Synergistic Enhancement of Sickle Red Blood Cell Adhesion to Endothelium by Hypoxia and Low Nitric Oxide Bioavailability
Authors: Gutsaeva Diana , Parkerson James , Yerigenahally Shobha , Ikuta Tohru , Head C. Alvin 
Published: October 2019

Abstract
Abstract 1637
The mechanisms underlying sickle red blood cell (RBC) adhesion to the endothelium, which constitutes a major pathologic event in sickle cell disease (SCD), are not fully understood. Adhesion of sickle RBCs to endothelial cells is believed to be regulated by multiple hematologic and physiologic factors including fetal hemoglobin levels, leukocyte numbers, oxygen tension, inflammatory cytokines, and nitric oxide (NO) bioavailability, but the extent to which each parameter contributes to sickle RBC adhesion remains unclear. Here we studied the effects of hypoxia and NO bioavailability on sickle RBC adhesion using mice deficient for endothelial nitric oxide synthase (eNOS). We found that these mice had NO metabolite levels comparable to those of SCD UAB mice (Ryan et al. Science 278:873, 1997). Sickle RBCs obtained from SCD UAB mice or control RBCs from control mice (C57BL/6J) were injected into eNOS-deficient or control mice. Sickle RBC adhesion in the skull bone marrow venules was studied by intravital microscopy. Control RBCs did not show any adherence to the endothelium in either control mice or eNOS-/- mice and only sickle RBCs adhered to the endothelium in eNOS-/- mice. Sickle RBCs adhered to endothelial cells in control mice under normoxia at an adhesion score of 0.6, which was enhanced to 2.7 by hypoxia, suggesting that hypoxia increases sickle RBC adhesion. In contrast, in eNOS-/- mice, the adhesion score of sickle RBCs was 2.5 under normoxia and increased to 13.7 under hypoxia, indicating a synergistic enhancement of sickle RBC adhesion under hypoxia and low NO availability. To examine whether increasing NO bioavailability in eNOS-deficient mice is sufficient for inhibiting sickle RBC adhesion, we treated eNOS-/- mice under hypoxia with low-dose NO (20 ppm). Inhalation of low-dose NO (20 ppm) restored reduced NO metabolite levels in eNOS-/- mice and decreased sickle RBC adhesion scores under hypoxia to levels which were statistically indistinguishable from those of eNOS-/- mice under normoxia and comparable to those of control mice under hypoxia. These results suggest that NO inhalation disrupted sickle RBC adhesion by hypoxia and low NO bioavailability. Moreover, we showed that inhaled NO induced only a marginal change in wall shear rates which exert effects on blood cell-endothelial cell interactions, indicating that inhaled NO inhibits sickle RBC adhesion under hypoxic conditions by both wall shear rate-dependent and -independent mechanisms. These in vivo studies demonstrate that sickle RBC adhesion is regulated in a synergistic manner by hypoxia and NO bioavailability, and that inhaled NO inhibits sickle RBC adhesion in part through modulation of wall shear rates. Inhalation of NO may alleviate the clinical severity of SCD patients by interrupting the synergy created by hypoxia and reduced NO availability for sickle RBC adhesion.

Disclosures:
No relevant conflicts of interest to declare.