Cell-Free Hemoglobin Increases Leukocyte Adhesion and Mitochondrial Oxidative Damage in the Pulmonary Microvascular Endothelium.

Sepsis is a critical problem in intensive care units globally, and accounts for 20% of all annual deaths. During septic shock, the highly inflammatory state leads to the release of cell-free hemoglobin (CFH) from lysed red blood cells. Once released into the vascular circulation, CFH can oxidize from the normal ferrous 2+ state to methemoglobin (3+, ferric). We have shown previously that CFH is a key driver of acute respiratory distress syndrome due to the breakdown of the alveolar capillary barrier. However, the underlying mechanism of how CFH disrupts the pulmonary endothelium remains unknown. Originally, it was believed hemoglobin acted only in an oxidative damage fashion. We hypothesized that oxidized CFH disrupts the pulmonary endothelium due to mitochondrial oxidative damage and leads to increased leukocyte adhesion. We utilized primary human lung microvascular endothelial cells (HLMVECs) to probe the mechanism underlying CFH-induced barrier dysfunction. Endothelial barrier function was analyzed using electric cell-substrate impedance sensing (ECIS) to quantify the transendothelial resistance changes in real-time following treatment with CFH and CFH (1.0 and 0.5 mg/mL). Permeability was assessed using the SynVivo idealized network coculture system using HLMVECs and small airway epithelial cells and was quantified using fluorescent 60 kDa dextrans mixed with CFH (1.0 mg/mL) in media flowed through the channels while images were taken every 15 minutes for 4 h. Cytokines were measured via ELISA post CFH treatment from conditioned culture media at 1, 3, 6, and 24 h. Adhesion of leukocytes was quantified using immunofluorescence by counting the number of fluorescent polymorphonuclear cells (PMNs) adhered to an endothelial monolayer following pretreatment with CFH of the endothelial cells, the PMNs, or both. Mitochondrial superoxide production was quantified by MitoSOX staining followed by flow cytometry analysis of CFH-treated HLMVECs. We saw a dose-dependent decrease in transendothelial resistance following oxidized CFH treatment but not with CFH treatment (-1270 vs 262 max TER drop, p<0.0001). Real-time permeability assays using the SynVivo system showed an increase in barrier permeability from CFH treatment compared to CFH (47 vs 6 relative permeability). Surface expression of E-selectin (p=0.037) and ICAM-1 (p=0.0497) was increased following oxidized CFH treatment versus vehicle. In addition, pretreatment of HLMVECs with CFH increased adhesion of polymorphonuclear cells (PMNs) to the endothelium (1.69 x 10 vs 7.81 x 10 GFP intensity, p=0.0014), while pretreatment of the PMNs did not increase adhesion. Treatment of HLMVECs with CFH for 24 h increased secretion of IL-1b, IL-6, and IL-8 (p<0.05). HLMVECs exposed to CFH for 6 h have significantly increased mitochondrial superoxide compared to vehicle treated cells (608 vs 283 adjusted MFI, p=0.0014). These data demonstrate that oxidized CFH decreases pulmonary endothelial function through both immune and oxidative damage pathways. We also show that CFH induces endothelial dysfunction in an oxidation-state dependent manner. In summary, these studies provide new evidence to the potential mechanism in which CFH harms the endothelium during sepsis beyond the conventionally attributed oxidative cycling of the heme moiety.