The Effect of IV Iron Therapy on HIF Pathway and HIF Regulated Genes in Elderly Patients with Unexplained Anemia
Authors: Katsakhyan Levon , Swierczek Sabina , Artz Andrew , Price Elizabeth A , Schrier Stanley L. , Cohen Harvey Jay , Prchal Josef T. , Gordeuk Victor R. 
Published: November 2019

Abstract
LK, SS Equal contributions
VG, JTP Equal credit as senior authors
Prolyl hydroxylase 2 (encoded by EGLN1), the principle negative regulator of HIF-1 and HIF-2 (encoded by EPAS1), is an iron dependent enzyme (Kaelin WG et al. Mol Cell, 2008); thus iron deficiency can augment hypoxic responses (Zhang X et al Blood Cells Mol Dis, 2014). Because EPAS1 mRNA has a 5' iron regulatory element, iron deficiency may inhibit the translation of HIF-2α and downregulate its target genes (Sanchez M et al Nat Struct Mol Biol, 2007). The Partnership for Anemia: Clinical and Translational Trials in the Elderly conducted a trial to determine the efficacy of IV iron in patients with borderline iron deficiency (Price E et al. Blood Cells Mol Dis, 2014). We measured expression levels of HIF1A, EPAS1, EGLN1 and HIF target genes in platelets and granulocytes. Nineteen patients were treated with 200 mg IV iron sucrose weekly for 5 weeks. Blood was obtained at screening, one week after the second dose of IV iron and 8 weeks after completion of iron therapy. Granulocytes and platelets were isolated and their RNA reverse transcribed. Transcripts were quantified by real-time qT-PCR, and normalized to HPRT gene. The threshold cycle for the expression of each gene was calculated and compared to 5 healthy controls.
We found a positive correlation between granulocyte and platelet mRNA of HIF1A, EPAS1 and EGLN1, and these relationships achieved a one-sided significance level of 0.1 or stronger for most data points for HIF1A and EPAS1 (Table 1). There was also a positive correlation for granulocyte and platelet mRNA of 5 of 7 hypoxia response genes (P of 0.1 or stronger in many comparisons; the strongest being for VEGF and PDK1).
Table 1. Spearman correlation rho between granulocyte and platelet mRNAs (N = 17). Gene Screen TV2 FU2 Regulators of the hypoxic response HIF1A .52** .21 .40* EPAS1 .40* .54** .67** EGLN1 .28 .30 .09 Responders to the hypoxic response GLUT1 .44** .00 .07 HK1 .25 .20 .36* PDK1 .24 .55** .41* VEGF .68** .33* .62** FOXO3 -.10 .22 .45** BNIP3 .03 -.19 -.08 BNIP3L .15 -.05 .20 *one-sided P <0.10; **P <0.05
There were positive relationships in the expression of the three hypoxic response regulating genes with lower iron stores, as reflected in three measures, and these tended to be strongest for the TfR to log ferritin index (Table 2). These relationships also tended to hold for HK1, PDK1, VEGF and BNIP3L in both types of cells, but the relationships were stronger in platelets.
Table 2. Spearman correlation for relationship between iron status and gene expression at screening. A negative correlation with ferritin and positive correlation with TfR and TfR/log ftn indicates increased gene expression with lower iron stores. Granulocytes Platelets Ferritin TfR TfR/log ftn Ferritin TfR TfR/log ftn Regulators of the hypoxic response HIF1A -.04 .05 .15 -.26 .41* .52** EPAS1 -.31 .090 .23 -.016 .15 .09 EGLN1 -.18 .26 .31 -.33* .57** .66** Responders to the hypoxic response GLUT1 .39* -.33 -.38* .27 .35* .23 HK1 -.20 .016 .07 -.22 .23 .23 PDK1 -.22 .31 .39* -.38* .16 .24 VEGF -.22 .12 .30 -.22 .48** .52** FOXO3 .10 -.097 -.061] -.19 -.15 -.14 BNIP3 -.04 -.10 -.007 -.18 -.30 -.25 BNIP3L -.08 .13 .21 -.36* .43** .50**
Trends for change in gene expression with iron therapy were also observed. Iron therapy led to decreased expression of HIF1A and EGLN1 in both cell types but increased expression of EPAS1; these relationships were stronger in granulocytes, where they achieved a one-sided significance of P <0.2. IV iron tended to be associated with reduced expression of a panel of 7 HIF target genes, more so in granulocytes than in platelets. Expression of FOXO3 did not decrease with IV iron, perhaps consistent with its being a target of HIF-2 as well as HIF-1.
Expression of hypoxic response regulating genes tended to correlate between platelets and granulocytes, as did expression of one-half of hypoxic response genes tested. Furthermore, expected relationships with iron status were observed for the expression of HIF1A, EPAS1 and EGLIN1. However, variable responses were found in the expression of certain HIF target genes in response to iron therapy that may be related to a balance of decreased HIF-a protein with enhanced translation of HIF-2a. Transcripts of these HIF regulated and HIF pathway genes are being compared to normals and to non-anemic elderly subjects to determine if anemia in the elderly is associated with a blunted hypoxic response.

Disclosures
No relevant conflicts of interest to declare.