Oxidized Hemoglobin Causes Human Lung Microvascular Endothelial Barrier Dysfunction

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Cell‐free hemoglobin is released into the circulation during conditions in which red blood cell lysis occurs such as hemolytic disorders and sepsis. In septic patients, increased levels of circulating cell‐free hemoglobin are independently associated with mortality and contribute to organ dysfunction, including acute respiratory distress syndrome (ARDS). Furthermore, during inflammatory conditions in which reactive oxygen species are high, cell‐free hemoglobin can be converted to higher oxidation states which enhances its damaging capacity. We previously showed that hemoglobin contributes to tissue edema in an ex vivo perfused human lung and microvascular endothelial barrier dysfunction in vitro; hemoprotein reductants (ascorbate and acetaminophen) protected against this phenomenon. Therefore, we hypothesized that higher oxidation states of hemoglobin are more injurious to the barrier function of lung microvascular endothelium. Utilizing primary human lung microvascular endothelial cells (HLMVEC), we found that stimulation with 0.5 mg/mL ferric (Hb3+; −380.2 Ω, p<0.001 vs. control) but not ferrous (Hb2+; +278.3 Ω, p=0.9893 vs. control) hemoglobin resulted in a drop in transendothelial electrical resistance (Figure ). The barrier‐disrupting effects of Hb3+ were reduced with haptoglobin (binds free hemoglobin; +208.5 Ω, p<0.0001 vs. Hb3+) and hemopexin (binds free heme), but not the iron chelator deferoxamine. The mechanism of Hb3+‐mediated microvascular endothelial barrier dysfunction involved disruption of adherens junction protein VE‐cadherin at cell‐cell borders and rearrangement of the actin cytoskeleton with intercellular gap formation. These data suggest that oxidized cell‐free hemoglobin causes microvascular endothelial barrier dysfunction to a greater degree than its non‐oxidized form.Support or Funding InformationThis work is funded by NIH HL135849, HL103836, and HL094296.Figure 1