Inhibition Of the Rho/Rho-Kinase Pathway Reduces In Vitro Adhesion Of Sickle Cell Anemia Eosinophils To Human Endothelial Cells
Authors: Pallis Flavia Rubia , Vital Daiana Morelli , Fertrin Kleber Yotsumoto , Saad Sara T O , Conran Nicola , Costa Fernando Ferreira , Franco-Penteado Carla Fernanda 
Published: October 2019

Abstract

Introduction
Sickle cell anemia (SCA) individuals present a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion. This process is a multi-step process that involves different cell types such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation. Leukocytes may have a propagating and, possibly, initiating role in SCA vaso-occlusion. In vivo and in vitro studies using animal models show that neutrophils play a key role in vaso-occlusion; however, eosinophils (EOs) may also participate in this phenomenon in humans. We have previously shown that SCA eosinophils demonstrating greater adhesion to fibronectin. A small GTPase, Rho, and its target molecule, Rho-kinase, play an important role in the cell functions, including contractility, chemotaxis, adhesion, and migration. Y-27632 and NSC23766 are Rho-kinase inhibitors, that reduce leukocyte infiltration in several models of EOs inflammation, such as endotoxic liver and lung injury. The objective of this study was to investigate the effect of Rho kinase pathway on the in vitro adhesion of sickle EOs to activated human endothelial cell layers (umbilical vein endothelial cells, HUVEC).

Methods
Eosinophils were isolated from peripheral blood of healthy controls (n=5), steady-state SCA patients (SCA, n=7) and SCA patients on HU therapy (SCAHU, n=5), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion (1x106 cell/ml in Ham’s F12 K) to cultured HUVEC grown to confluence was assessed using static adhesion assays. HUVEC cells were treated, or not, with 100 µM NSC23766 or Y-27632 for 1h before incubation with an inflammatory stimulus (10nM TNF-alpha – 3 h), subsequently Eos were added to the cell layer (1 hour, 37°C, 5%CO2). For adhesion under flow conditions, biochips with microchannels coated with HUVEC were pre treated or not with Y-27632 or NSC23766 per 1 h before the TNF-alphastimulus. Purified eosinophils was added to HUVEC-coated microchannels at a flow rate of 0.5 dynes/cm2. Images of adherent eosinophils were recorded using real-time video microscopy and analysed using Duco Cell software.

Results
EOs from SCA patients demonstrated a significantly greater adhesion to HUVEC than healthy controls (14.42 ± 1.8% vs 9.35 ± 1.7 %; p<0.02); no significant difference was observed between the SCAHU group, compared to the healthy control and SCA group (10.5 ± 1.6%). Pre treatment of HUVEC with TNF-alpha significantly augmented the adhesion of healthy control, SCA, and SCAHU eosinophils to HUVEC (16.1 ± 3.2%, 33.1 ± 5.1%, 17.9 ± 3.5%, P<0.01, respectively). However, this adhesion was significantly higher for SCA EOs compared to healthy control and SCAHU EOs (p<0.05). Interestingly, when the HUVEC was pre-treated with NSC23766 then stimulated with TNF-alpha, adhesion of EOs from SCA and SCAHU was significantly reduced (22.9 ± 5,6%, 17.2 ± 6.1%, respectively, p<0.05). However, when HUVEC was pre-treated with Y-27632 no significant difference was found. In addition, EOs from SCA patients demonstrated greater adhesion to HUVEC under flow conditions when compared to healthy control and SCAHU. EOs when the HUVEC was protect with Rho inhibitors (Y-27632 and NSC23766) before stimulation with TNF-alpha, EOs adhesion under physiological shear stress was diminished in all groups investigated (healthy control, SCA and SCAHU).

Conclusion
Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion, in an in vitro model. Therapy with such Rho-kinase inhibitors could be used in patients with SCA, with beneficial effects on the vascular occlusion process.
Financial Support: FAPESP/CNPq/INCTS

Disclosures:
No relevant conflicts of interest to declare.