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

Poster Board Number: E110

Kyle Riedmann (Vanderbilt University, Vanderbilt University Medical Center), Adrienne Conger (Vanderbilt University Medical Center), Jamie Meegan (Vanderbilt University Medical Center), Lorraine Ware (Vanderbilt University Medical Center), Julie Bastarache (Vanderbilt University Medical Center)

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 CFH2+ and CFH3+ (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 CFH2+,3+ (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 CFH3+ 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 CFH3+ 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 CFH3+ treatment but not with CFH2+ treatment (-1270 vs 262 max TER drop, plt;0.0001). Real-time permeability assays using the SynVivo system showed an increase in barrier permeability from CFH3+ treatment compared to CFH2+ (47 vs 6 relative permeability). Surface expression of E-selectin (p=0.037) and ICAM-1 (p=0.0497) was increased following oxidized CFH3+ treatment versus vehicle.  In addition, pretreatment of HLMVECs with CFH3+ increased adhesion of polymorphonuclear cells (PMNs) to the endothelium (1.69 x 108 vs 7.81 x 109 GFP intensity, p=0.0014), while pretreatment of the PMNs did not increase adhesion. Treatment of HLMVECs with CFH3+ for 24 h increased secretion of IL-1b, IL-6, and IL-8 (plt;0.05). HLMVECs exposed to CFH3+ 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.

Support or Funding Information

Research reported was supported by NHLBI of the National Institutes of Health under award number HL150783.

Research reported was supported bylt;igt; lt;/igt;NHLBI of the National Institutes of Health under award number lt;igt;HL150783.lt;/igt;



Oxidized hemoglobin increases mitochondrial superoxide and leukocyte adhesion. A) Fluorescent images of PMNs (green) adhering to the endothelial monolayer (red) when treated with hemoglobin; B) Quantification of PMN adherence expressed as GFP fluorescent intensity; C) Quantification of mitochondrial superoxide by MitoSOX staining via flow cytometry expressed as adjusted MFI.