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Negin Ashki, Ann Chan, Yu Qin, Meagan Kiyohara, Lynn Gordon; Human Corneal Limbal Epithelial Cells Up-regulate Angiogenic Factors Following Exposure to Peroxynitrite. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2560. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal neovascularization (NV) is a sight threatening condition that may be associated with infection or inflammation. Macrophages recruited to the cornea may release various proinflammatory cytokines, and reactive oxygen and nitrogen species, such as nitric oxide (NO) and superoxide anion (O2), which react together to form the highly toxic molecule peroxynitrite (ONOO-). We investigated the potential role of ONOO- in up-regulating angiogenic factors, vascular endothelial growth factor (VEGF), hypoxia-inducible factors-1 alpha (HIF-1 alpha) and HIF-2 alpha, and fibroblast growth factor (FGF) in human corneal limbal epithelial cells (HCLE).
Confluent HCLE cells, incubated in serum-free keratinocyte media, were exposed to 500 µM of ONOO-donor for 0,2,4,6, 8, 12, 18, and 24 hours at 37 oC. Angiogenic proteins, VEGF, FGF, HIF-1 and HIF-2 alpha were analyzed via western blot, RT-PCR for VEGF, and VEGF functional assays. Conditioned media from HCLE cells treated with ONOO- were collected and secreted VEGF detected and analyzed using enzyme-linked immunosorbent assay (ELISA) kit. Functional assays consisting of migration and tube formation of human umbilical vein endothelial cells (HUVEC) were also performed using the conditioned media. Quantification of the effect was done in a masked fashion. All data was analyzed for statistical significance using Prism statistical software.
Increased HIF-1 expression was seen at 4,6, and 8 hours post-ONOO- exposure (p<0.05). FGF expression was elevated at 4 hrs and peaked at 8 hrs following treatment (P<0.05). Similarly, increased VEGF expression, by both ELISA and functional assays, was observed at 4,6, and 8 hours post-ONOO- treatment. When the ONOO- donor was completely depleted of its ONOO- concentration or when HCLE cells were treated with media only, there were no changes in expression of the above angiogenic factors.
VEGF and FGF are key angiogenic factors found in the cornea and involved in regulating angiogenesis. It has been well established that HIF-1 alpha is a potent regulator of VEGF. Exposure of HCLE cells to ONOO- causes an up-regulation of angiogenic factors including VEGF, FGF, and HIF-1. This may be an important mechanism for inflammation-associated corneal NV.
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