Redox active hemoglobin enhances lipopolysaccharide-induced injury to cultured bovine endothelial cells

Abstract

The interaction of cell-free hemoglobin with lipopolysaccharide (LPS) is thought to aggravate the pathophysiology of sepsis and/or septic shock. This study examines the possible modulatory role of cell-free hemoglobin on LPS-induced apoptosis of cultured bovine aortic endothelial cells. Experiments were performed with or without fetal bovine serum, a source of LPS-binding protein and soluble CD14. In the absence of serum, LPS alone or coincubated with purified bovine hemoglobin (BvHb), human hemoglobin (Hb), or α-cross-linked Hb (ααHb) did not induce apoptosis. In the presence of serum, LPS induced significant apoptosis. LPS combined with BvHb, Hb, or ααHb produced the same extent of apoptosis as LPS alone. To examine whether the H2O2-driven redox activity of hemoglobin alters LPS-induced apoptosis, glucose oxidase was added to the system to generate a subtoxic flux of H2O2. The combined treatment of LPS, glucose oxidase, and BvHb, Hb, or ααHb enhanced apoptosis compared with LPS alone. These findings support a possible mechanism whereby the redox cycling of hemoglobin, and not its direct interaction with LPS, contributes to the hemoglobin-mediated enhancement of LPS-related pathophysiology.