Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease

Abstract

Sickle cell disease (SCD) is caused by genetic defects in the β-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology.

Keywords: HIF-2α; anemia; hypoxia; hypoxia-inducible factor (HIF); intestinal epithelium; iron; iron homeostasis; iron metabolism; sickle cell disease.

FIGURE 1.

Disruption of intestinal HIF-2α improves SCD-induced tissue iron overload. A–C, Hif2α^F/F and Hif2α^ΔIE mice were lethally irradiated and transplanted with Hb-WT or Hb-SS bone marrow (A) and analyzed 6 months later for HIF-2α protein expression in the duodenal mucosa (B) and gene expression analysis of duodenal Dcytb, Dmt1, Fpn1, Bcl2, Bcl-xL, Casp1, Glut1, PDK1, PGK1, and VEGF1 (normalized to β-actin) (C). D, serum (μg/dl) and tissue iron content (calculated as μg per gm of wet weight). E, Perls' iron stain of the spleen and liver. F, gene expression analysis of liver hepcidin (Hepc) normalized to β-actin. G, H&E staining of spleen and liver. Bar graph represents the mean value ±S.D. ****, p < 0.0001; **, p < 0.01; *, p < 0.05; ns, not significant.

FIGURE 2.

Disruption of intestinal HIF-2α improves Sickle cell anemia. A, representative blood smear of Hif2α^F/F-SS and Hif2α^ΔIE-SS mice. B, hematological analyses (Hb, RBC number, hematocrit value (HCT), and mean corpuscular volume (MCV)). C and D, total and indirect bilirubin measurement (C) and reticulocyte count (D). E, serum erythropoietin (EPO). Each bar graph represents the mean value ±S.D. ****, p < 0.0001; **, p < 0.01; *, p < 0.05; ns, not significant. The data represent 10 mice per group.