H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice

Abstract

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe(2+) from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes Fe(2+) to catalytically inactive Fe(3+) inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic SCD mice in response to plasma hemoglobin. We utilized a Sleeping Beauty (SB) transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections into NY1DD SCD mice. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or a human triple missense FHC (ms-hFHC) plasmid with no ferroxidase activity. 8 weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) 1 h after infusion of stroma-free hemoglobin, while mice overexpressing wt-hFHC had only 5% stasis (p < 0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, ferritin light chain (FLC), 5-aminolevulinic acid synthase (ALAS), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 activity and protein. There was also a decrease in hepatic activated nuclear factor-kappa B (NF-κB) phospho-p65 and vascular cell adhesion molecule-1 (VCAM-1). Inhibition of HO-1 activity with tin protoporphyrin demonstrated HO-1 was not essential for the protection by wt-hFHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, adhesion molecules, and microvascular stasis in transgenic SCD mice.

Keywords: H-ferritin; endothelium; inflammation; sickle cell disease; vaso-occlusion.

FIGURE 1

Human ferritin heavy chain (FHC) expression is increased in transgenic sickle mice. NYDD1 sickle mice were injected hydrodynamically with Sleeping Beauty (SB) transposase vector with either a wild-type (wt) human fTH-1 gene or a triple missense (ms) fTH-1 gene devoid of ferroxidase activity. Control NY1DD sickle mice were infused hydrodynamically with lactated Ringer’s solution (LRS). 8 weeks after hydrodynamic infusion, the spleens and livers were removed and flash frozen. (A) Total mRNA (n = 4 mice/treatment group) was isolated from the spleens and transcription of the human wt- and ms-fTH transgene mRNA was demonstrated by qRT-PCR, values are expressed as mean + SD of the ratio of FHC to GAPDH mRNA. (B) Western blots of liver microsomes and cytosol from treated sickle mice were immunostained for FHC expression. Note that because the third mutation at residue 86 is in a loop section suspected to be a conformational epitope (Addison et al., 1984), it is likely that our primary anti-FHC antibody did not bind our triple ms-hFHC protein on western blots.

FIGURE 2

wt-hFHC inhibits hemoglobin-induced stasis in NY1DD sickle mice. Microvascular stasis was measured in a dorsal skin-fold chamber model 8 weeks after hydrodynamic infusion of LRS, ms-hFHC, or wt-hFHC SB vectors. Microvascular stasis was measured 1 and 4 h after infusion of stroma-free hemoglobin (8 μmol/kg) via the tail vein (n = 8 per group). Values are mean % stasis ± SD, *p < 0.05 for wt-FHC group vs. both other groups, as calculated by one-way ANOVA.

FIGURE 3

Heme oxygenase-1 (HO-1) is up-regulated in NY1DD sickle mice expressing human wt-FHC. (A) 8 weeks after hydrodynamic infusions, liver HO-1, and GAPDH mRNA ratios were measured by qRT-PCR (n = 4 mice per treatment group). Values are means ± SD, ^†p < 0.05 for wt-FHC and ms-FHC vs. LRS, as calculated by one-way ANOVA. (B) Microsomal membranes (n = 4 mice per treatment group) were isolated from the livers, run on a western blot (30 μg of microsomal protein per lane), and immunostained for HO-1 and GAPDH protein expression. (C) Heme oxygenase (HO) enzymatic activity was measured by measuring bilirubin production using 2 mg protein of liver microsomes per reaction (n = 4 mice per treatment group). HO activity in mice expressing human wt-FHC treated with the HO inhibitor, tin protoporphyrin (SnPP; 40 μmol/kg/day × 3 days, intraperitoneally), was also measured. Values are means ± SD, *p < 0.01 for wt-FHC vs. LRS, ms-FHC, and wt-FHC + SnPP, as calculated by one-way ANOVA. (D) Microvascular stasis was measured 1 and 4 h after infusion of stroma-free hemoglobin (8 μmol/kg) via the tail vein in mice treated with LRS, ms- and wt-FHC (n = 4 mice per group) as seen in Figure 2. Inhibition of HO activity with SnPP (40 μmol/kg/day × 3 days, intraperitoneally) did not block the inhibitory effect of wt-FHC on stasis. Values are mean % stasis ± SD, p < 0.05 for wt-FHC mice vs. LRS and ms-FHC, as calculated by one-way ANOVA.

FIGURE 4

Liver heme content and nuclear Nrf-2 and Bach-1 levels are elevated in sickle mice expressing human wt-FHC. (A) The heme content of liver microsomes from sickle mice treated with wt-FHC, ms- or LRS was determined by the pyridine hemochromogen method (n = 4).Values are means ± SD, *p < 0.05 by one-way ANOVA for wt-FHC vs. other groups. Nuclear extracts were isolated from livers, and 30 μg of nuclear extract protein from each liver was run on a western blot and immunostained for Bach-1 (B) and Nrf-2 (C). The GAPDH loading control at the bottom applies to both (B) and (C).

FIGURE 5

Decrease in nuclear phospho- and total NF-κB p65 and VCAM-1 in sickle mice overexpressing wt-FHC. (A) Nuclear extracts were isolated from livers, and 30 μg of nuclear extract protein from each liver was run on a western blot and immunostained for phospho- and total NF-κB p65 in wt-, ms-, and LRS-treated mice (n = 4). (B) Microsomal membranes were isolated from livers, and 30 μg of microsomal protein from each liver was run on a western blot and immunostained for VCAM-1.

FIGURE 6

Human FHC is expressed in the nuclei of mice expressing wt- and ms-FHC. (A) Immunofluorescence for human FHC (red) in HEK-293 cells untreated or transfected with wt-FHC or ms-FHC constructs. Nuclei were stained with DAPI (blue). (B) Nuclear extracts from the livers of LRS-, ms-, and wt-FHC treated mice (n = 4) were isolated; 30 μg of nuclear extract protein from each liver was run on western blot and immunostained for human FHC. Note: different primary antibodies to FHC were used for (A) and (B).

FIGURE 7

Mouse ferroportin, 5-aminolevulinic acid synthase (ALAS), and ferritin light chain (FLC) proteins are increased in sickle mice overexpressing human wt-FHC. Proteins of subcellular fractions isolated from livers of wt-, ms-, and LRS-treated mice (n = 4) were run on a western blot (30 ug protein/lane) and immunostained for (A) microsomal ferroportin, (B) mitochondrial ALAS, and (C) cytosolic FLC.