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Patrick Cammarata, Sudha Neelam, Morgan Brooks; The role of HIF-1α/-2α and VEGF in lens epithelial cell survival in hypoxia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1261.
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© ARVO (1962-2015); The Authors (2016-present)
The prosurvival signaling cascades that mediate the unique ability of human lens epithelial (HLE) cells to survive in their naturally hypoxic environment are not well defined. The aim of this study was to identify the functional expression of the hypoxia inducible factors HIF-1α and HIF-2α and establish their role in regulating VEGF expression. Furthermore, we demonstrate a link between sustained VEGF expression and the ability of the hypoxic HLE cells to thrive in low oxygen conditions and resist mitochondrial membrane permeability transition.
HIF translation inhibitors were used to demonstrate the role of HIF-1α and HIF-2α in regulating VEGF and Bcl-2 expression. Axitinib, was employed to demonstrate a role for the VEGF-VEGFR2 receptor complex in regulating Bcl-2 expression. Western blot analysis was used to detect the protein levels of HIF-1α and HIF-2α, as well as the proapoptotic protein, BAX and the prosurvival protein, Bcl-2. VEGF levels were analyzed with ELISA. The potentiometric dye, JC-1, was used to determine the effect of the inhibitors on mitochondrial membrane permeability transition.
Cultured HLE cells (B3) maintained under hypoxic condition (1% oxygen) displayed consistent accumulation of VEGF throughout the 72 h incubation period. Using HIF translation inhibitors targeting HIF-1α or HIF-2α, the specific inhibition of each protein did not diminish VEGF synthesis. The combined inhibition of HIF-1α and HIF-2α expression, using a double HIF translation inhibitor, markedly decreased the level of VEGF. The inhibition of VEGF synthesis was associated with a profound deficiency in the level of the prosurvival protein, Bcl-2. Axitinib also prevented the VEGF-mediated expression of Bcl-2. The loss of VEGF coupled with the decrease in intracellular Bcl-2 correlated with marked mitochondrial depolarization, an early predictor of cellular apoptosis.
Our data support a model in which the sustained synthesis of VEGF in human lens epithelial cells, maintained under hypoxic condition, is regulated by a compensatory interrelationship between HIF-1α and HIF-2α. VEGF acts as a prosurvival factor in hypoxic lens epithelial cells by maintaining consistent expression of the antiapoptotic protein, Bcl-2, which likely prevents the translocation of cytosolic BAX to the outer mitochondrial membrane, thus preventing the initiation of mitochondrial depolarization.
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