Synergistic role of hemin in endothelial cell activation in the lungs of sickle cell mice

Although sickle cell disease (SCD) has long been characterized by typical clinical picture of sickle hemoglobin, chronic hemolytic anemia, and recurring episodic vaso‐occlusive crisis, mechanistic cascades linking hemolysis to vaso‐occlusion still remain largely unclear. Notably, the vascular endothelium in patients with SCD exhibits a consecutively activated, pro‐inflammatory phenotype even under steady state conditions, and a further enhanced inflammatory response occurs during vaso‐occlusive crisis. Recent studies have suggested that the hemolytic products, including heme and its oxidized form hemin, may directly target the endothelium, promote endothelial cell pro‐inflammatory activation, and consequently trigger vaso‐occlusion. Thus, the present study examined a potential mechanism of hemin in promoting pro‐inflammatory lung endothelial cell activation in SCD. Fluorescence microscopy was first performed in cultured rat pulmonary microvascular endothelial cells (PMVECs) that were stably transduced with a GFP‐tagged P‐selectin to examine the dynamics of P‐selectin translocation following stimulation with hemin (400 nmol/L). Indeed, hemin markedly stimulated P‐selectin translocation from cytosol to the plasma membrane leading to P‐selectin surface expression. Such hemin‐induced P‐selectin surface expression was nearly abolished by the membrane‐permeable radical scavenger tempol (20 μmol/L), indicating that the stimulative effect of hemin is mediated by cell oxidative stress. Next, an ex vivo polymorphonuclear neutrophil (PMN)‐perfused lung model was employed to determine whether hemin induces potentiated intracapillary neutrophil retention in SCD lungs. Lungs isolated from 12‐week‐old transgenic humanized homozygous SCD (SCD−/−) and heterozygous (SCD+/−) mice were assessed for intravascular PMN sequestration using immunohistochemistry for leukocyte myeloperoxidase following hemin perfusion (10 μmol/L, 15 min) followed by perfusion of non‐stimulated rat PMNs (1.0 × 10 5 /mL, 20 min). Hemin‐stimulated intracapillary PMN sequestration was observed to be more pronounced in SCD−/− lungs as compared to SCD+/− lungs. The potentiated hemin‐induced PMN sequestration in SCD−/− lungs was dramatically attenuated when the lungs were perfused with a P‐selectin antibody (0.5 mg per lung set, 5 min), tempol (150 μmol/L, 5min), or, to a lesser degree, endothelin receptor A (ET A ) antagonist BQ‐123 (1 μmol/L, 5 min), but not endothelin receptor B (ET B ) antagonist BQ‐788 (1 μmol/L, 5 min), prior to hemin perfusion. Taken together, these observations suggest that hemin, by activating P‐selectin surface expression via intracellular reactive oxygen species generation, acts additively or synergistically to promote alveolar capillary endothelial pro‐inflammatory phenotype in SCD lungs. Support or Funding Information HL‐117684 (to S. Meiler) and HL‐066299 (to S. Wu)