Hypoxic Regulation of Vascular Endothelial Growth Factor in Retinal Cells

TLDR

VEGF is an angiogenic protein whose intraocular concentrations closely track active neovascularization in diabetic retinopathy, central retinal vein occlusion, retinopathy of prematurity, and rubeosis iridis. The study aimed to determine whether hypoxia induces VEGF expression in retinal cells and thereby promotes retinal endothelial cell proliferation. Retinal pigment epithelial cells, pericytes, and microvascular endothelial cells were exposed to hypoxia in vitro; VEGF RNA was quantified by Northern blot, and conditioned medium was tested on endothelial growth with a VEGF‑neutralizing receptor IgG chimeric protein. Hypoxia rapidly increased VEGF RNA (3–30× after 18 h), the effect was oxygen‑dependent and reversible, and conditioned medium from hypoxic pericytes and RPE stimulated endothelial growth by 20 %—an effect entirely blocked by VEGF neutralization, confirming VEGF mediates hypoxia‑induced proliferation.

Abstract

<h3>Background:</h3> Vascular endothelial growth factor (VEGF) is an angiogenic protein and vasopermeability factor whose intraocular concentrations are closely correlated with active neovascularization in patients with diabetes mellitus, central retinal vein occlusion, retinopathy of prematurity, and rubeosis iridis. <h3>Objective:</h3> To determine whether hypoxia could induce expression of VEGF in retinal cells, which then promotes retinal endothelial cell proliferation. <h3>Methods:</h3> Retinal pigment epithelial cells, pericytes, and microvascular endothelial cells were exposed to hypoxic conditions in vitro, and RNA expression of VEGF was evaluated by Northern blot analysis. The VEGF-specific proliferative potential of the medium was measured by means of retinal endothelial cell growth assays and VEGF-neutralizing VEGF receptor IgG chimeric protein. <h3>Results:</h3> The VEGF RNA levels increased within 4 hours and reached elevations of threefold to 30-fold after 18 hours of hypoxia (0% to 5% oxygen, 5% carbon dioxide, 90% to 95% nitrogen) in all cell types (.01&lt;<i>P</i>&lt;.03). Stimulation was dependent on oxygen concentration. The VEGF RNA levels were normalized by reinstitution of normoxia for 24 hours (<i>P</i>&lt;.004). Medium conditioned by hypoxic retinal pericytes and retinal pigment epithelial cells stimulated retinal endothelial cell growth by 20% (<i>P</i>=.04), and this stimulation was entirely inhibited by VEGF-neutralizing receptor chimeric protein (<i>P</i>=.02). <h3>Conclusion:</h3> Hypoxia increases VEGF expression in retinal cells, which promotes retinal endothelial cell proliferation, suggesting that VEGF plays a major role in mediating intraocular neovascularization resulting from ischemic retinal diseases.