occlusion process in SCA, limiting the infiltration of leukocytes, and reducing vascular inflammation. Support or Funding Information FAPESP This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .