Hypoxia Stimulates Retinal Cell Type-Specific Differences in the Expression of VEGF Splice Variants

Abstract

Purpose: : Vascular endothelial growth factor (VEGF) is a potent mitogen that acts on microvascular endothelial cells to promote vasculogenesis and angiogenesis and is a principle mediator of pathological ocular neovascularization. In the rat, alternative mRNA splicing yields distinct VEGF isoforms, notably VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈, which differ in their ability to bind heparin. Our knowledge of the relative expression of the different VEGF splice variants under normal and pathological conditions is limited. The purpose of this study was to investigate the effect of hypoxia on splice variant-specific VEGF production by retinal cells.

Methods: : Confluency-matched primary cultures of rat retinal microvascular endothelial cells (RMEC), retinal astrocytes, and Müller cells were subjected simultaneously to normoxia or hypoxia for 6 hrs. RNA was isolated from cell lysates, and cDNA was produced using a random primer method. Relative amounts of VEGF₁₂₀, VEGF₁₆₄, VEGF₁₈₈, and VEGF_Total were measured by RT-PCR using sequence specific TaqMan Gene Expression primers. All data was normalized to the house-keeping gene, β-actin.

Results: : In response to hypoxia, astrocytes demonstrated a 3-fold increase in VEGF₁₂₀ (p < 0.0001) and nearly a 4-fold increase in VEGF₁₆₄ (p < 0.0001). VEGF₁₆₄ was the only splice variant demonstrating a significant hypoxic response in RMEC, with a 2.5-fold increase (p = 0.0017). VEGF₁₈₈ did not exhibit a significant hypoxic response in any of the cell types studied. In normoxia, the relative production of individual splice variants between all cell types studied remained consistent; VEGF₁₆₄ was the most abundant, followed by VEGF₁₂₀ and VEGF₁₈₈. This pattern of expression was unaffected by hypoxic stimulation.

Conclusions: : This in vitro study is the first to explore differential cell type-specific up-regulation of VEGF splice variants in response to hypoxia. These findings suggest that VEGF may play multiple key roles in promoting normal and pathological retinal angiogenesis. Additional experiments are being conducted using imaging mass spectrometry to study the spatial and temporal distribution of these splice variants in vivo.

Keywords: vascular endothelial growth factor • retinopathy of prematurity • retinal neovascularization