338 - The effect of iron deficiency and iron treatment on angiogenesis in human retinal microvascular endothelial cells

Publication Number: 3325.338

Madeline Klein, University of Minnesota Masonic Children's Hospital, Minneapolis, MN, United States; Kaoru Terai, University of Minnesota, Minneapolis, MN, United States; Timothy Monko, University of Minnesota Medical School, Minneapolis, MN, United States; Rehan Krishnan, University of Minnesota Medical School, Collierville, TN, United States; Thomas Bastian, University of Minnesota Medical School, Minneapolis, MN, United States; Ellen Ingolfsland, University of Minnesota, Minneapolis, MN, United States

Background: Retinal vascular development is influenced by oxidative stress and iron-dependent angiogenesis. Iron deficiency (ID) and iron treatment (IT) are both associated with reactive oxygen species (ROS) production, which is linked to the development of retinopathy of prematurity (ROP). ID promotes hypoxia-inducible factor 1α (HIF-1α) nuclear translocation and vascular endothelial factor (VEGF) production, a key driver of ROP pathogenesis. However, the effects of ID and IT on the development of ROP are understudied.

Objective: To determine how ID and IT affect angiogenesis in human retinal microvascular endothelial cells

Design/Methods: Human retinal microvascular endothelial cells (HRMECs) were plated and exposed to three conditions: vehicle (water treated), iron deficiency (10 µM deferoxamine (DFO) treated) and iron treatment (50 µM ferric ammonium citrate (FAC) treated). Iron status and HIF-1α- VEGF pathway molecules were measured by qPCR or western blot after 48h exposure. Cell migration was studied by scratch assay: after reaching confluence, a scratch was made with a 200 µL pipette tip and the cells were photographed using a Celldiscoverer 7 automated microscope every 4 hours for 72 hours and rate of cell migration calculated and normalized to vehicle treatment (ImageJ). One-way ANOVA was performed between groups (GraphPad Prism).

Results: DFO treatment induced iron deficiency as indicated by decreased ferritin and FerroOrange intensity, and FAC treatment increased iron relative to control as indicated by low transferrin receptor and high ferritin (Fig 1A-D). In the scratch assays, there was a trend towards faster rate of cell migration among DFO treated cells compared to FAC treated cells (Fig 2, p=0.06). Further angiogenic assays are ongoing to determine if trends toward increased angiogenic markers (EpoR, Egln3, Egln1, Fig 1E-H) among iron deficient HRMECs are consistent with increased functional angiogenesis.

Conclusion(s): Iron deficiency may lead to faster rates of cell migration of HRMECs compared to control or iron treatment. This suggests that iron deficiency promotes angiogenesis, consistent with our published data of more normalized retinal vascularization in iron deficient, anemic rats. Further studies are ongoing to determine if iron deficiency directly regulates the HIF-1α-VEGF pathway of angiogenesis as the primary mechanism of action. Together, these results may help guide future management of iron status in premature infants in the Neonatal Intensive Care Unit.

Figure 1. Evidence of iron status and angiogenic markers
Iron status as indicated by transferrin receptor (TfR) A) mRNA expression by qPCR and B) protein level by western blot, C) ferritin protein by western blot, and D) FerroOrange intensity. Angiogenic markers E) erythropoietin receptor (EpoR), F) prolyl hydroxylase domain (PHD) containing (Egln) 3, and G) Egln1 mRNA expression by qPCR and by H) PHD protein level by Western blot. ** signifies < 0.01.

Figure 2. Scratch assay of HRMECs treated with vehicle, deferoxamine (DFO) or ferrous ammonium citrate (FAC)
A) Linear regression model of scratch assay by treatment group, shown as scratch distance/ original scratch distance. B) Time to scratch closure by treatment group normalized to vehicle (p=0.06). C-E) Representative images of scratch assay under treatment with C) Vehicle, D) DFO, and E) FAC.