H-Ferritin Ferroxidase Activity Induces Cytoprotective Pathways and Inhibits Microvascular Stasis in Transgenic Sickle Mice

Abstract 378

Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We hypothesize that intravascular heme, liberated from hemoglobin S derived from hemolyzed sickle red blood cells, is fundamental to inflammation and vaso-occlusion in SCD. Heme can be degraded by heme-oxygenase-1 (HO-1), releasing Fe²⁺, carbon monoxide (CO), and biliverdin/bilirubin. We have previously shown that increases in HO-1 activity inhibit vascular inflammation and vaso-occlusion in transgenic mouse models of SCD. HO-1 products, CO and biliverdin/bilirubin, also are protective in SCD, but Fe²⁺ released from the heme ring requires further processing. The released Fe²⁺ from heme is oxidized by ferritin heavy chain (FHC) ferroxidase activity and safely stored as catalytically-inactive Fe³⁺ inside ferritin clusters. FHC overexpression has been shown to be cytoprotective in response to inflammation and oxidative stress in vivo and in vitro. In this study, we hypothesize that overexpression of FHC with its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wt-ferritin heavy chain (wt-FHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer's solution LRS) or a triple missense (ms-) human FHC plasmid encoding no ferroxidase enzyme activity. Eight weeks after injection, the mice were implanted with dorsal skin-fold chambers to access microvascular blood flow. LRS-treated mice had 40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin, while wt-FHC overexpressing mice had only 5% stasis (p<0.05), suggesting vascular protection by wt-FHC. Ferroxidase activity was critical in this protection as mice overexpressing ms-FHC were not protected from microvascular stasis (33%). The wt-FHC SCD mice had marked increases in FHC mRNA and protein, light chain ferritin, 5-aminolevulinic acid synthase (5-ALA-synthase), cellular heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear FHC and microsomal HO-1 activity and protein, and a decrease in activated phosho- and total nuclear factor-kappa B (NF-κB) p65 in the nuclei of the livers. We conclude that wt-FHC ferroxidase activity enhances 5-ALA-synthase activity and cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-κB-activation, inflammation and microvascular stasis in transgenic SCD mice. Therapies directed at increasing ferritin heavy chain production and decreasing reactive iron availability may be beneficial in limiting sickle cell crises and end organ damage.