Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Abstract

Background: HbS oxidation is recognized as an important element in the pathophysiology of sickle cell disease. Results: The ferric/ferryl redox cycle of HbS is compromised. Conclusion: The inability of ferryl HbS to revert back results in oxidative damage and mitochondrial dysfunction in lung epithelial cells. Significance: These oxidative pathways may contribute to the vasculopathy in sickle cell disease and can be targeted with antioxidants.

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase‐1

“…Following incubations with various Hb species at equimolar (100 μ m ) concentration for 12 h, cells were fixed in 4% paraformaldehyde. Immunocytochemical staining was performed with mouse anti‐HO‐1 primary antibody (Abcam, Cambridge, MA, USA) followed by secondary goat anti‐mouse Alexa Fluor 488 conjugated antibody for HO‐1 as described earlier 6. Cellular actin cytoskeleton was counterstained with Phalloidin Alexa‐647 (red) to visualize the cellular morphology.…”

Intravascular hemolysis and the pathophysiology of sickle cell disease

“…This is also correct. Published data evidence the involvement of oxidative stress (79,100,105,108,113,115,116,121,123,132,134,135), inflammation (9, 22, 27, 32, 101, 105-108, 115, 124-127, 134), dyslipidemia (135)(136)(137)(138), microparticles (89,122,123,139), and vasoactive peptides (110,111,(140)(141)(142)(143). These additional pathways (depicted in Figure 2) are potentially additive or synergistic to intravascular hemolysis-like mechanisms.…”

“…Hemoglobin oxidation is driven not only by reaction with NO (112), but also by reaction with a host of additional physiological oxidants (113)(114)(115)(116)(117)(118). Ferric and ferryl forms of hemoglobin produced in SCD and other forms of hemolysis are highly reactive in promoting oxidation (119)(120)(121). Hemolysis also produces red cell microparticles that can deliver toxic heme to endothelial cells (122,123).…”

Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells

“…However, mitochondrial bioenergetic analyses have not been explored extensively in the context of Hb cytotoxicity. Recently, however, heme and cell-free Hb have been shown to disrupt mitochondrial bioenergetic function in different cell types (22)(23)(24). Using lung epithelial cells (E10) as an AT1 cell model, we investigated the differential toxicity of acellular Hb and its oxidized species on the mitochondrial transmembrane potential and also on oxygen consumption rate (OCR).…”

“…The cells were exposed to the Hb proteins, as indicated earlier for 24 hours. The cells were thoroughly washed four to five times in prewarmed PBS to remove excess unbound Hb, then loaded with JC-1 dye (8 mM) for 30 minutes as described previously (24). After removal of excess dye, cells were detached using 0.025% trypsin-EDTA and washed in PBS.…”