A Novel Hemoglobin Binding Peptide Increases Intracellular Heme and Potentiates Hemoglobin-Induced HO-1 Levels in Endothelial Cells

Abstract 1065

The hemolysis that occurs in many forms of hereditary and acquired hemolytic anemia, including sickle cell disease, saturates the hemoglobin/heme scavenging system resulting in increased levels of cell-free hemoglobin circulating in the plasma. Several recent studies have suggested a central role for intravascular hemolysis and cell-free hemoglobin in the development of vascular dysfunction, including pulmonary hypertension, in affected humans potentially by imposing oxidative and inflammatory stress. In agreement, mouse models of sickle cell disease and severe hereditary spherocytosis also develop vascular dysfunction and pulmonary hypertension. However, the role of intravascular hemolysis and cell-free hemoglobin in vascular dysfunction has proved controversial and a resolution of this important issue requires new experimental tools. This controversy highlights the importance of understanding if cell-free hemoglobin does indeed contribute to vascular complications associated with sickle cell disease. To address the role of cell-free hemoglobin in vascular pathology, we have synthesized a novel hemoglobin-binding peptide, hE-Hb-B10. This peptide is linked to a small fragment of apolipoprotein-E (apoE) to facilitate the endocytic clearance of cell-free hemoglobin through the ubiquitous heparin sulfate proteoglycan (HSPG)-associated lipoprotein pathway versus hemoglobin/heme scavenging system. We have shown previously that hE-Hb-B10 reduces cell-free hemoglobin levels and restores NO-dependent vascular function in murine models of hemolytic anemia. In the current studies, we investigate the cellular response of endothelial cells to hemoglobin uptake facilitated by hE-Hb-B10. We show that treatment of bovine aortic endothelial cells (BAECs) with oxyhemoglobin in the presence of hE-Hb-B10 augments intracellular heme concentration compared to oxyhemoglobin alone. Additionally, incubation of BAECs with methemoglobin increases heme oxygenase-1 (HO-1) protein levels and this induction is potentiated by hE-Hb-B10. hE-Hb-B10 also augments HO-1 induction by oxyhemoglobin, suggesting that hemoglobin uptake facilitated by hE-Hb-B10 is not dependent on the oxidation state of hemoglobin. In contrast, both Hb-B10, a peptide lacking the apoE fragment, and the scrambled hE-Hb-sB10 peptide in which the hemoglobin-binding sequence is scrambled, inhibit HO-1 induction caused by hemoglobin. Taken together, these data suggest that hE-Hb-B10 facilitates hemoglobin uptake into endothelial cells, augmenting both intracellular heme concentration and the induction of HO-1 by hemoglobin. While HO-1 expression is indicative of oxidative stress, enzymatic products of HO-1 can provide important protective functions against oxidative stress and iron overload. Therefore, altering HO-1 expression in SCD could potentially improve or worsen the severity of this disease. Indeed, potentiating HO-1 levels in models of SCD has been shown to be protective in murine models of SCD. Overall, our findings demonstrate that hE-Hb-B10 is a useful tool in determining the role of Cell-free hemoglobin in SCD pathology and suggests a mechanism by which this novel peptide could impact disease outcome.