Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin

Eph receptor tyrosine kinases and their ligands ( ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up- regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/ pyruvate ratio, time course of hypoxia- inducible factor- 1 alpha ( HIF- 1 alpha) induction, and localization of stabilized HIF- 1a by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up- regulates not only HIF- 1 alpha and vascular endothelial growth factor ( VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC- 3 cells, the hypoxia- induced up- regulation of Ephs and ephrins is abrogated by small interfering RNA- mediated down- regulation of HIF- 1 alpha. These novel findings shed light on the role of this versatile receptor/ ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene- targeted mice.