Constitutive and hypoxia-induced VEGF production by cultured uveal melanocytes and retinal pigment epithelial cells

Abstract

Constitutive secretion of VEGF is crucial for maintaining ocular circulation while hypoxia-induced VEGF secretion plays an important role in pathological neovascularization. Previous studies have highlighted the critical function of RPE cells in these situations. The role of uveal melanocytes (UM) in VEGF production, however, has not been well described. The aim of this study was to compare VEGF production from human RPE and UM cell lines obtained in pairs from 3 donors to minimize individual variability in cellular function. Cells were subjected to hypoxia, (1% oxygen environment) or chemical hypoxia (cobalt chloride, CoCl₂) at different times or dosages, respectively. The effects of these treatments on the cell viability and cell proliferation were tested using MTT and cell counting with trypan blue testing. The production of VEGF and its main upstream factor (hypoxia-inducible factors-1α, HIF-1α) were measured in the conditioned culture medium and cellular extracts, by using ELISA analysis. Additionally, mRNA levels of VEGF and HIF-1α were quantified through real-time PCR analysis. The effects of CoCl₂ on the expression of VEGF and HIF-1α in UM and RPE cells were also examined using flow cytometry. Hypoxia and COCL₂ exposure did not affect cell viability and cell proliferation. This study revealed that the constitutive production of VEGF by RPE cells is significantly greater than from the UM. However, UM demonstrated a more robust response to high hypoxia or chemical hypoxic stimulation compared to RPE cells. The data suggests that while RPE cells play a critical role in constitutive VEGF production under normal conditions, UM may contribute significantly to the pathological increase in VEGF under severe ocular hypoxia. The observation that intraocular injection of CoCl₂ to produce local chemical hypoxia, results in a significant increase of VEGF levels in intraocular fluids and tissues, has not been reported previously. While this model cannot currently test the in vitro results, it may help further our understanding of UM and RPE cells' roles in VEGF production in future studies using more advanced technologies in a well-established in vivo model.