Abstract
Cell free hemoglobin (Hb), becomes oxidized in the circulation during hemolytic episodes in sickle cell disease (SCD) or thalassemia and may potentially cause major complications that are damaging to the vascular system. Hemolytic anemias are commonly associated with pulmonary hypertension (PH) and often result from dysfunction of lung endothelial cells. The aim of this study was to determine the effect of different Hbs on cultured human lung endothelial function. Toward this goal, endothelial permeability, oxidative stress response parameters, glycolytic and mitochondrial bioenergetic functions were monitored in cultured human pulmonary arterial endothelial cells (HPAEC) following incubation with human adult Hb (HbA), and Hb isolated from patients with sickle cell Hb (HbS, βV6E) and HbE (βE26K) that commonly co-exist with β-thalassemia. These mutant Hbs are known for their distinct oxidative profiles. HPAEC treated with the ferrous forms of HbE, HbS for 24 h showed higher loss of endothelial monolayer integrity with concomitant rise in reactive oxygen radical production, lipid hydroperoxide formation and higher expressions of oxidative stress response proteins including heme oxygenase-1 (HO-1) accompanied by a rise in uncoupled mitochondrial respiration. Loss of membrane permeability was diminished in part by haptoglobin (Hp, protein scavenger), hemopexin (Hpx, heme scavenger) or ascorbate (reducing agent). To understand the role of Hb oxidation, HPAEC were exposed to ferric or ferryl states of the mutant Hbs. Ferryl forms of all proteins caused a significant damage to the endothelial monolayer integrity at a higher degree than their respective ferric Hbs. Ferryl forms of HbS and HbE also caused a loss of respiratory chain complex activities in isolated endothelial mitochondria and basal oxygen consumption in HPAEC. However, longer incubation with ferryl Hbs produced bioenergetic reprogramming including higher degree of uncoupled respiration and glycolytic rate. The data in this report collectively indicate that higher oxidation forms of HbS and HbE cause endothelial dysfunction through distinct damaging mechanisms involving mitochondrial bioenergetic function.
Highlights
Hb constitutes almost 95% of all the proteins within red blood cells (RBC); it is a tetramer of two α and two β subunits (α2β2), with a heme (Fe+2) moiety bound to each subunit
The byproduct of Hb oxidation is recognized as a damage-associated molecular pattern (DAMP) molecule (Gladwin et al, 2010)
Studies in a sickle cell mice model showed unequivocally that heme released from plasma oxidized Hb binds to the toll-like receptor-4 (TLR-4) on macrophages that can activate the innate immune system (Belcher et al, 2014)
Summary
Hb constitutes almost 95% of all the proteins within red blood cells (RBC); it is a tetramer of two α and two β subunits (α2β2), with a heme (Fe+2) moiety bound to each subunit. The cycle consists of three distinct steps: (1) initial oxidation of HbFe2+ to HbFe4+, (2) autoreduction of the HbFe4+ intermediate to HbFe3+, and (3) reaction of ferric/metHb with an additional H2O2 molecule to regenerate the ferryl intermediate/ferryl protein radical (·HbFe4+ = O) (Alayash, 2004, 2014) This radical may migrate to the oxidation “hotspot” to further damage the protein, including the irreversible oxidation of βCys and subsequent dimerization of the protein (Alayash, 2004). Because this surface amino acid is an important endpoint for free radical induced oxidation (Jia et al, 2007; Pimenova et al, 2010) our group has developed βCys as a quantitative reporter for H2O2 induced oxidation (Kassa et al, 2017; Strader and Alayash, 2017) Both ferric and ferryl species are known to release highly toxic heme moieties in the circulation from the parent Hb molecule (Kassa et al, 2016). Dysfunction of lung endothelial cells leading to pulmonary arterial hypertension (PAH) has increasingly been recognized in severe β-thalassemic or SCD patients and in other hemolytic conditions (Gladwin et al, 2010; Morris and Vichinsky, 2010)
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