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Sudha Neelam, Morgan M. Brooks, Patrick R. Cammarata; Lenticular Mitochondrial Protection. Part 1: BAX/VEGF Regulates Mitochondrial Depolarization in Hypoxia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1060.
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Human lens epithelial (HLE) cells thrive in 1-3% oxygen, normally pathological for most tissues. The signal transduction pathways involved with HLE survival in hypoxia (Hyp) are not well established. Cellular apoptosis is regulated by the relative levels of pro and anti-apoptotic proteins. BAX is a pro-apoptotic member of the Bcl-2 family. Activation of BAX causes its translocation to the mitochondrial outer membrane causing mitochondrial permeability membrane transition (mPMT) by opening of the mitochondrial permeability transition pore. The role of vascular endothelial growth factor (VEGF) in HLE survival under low oxygen conditions is unknown. ERK plays a crucial role in mitochondrial protection (mitoprotection) of cultured HLE. In this study we examined the effects of ERK phosphorylation on relative VEGF and BAX levels in cultured HLE-B3 cells in 1% oxygen (Hyp) vs. atmospheric oxygen (Norm).
Specific antisera and western blot analyses was used to detect ERK1/2, pERK1/2, BAX and Bcl-2 expression. ELISA was performed with HLE-B3 supernatants to detect VEGF levels under Hyp and Norm. JC-1 fluorescent dye analysis was employed to determine the extent of mitochondrial depolarization.
No significant difference in VEGF levels was observed at 3h or 8 h of Hyp compared with Norm control. Subsequent to 3h of initial Hyp treatment, cells were reintroduced to Norm for an additional 2, 4 and 24 h, whereupon VEGF levels were evaluated. No significant differences in the VEGF levels were observed as a result of prior Hyp treatment or reintroduction of oxygen relative to cells maintained under Norm throughout the incubation period. MEK inhibitor, UO126-treated (10µM) cells maintained in Hyp (8h) showed no change in VEGF or BAX compared to Hyp non-treated cells. In contrast, UO126 addition to reoxygenated cells elicited profoundly reduced VEGF levels while at the same time increasing BAX levels, at 4h and 24h, relative to non-treated control cells. Under no experimental conditions did Bcl-2 levels significantly deviate from control cells. Whereas, UO126 did not elicit mPMT with Hyp cells, UO-treated cells, reintroduced to oxygen, prompted profound mPMT.
Regulation of synthesis and/or accrual of VEGF and BAX in HLE-B3 cells appear to be independent of the ERK pathway in Hyp. In contrast, inhibition of ERK phosphorylation upon reoxygenation elicits profound loss of VEGF, increases BAX and brings about considerable mPMT. VEGF may play a significant role in hypoxic lenticular mitochondrial protection by suppression of BAX levels. Lowered BAX in Hyp in contrast to markedly increased BAX in Norm provides the causal foundation for lenticular epithelial cell mitoprotection in low oxygen.
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