Endothelin a Receptor (ETR_A) Blockade Inhibits Leukocyte Adhesion to the Vascular Endothelium in Sickle Cell Disease

Objective:Recurrent acute episodes of vaso-occlusion (VOC) are one of the hallmarks of sickle cell disease (SCD). Leukocytes are in an activated state in SCD and their recruitment to the vascular endothelium marks a critical event in the pathophysiology of the vaso-occlusive process. Endothelin-1 (ET-1), a potent vasoconstrictive and pro-inflammatory endogenous peptide, is elevated in steady state SCD, increases during acute VOC crisis, and remains elevated post crisis. This study asked whether the ET-1-ETR_A signaling pathway is involved in promoting leukocyte adhesion in a hypoxia-reoxygenation challenged mouse model of SCD. Secondly, we wondered whether ET-1 is able to directly activate and increase the proadhesive phenotype of the peripheral leukocyte population.

Methods: Animals:We used the UAB knockout-transgenic mouse model of SCD for our studies (Ryan, 1997). Experimental groups consisted of homozygous (SCD^-/-), heterozygous (SCD^+/-), and C57BL/6 control mice. Intravital Microscopy:Bosentan (BOS; dual ETR_A/B antagonist) or Ambrisentan (AMB; specific ETR_A antagonist) was administered by medicated chow for 8 weeks. Prior to imaging, animals were exposed to 5 hrs of 8% O₂ followed by 2 hrs of reoxygenation in room air. Leukocytes were stained in vivo with a PE-conjugated anti-CD45 antibody. Mice were anesthetized and the calvarial bone marrow microvascular network was observed using an intravital microscope with water-immersion objectives. Images were analyzed off-line for leukocyte velocity, adhesion, and flow dynamics (Image Pro-Plus 5.0). Superoxide/Cytokine/Integrin measurements: Leukocytes were isolated from freshly harvested peripheral blood (SCD^-/- and controls) and exposed to ET-1 or vehicle. ROS production was measured by luminescence (Lumistar Galaxy Luminometer). Intracellular cytokine and surface integrin expression was analyzed by flow cytometry. Neutrophil Adhesion Assay: Peripheral neutrophils were purified by a Ficoll gradient from SCD^-/- mice and controls. Following ex vivo incubation with ET-1 or vehicle, the purified neutrophils were added to pre-activated (thrombin) rat PMVECs. After washing with PBS, the cells were fixed and stained with MPO to identify adherent neutrophils.

Results: Intravital microscopy revealed that ETR blockade with BOS, but not AMB, enhanced WBC rolling and adhesion at steady state without altering flow velocity, suggesting an important role for ETR_B signaling at baseline in SCD^-/- animals. Following a hypoxia/reoxygenation protocol, both antagonists were equally effective in decreasing WBC rolling and adhesion in SCD^-/- animals. Both ETR antagonists were able to correct H/R-induced decreases in blood flow velocity to near baseline values. Next we asked if ET-1 is capable of activating WBCs by first measuring levels of receptor mRNA in sorted peripheral neutrophils and monocytes. Next we measured intracellular cytokine, surface integrins and ROS production following ex vivo exposure to ET-1. Results indicated a very low amount of ETR_A mRNA in neutrophils of SCD^-/- mice and controls. ETR_B mRNA was predominantly expressed in monocytes of SCD^-/- mice. ET-1 ex vivo stimulation failed to increase cytokine, integrin and ROS levels beyond baseline in sickle neutrophils and monocytes. Functionally, more neutrophils from SCD^-/- mice adhered to pre-activated endothelial cells than controls; however, ex vivo ET-1 stimulation failed to increase adhesion.

Summary and Conclusions: Our results demonstrate that selective ETR_A blockade is sufficient to abrogate H/R-induced leukocyte-endothelial interactions in a preclinical model of SCD. While the dual ETR_A/B antagonist produced similar results after H/R, its proadhesive effect at steady state suggests an important role for the endothelin B receptor under baseline conditions. Although the long-term significance of these findings remains to be elucidated, at this early stage our results suggest a potential therapeutic advantage for a selective ETR_A treatment strategy in SCD. The lack of response of the leukocyte population to direct ET-1 stimulation suggests that ET-1 targets primarily the vascular endothelium to promote leukocyte adhesion. In conclusion, this study established a potentially important role for clinically available ETR_A antagonists in the treatment portfolio for patients with SCD.