Human Lung Microvascular Endothelial Cell Death in Response to Cell‐free Hemoglobin

During sepsis, circulating levels of cell‐free hemoglobin (CFH) are often elevated due to the lysis of red blood cells, and higher concentrations are associated with worse clinical outcomes. Sepsis is caused by a widespread inflammation response, which leads to the release of pro‐inflammatory molecules and cytokines into the circulation. These compounds can prime and activate the endothelium rendering it more susceptible to damage from other circulating molecules. Sepsis is a leading cause of acute respiratory distress syndrome (ARDS), which is characterized by vascular leak and fluid accumulation in the lungs. In our previous studies in murine models of sepsis, lung endothelial cell death was observed, which may contribute to the alveolar capillary barrier dysfunction that characterizes ARDS. Here we investigate whether CFH can initiate cell death in vitro and whether this is potentiated by exposure to pro‐inflammatory signals. Primary human lung microvascular endothelial cells (HLMVECs) were cultured to confluence and exposed to various clinically relevant concentrations of CFH diluted in complete media for up to 24 hours or pre‐treated for 4 hours with 5 ng/ml Cytomix (equal parts IL‐1β, TNF‐α, and INFγ), followed by treatment with 1.0 mg/ml CFH for 24 hours. Cell viability was determined using Draq7, a cell membrane impermeable nuclear stain. Cell death was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, which detects DNA fragmentation from late apoptosis or necrosis. While there was a time‐ and concentration‐dependent trend, no significant differences between CFH treated and untreated cells were seen in the absence of cytomix, even at the highest concentration of 1.0 mg/ml CFH at 24 hours for the TUNEL assay (57 TUNEL + cells per FOV in CFH‐treated vs. 17 in control, p=0.8245). However, there was a significant increase in Draq7 staining with 1.0 mg/ml CFH treatment after 24 hours (7.9% Draq7 + cells in CFH‐treated vs. 2.3% in control, p=0.0240). Exposure to cytomix augmented the decrease in viability from CFH as measured by Draq7 assay (32.4% Draq7 + cells in Cyto + CFH‐treated cells vs. 14.5% in CFH‐treated cells, p<0.0001). These findings indicate that CFH alone has modest effects on lung endothelial cell viability. However, during sepsis when circulating cytokine levels are increased, the effect of CFH on endothelial cell death is enhanced, which may contribute to the microvascular leak and alveolar fluid accumulation that are characteristic of acute lung injury. Support or Funding Information This work is funded by NIH HL135849, HL103836, and HL094296. Figure 1