Overexpression of Oxygen-Stable Hif-1 in the Corneal Epithelium Results in "Physiologic" Neovascularization of the Corneal Stroma, Independent of VEGF Isoform

Abstract

Purpose: : Vascular endothelial growth factor (VEGF-A) is a potent angiogenic factor. It is required for normal vascular development, but also contributes to pathologic neovascularization resulting from tissue hypoxia. The hypoxia-regulated transcription factor, HIF-1, regulates VEGF expression and contributes to VEGF-induced angiogenesis and neovascularization. We determined the effect of over expressing a stable, oxygen-independent form of HIF-1 in the corneal epithelium and tested whether different VEGF isoforms altered the pattern of neovascularization or the functional status of the vessels that formed in these corneas.

Methods: : Lines of transgenic mice were generated that over expressed human HIF-1α under control of the keratin-14 (K14) promoter. In one line, HIF-1α was mutated at two proline residues that are required for the oxygen-dependent degradation of the protein (K14/Hif-1α double-proline [dp] mice). K14/Hif-1α^dp mice were mated to mice with germline deletion of VEGF splice variants to generate offspring expressing all VEGF-A isoforms or only VEGF-A₁₆₄ or VEGF-A₁₈₈. Corneal vessels were examined by light microscopy. The vasculature was perfused with fluorescein dextran or Evans Blue through the femoral vein to visualize the corneal blood vessels and to test their permeability.

Results: : Unlike transgenic mice that over expressed wild type Hif-1α, K14/Hif-1α^dp mice had blood vessels in the sub-epithelial corneal stroma. The vessels connected to the limbal vasculature, spanned the cornea and appeared morphologically normal. The vascularized corneas remained transparent, with no evidence of edema or corneal lesions. No leakage was detected when the vasculature was injected with fluorescein-dextran or Evans Blue dye. K14/Hif-1α^dp mice expressing only VEGF-A₁₆₄ or VEGF-A₁₈₈ also had blood vessels in the corneal stroma. These appeared similar to the vessels in the corneas of K14/Hif-1α^dp mice that expressed all VEGF-A isoforms. These vessels were not leaky and the corneas remained clear.

Conclusions: : Although the cornea is normally avascular, normal-appearing blood vessels can be produced there by over expressing stable HIF-1α in the epithelium. Different VEGF-A isoforms support normal neovascularization in this context. The K14/Hif-1α^dp mice may be a good model to identify the factors that determine whether hypoxia and VEGF-A promote the formation of normal or pathologic blood vessels.

Keywords: growth factors/growth factor receptors • transgenics/knock-outs • vascular endothelial growth factor