ferric-ammonium-citrate and Hypoxia

Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis.

Topics: Autocrine Communication; Basic Helix-Loop-Helix Transcription Factors; Carcinogenesis; Cell Membrane Permeability; Cell Proliferation; Endothelial Cells; Ferric Compounds; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Iron; Models, Immunological; Neoplasm Invasiveness; Neovascularization, Pathologic; Prolyl Hydroxylases; Quaternary Ammonium Compounds; Receptor Protein-Tyrosine Kinases; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

The regulation of heme oxygenase (HO) activity and its dependence on iron was studied in bovine aortic endothelial cells (BAEC) subjected to hypoxia-reoxygenation (H/R). HO activity was induced by hypoxia (10 h) and continued to increase during the reoxygenation phase. HO-1 protein levels were strongly induced by hypoxia from undetectable levels and remained elevated at least 8 h postreoxygenation. Addition of the Fe(3+) chelator desferrioxamine mesylate (DFO) or the Fe(2+) chelator o-phenanthroline during hypoxia alone or during the entire H/R period inhibited the induction of HO activity and HO-1 protein levels. However, DFO had no effect and o-phenanthroline had a partial inhibitory effect on HO activity and protein levels when added only during reoxygenation. Loading of BAEC with Fe(3+) enhanced the activation of the HO-1 gene by H/R, whereas loading with L-aminolevulinic acid, which stimulates heme synthesis, had little effect. These results suggest that chelatable iron participates in regulating HO expression during hypoxia.

Topics: Acetylcysteine; Aminolevulinic Acid; Animals; Aorta, Thoracic; Carbon Monoxide; Cattle; Cell Hypoxia; Cells, Cultured; Deferoxamine; Endothelium, Vascular; Enzyme Activation; Ferric Compounds; Free Radical Scavengers; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hypoxia; Iron Chelating Agents; Male; Oxidative Stress; Phenanthrolines; Photosensitizing Agents; Quaternary Ammonium Compounds