Endothelin‐receptor A (ETA) mediates enhanced agonist‐induced intracapillary neutrophil retention in lungs of sickle cell disease
Authors: Zheng Zhen , Hu Xingang , Dickerson Carol , Meiler Steffen E , Wu Songwei 
Published: November 2023

Whether or not in an acute crisis, the vascular endothelium in patients with sickle cell disease (SCD) exhibits an activated pro‐inflammatory state, which undoubtedly influences the complex pathophysiology of the disease. Despite this significance, molecular mechanisms underlying such endothelial cell activation remain incompletely understood. Nonetheless, recent studies have implicated endothelin‐1 (ET‐1) as an important mediator for adverse vascular pathologies in patients with SCD. Here we investigated the role of ET‐1 and its receptors in the pro‐inflammatory lung endothelial cell activation in SCD. We first examined the presence of endothelin‐receptors A (ETA) and B (ETB) in rat and mouse pulmonary microvascular endothelial cells (PMVECs). Using sequence‐specific primers to target ETA and ETB, a single product of predicted size for each receptor was amplified from the cDNA from PMVECs; sequence analysis revealed that the cloned product was virtually identical to the previously reported ETA and ETB. RT‐PCR showed that direct adenylyl cyclase activation by forskolin (10 μmol/L), which was reported to increase ETA mRNA in smooth muscle cells, increased both ETA and ETB mRNA in PMVECs following a 6‐hr exposure. Further, RT‐PCR demonstrated an approximately 4‐fold increase in ETA and 6‐fold increase in ETB mRNA in PMVECs following chronic ET‐1 stimulation (20 nmol/L, 6 hr). This ET‐1‐induced increase in ETA and ETB mRNA was partially blocked by ETA antagonist BQ‐123 (1 μmol/L) but not ETB antagonist BQ‐788 (1 μmol/L), indicating that this endothelin receptor upregulation requires an ETA signaling pathway in PMVECs. Finally, we employed an ex vivo polymorphonuclear neutrophil (PMN)‐perfused lung model to determine whether the ET‐1‐ETA signaling pathway mediates an augmented agonist‐stimulated intracapillary neutrophil retention in SCD lungs. Lungs isolated from 12‐week transgenic humanized homozygous SCD (SCD−/−) and heterozygous (SCD+/−) mice were assessed for intravascular PMN sequestration using immunohistochemistry for leukocyte myeloperoxidase following either thrombin perfusion (3 unit/mL, 15 min) or hypoxic ventilation (5% O2, 30 min) followed by perfusion of non‐stimulated rat PMNs (1.0 × 105/mL, 20 min). Thrombin‐ and hypoxia‐stimulated intracapillary neutrophil retention was observed to be more pronounced in both groups of SCD−/− lungs. To examine which endothelin receptor pathway is involved in this process, we treated both groups with antagonists to either ETA, ambrisentan (20 mg/kg/i.p., 3 doses with each at a 24‐hr intervals), or ETA/B, bosentan (20 and 50 mg/kg/i.p., 3 doses with each at a 24‐hr intervals) prior to lung isolation. ETA or ETA/B blocking comparably reduced thrombin‐ and hypoxia‐stimulated intracapillary neutrophil retention in SCD−/− lungs, but had a minimal effect on SCD+/− lungs. Altogether, these results suggest site‐specific endothelial ETA expression and an important functional role of the ET‐1‐ETA signaling pathway in mediating an alveolar capillary endothelial pro‐inflammatory phenotype in SCD lungs.Support or Funding InformationHL‐117684 (to S. Meiler) and HL‐066299 (to S. Wu)