Evaluation of the role of eosinophils in the initial vaso-occlusion process using transgenic mice for sickle cell disease: participation of Rho kinase pathway

Author(s)

Athos Rodrigues de Moraes

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2018-08-31

Examining board members

Carla Fernanda Franco Penteado; Anderson Ferreira da Cunha; Marcus Alexandre Finzi Corat

• Advisor: Carla Fernanda Franco Penteado

Abstract

In sickle cell disease, leukocytes play an important role in the pathophysiology of the disease and are considered the initiating cells of the vaso-occlusive process. Sickle cell disease (SCD) vaso-occlusion results from a complex process involving different mechanisms and interactions of red blood cells, leukocytes, endothelial cells, platelets, and plasma proteins. The Rho GTPases pathway seems to play an important role in the molecular mechanisms of chemotaxis, adhesion and migration of various inflammatory cells and little is known about the role of these Rho GTPases pathways in the rolling, vascular endothelial adhesion and leukocyte extravasation. The objective of this study was to evaluate in vivo the participation of eosinophils in the process of eosinophil-endothelium interaction, and in vaso-occlusion in SCD, besides the involvement of the Rho GTPases pathway in the inflammatory response induced by OVA and challenged with eotaxin in sensitized or not sensitized control animals, and transgenic animals for sickle cell anemia. We used male mice of the C57BL/6 J (control) lineage and sickle cell disease Townes model mice (SS). The results obtained by intravital microscopy of the cremaster muscle of the mice showed that after 4 hours of the challenge with eotaxin it was observed an increase in the number of leucocytes in rolling and adhered to the vascular endothelium in all sensitized groups investigated, however, this inflammation is potentiated when we compare the SS animals to the control group. Similar results were observed in relation to extravasation of leukocytes. Treatment with the specific ROCK inhibitor, Fasudil, was able to significantly reduce the rolling and adhesion of leukocytes to the vascular endothelium of the cremaster muscle in all groups, but was more significant in sickle cells disease group. In contrast to endothelial transmigration, treatment with Fasudil was able to significantly reduce leukocyte extravasation in SS animals, not reducing in control animals. With the objective to evaluate the participation of eosinophils in the initial processes of vaso-occlusion, confocal intravital microscopy was performed for the differentiation of total leukocytes in the cremaster muscle of controls and sickle cells diseases mice in a model of chemokine-induced allergic inflammation. We observed that the number of eosinophils in rolling and adhered to the vascular endothelium in the control and SS animals sensitized with OVA were significantly higher when compared to their respective non sensitized groups. However, adhesion and rolling parameters were significantly higher in sickle animals when compared to control animals. In relation to the transendothelial extravasation of eosinophils, we did not observe significant differences between the non sensitized and sensitized SS groups, however, in the control animals we observed a significant increase of extravasated eosinophils in the sensitized animals compared to the non sensitized animals. Fasudil treatment was able to significantly reduce the rolling and adhesion of eosinophils to the endothelium in animals of the sickle cell disease group, and the specific ROCK inhibitor significantly reduced eosinophil extravasation to the vascular endothelium of the cremaster muscle in sickle cells mice. Taken together, treatment with the ROCK inhibitor is able to reduce the interaction of leukocytes and eosinophils with the vascular endothelium in the allergic inflammation model. Thus, we suggest that inhibitors of this pathway may be new therapeutic agents in the treatment of clinical complications of sickle cell disease (AU)

• FAPESP's process: 15/26929-1 - In vivo evaluation of the role of eosinophil in the vaso-occlusion process using sickle cell transgenic mice: Involvement of Rho-quinase pathway
• Grantee: Athos Rodrigues de Moraes
• Support Opportunities: Scholarships in Brazil - Master