Abstract
Sickle cell disease (SCD) patients have low serum hemopexin (Hpx) levels due to chronic hemolysis. We hypothesize that in SCD mice, hepatic overexpression of hemopexin will scavenge the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis. To examine the protective role of Hpx in SCD, we transplanted bone marrow from NY1DD SCD mice into Hpx™/™ or Hpx+/+ C57BL/6 mice. Dorsal skin fold chambers were implanted in week 13 post-transplant and microvascular stasis (% non-flowing venules) evaluated in response to heme infusion. Hpx™/™ sickle mice had significantly greater microvascular stasis in response to heme infusion than Hpx+/+ sickle mice (p<0.05), demonstrating the protective effect of Hpx in SCD. We utilized Sleeping Beauty (SB) transposon-mediated gene transfer to overexpress wild-type rat Hpx (wt-Hpx) in NY1DD and Townes-SS SCD mice. Control SCD mice were treated with lactated Ringer's solution (LRS) or a luciferase (Luc) plasmid. Plasma and hepatic Hpx were significantly increased compared to LRS and Luc controls. Microvascular stasis in response to heme infusion in NY1DD and Townes-SS mice overexpressing wt-Hpx had significantly less stasis than controls (p<0.05). Wt-Hpx overexpression markedly increased hepatic nuclear Nrf2 expression, HO-1 activity and protein, the heme-Hpx binding protein and scavenger receptor, CD91/LRP1 and decreased NF-κB activation. Two missense (ms)-Hpx SB-constructs that bound neither heme nor the Hpx receptor, CD91/LRP1, did not prevent heme-induced stasis. In conclusion, increasing Hpx levels in transgenic sickle mice via gene transfer activates the Nrf2/HO-1 anti-oxidant axis and ameliorates inflammation and vaso-occlusion.
Highlights
Sickle cell disease (SCD) is an unrelenting hemolytic disease caused by a single base pair mutation in the β-globin chain of hemoglobin
Our results suggest that hepatic overexpression of Hpx in sickle mice inhibits inflammation and microvascular stasis in the dorsal skin-fold chamber (DSFC) model by delivering heme to CD91/LRP1 on the liver and increasing nuclear Nrf2 activation and heme oxygenase-1 (HO-1) expression in the liver [24,36,37]
To test whether Hpx is protective in SCD against heme toxicity, we transplanted NY1DD bone marrow (BM) into Hpx+/+ or Hpx-/- C57BL/6 mice
Summary
Sickle cell disease (SCD) is an unrelenting hemolytic disease caused by a single base pair mutation in the β-globin chain of hemoglobin. Heme derived from sickle red blood cells (RBCs) acts as a damage-associated molecular pattern (DAMP) that can activate toll-like receptor 4 (TLR4) of the innate immune system, independent of its cognate ligand lipopolysaccharide (LPS) [9], leading to oxidant production, inflammation, vaso-occlusion (VO) ischemia and tissue injury, including acute chest syndrome [2,4,10]. In SCD mice, the combination of heme-induced oxidative stress, inflammation and adhesion of circulating blood cells to vascular endothelium is a key driver of the proinflammatory and prothrombogenic vasculature that promotes sludging and stasis of blood flow in the postcapillary venules [11,12,13,14,15,16,17].
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