A Novel Role for P-Selectin in the Pathogenesis of Acute Chest Syndrome
Authors: Ghosh Samit , Weidert Frances , Flage Bethany , Sundd Prithu , Ofori-Acquah Solomon F 
Published: October 2019

Abstract
Acute chest syndrome (ACS) is a potentially fatal lung complication of sickle cell disease (SCD). There are currently no mechanistic based therapies for ACS and it continues to be a leading cause of death in SCD. Recent studies have identified independent links between extracellular heme with ACS, and with vaso-occlusive crisis (VOC) a common prodrome of ACS. Our experimental ACS model involves intravenous injection of purified hemin typically 35 µmoles/kg, which raises total plasma heme to a clinically relevant pathophysiologic level equivalent to ~0.5g/dl of hemoglobin, and causes respiratory failure exclusively in transgenic SCD mice of both the Townes and Berkeley strains (Ghosh et al., J Clin Invest, 2013). We have determined using time-lapse video microscopy and electrical cell impedance that hemin rapidly disrupts the pulmonary endothelial barrier in vitro. Hemopexin blocks hemin-induced pulmonary endothelial barrier disruption in vitro and respiratory failure in hemin-challenged SS mice. Importantly, genetic polymorphisms in the gene encoding heme oxygenase-1 the rate-limiting heme degradation enzyme are associated with ACS risk in the Cooperative Study of Sickle Cell Disease and the Silent Infarct Transfusion cohorts. These human data and our mechanistic results in SS mice support the heme hypothesis for ACS pathogenesis. P-selectin is a cell adhesion molecule involved in vascular inflammation. It is normally sequestered inside quiescent endothelium. However, recent evidence indicates extracellular heme activates the release of P-selectin unto the endothelial surface wall. Thus, in this study, we tested the hypothesis that P-selectin promotes ACS development. Infusion of a function blocking anti-P-selectin antibody protected SS mice (n=3) from hemin-induced ACS while all control SS mice (n=3) pretreated with IgG before the hemin challenge died (p=0.03) with severe hypoxemia and postmortem evidence of alveolar flooding. The apparent requirement for P-selectin in heme-induced lethal acute lung injury was confirmed by a 100% survival of P-selectin-/- (n=5) and 100% lethality of congenic P-selectin+/+ (n=5) mice challenged with hemin infusions (p=0.005). To identify the cell population involved in this disease process we generated bone marrow chimeric C57BL/6 mice lacking P-selectin in hematopoietic (P-selectinPLT-/-) and non-hematopoietic (P-selectinEC-/-) compartments. Seventy-five percent of P-selectinEC-/- mice were protected while all P-selectinPLT-/- congenic controls succumbed to hemin (n=3-4). The severity of lung injury in the P-selectinPLT-/- C57BL/6 mice was reflected by severe hypoxemia (SpO2: 82.75±2.14%) and a significantly higher lung wet/dry weight ratio compared to the ratio of the P-selectinEC-/- mouse lungs (p<0.05). Next, we transplanted congenic P-selectin-/- and P-selectin+/+ mice with SS mouse bone marrow to generate chimeric SS mice lacking endothelial P-selectin or expressing endothelial P-selectin respectively. Induction of ACS resulted in 40% lethality in the SS/P-selectinEC-/- mice (n=7) and 100% lethality in SS/P-selectinEC+/+ mice (n=7; p<0.01). The development of respiratory failure in the hemin modelinvolves de novo heme release from acute intravascular hemolysis. In this study, we found that SS/P-selectinEC-/- mice cleared ~70% of the hemin bolus within 30 min, which attenuated intravascular hemolysis and damped de novo heme release, while total plasma heme increased over 2-fold in the SS/P-selectinEC+/+ mice during the same time interval. Together these results demonstrate a critical intermediary role for P-selectin in the ACS triggered by acute intravascular hemolysis and elevated extracellular heme. In addition, we provide proof-of-principle in mice that antagonists of P-selectin can prevent and potentially treat ACS.

Disclosures
No relevant conflicts of interest to declare.