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C. Abbondandolo, S.-W. M. Koh; Anti-Apoptotic Bcl-2 mRNA Expression and Long-Term Corneal Endothelial Cell Survival Against Oxidative Stress ex vivo: Effect of VIP. Invest. Ophthalmol. Vis. Sci. 2007;48(13):550.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial (CE) cell acutocrine vasoactive intestinal peptide (VIP) protects CE cells against the acute killing effects of H2O2 ex vivo1 and switches the death mode from necrosis to apoptosis. The purposes were to demonstrate that the anti-apoptotic Bcl-2 gene expression and the long-term survival of CE cells injured by oxidative stress were promoted by VIP pretreatment, which increased the level of CREB phosphorylation.
Bovine corneoscleral explants were conditioned in Eagle's minimal essential medium/ 20 mM HEPES (EMEM/HEPES) at 37oC for 60 min before CE cells in corneal cups were treated with VIP (0 [control], 10-10, 10-8, and 10-6 M; 15 min). The explants were then treated with 1.4 mM H2O2/PBS (30 min, 37 oC) and incubated in EMEM/HEPES plus antibiotics for either 26 h, for Bcl2-mRNA quantification, or 64 h, for cell number quantification. Bcl-2 mRNA quantification was done by RTPCR using the Bcl-2 primers (Forward: GAGATGTCCAGTCAGCTGCACC, Reverse: ATAGGCACCCAGGGTGATGC) and kits containing the 18S rRNA primers for normalization of the mRNA levels (Ambion, Catalog #’s 1716 and 1718). The cell number quantification was done by genomic DNA levels using a kit (BioRad). The viability of CE cells in corneoscleral explants was examined by fluorescence microscopy (LIVE/DEAD cytotoxicity assay; Molecular Probes). The state of CREB phosphorylation in CE cells immediately following the VIP pretreatment was demonstrated by Western blot analysis using phosphoCREB antibody (UBI).
Bcl-2 mRNA levels increased in a VIP-concentration-dependent manner. The ratios of bcl-2:18S were (mean ± SEM): 0.207 ± 0.040, 0.192 ± 0.037, 0.335 ± 0.044*, and 0.456 ± 0.122* in 0, 10-10, 10-8, and 10-6 M VIP-pretreated CE cells, respectively (p<0.05, ANOVA; *: p<0.05 in control vs. VIP-pretreated). CE cells in explants recovering from oxidative stress injury increased in a VIP-concentration-dependent manner and were (in %)100 ± 9, 144 ±26, 190 ± 23*, 166 ± 18*, respectively (p<0.05, ANOVA). VIP dose-dependently increased the level of CREB phosphorylation.
Through up-regulation of Bcl-2 gene expression, VIP promoted the long-term survival of those CE cells that have survived the initial killing effect of severe oxidative stress.1. Koh and Waschek, (2000) IOVS, 41:4085-4092.
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