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F Seta, A Mezentsev, MW Dunn, M Laniado Schwartzman; 12(R)-Hydroxyeicosatrienoic Acid, a Corneal Epithelial Derived Eicosanoid, Stimulates Angiogenesis via ERK1/2 Activation of VEGF Induction . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2751.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In response to hypoxic injury, corneal epithelial cells synthesize an eicosanoid via cytochrome P450. This eicosanoid, 12(R)-hydroxyeicosatrienoic acid [12(R)-HETrE], then induces limbal vascular endothelial cells to proliferate and migrate into the cornea and begin neovascularization. We further investigated the cellular mechanisms underlying its angiogenic activity using endothelial cells derived from rabbit limbal microvessels (RLMVE cells). Methods: RLMVE cells were grown until 70% confluent and then quiesced for 36 hours. The cells were treated with 12(R)-HETrE (0.1nM) for various times. Northern or slot blot hybridization was used to determine VEGF mRNA levels and ELISA was used for measuring VEGF protein. Western blot analysis and in vitro kinase assays were used to determine MAPK activation. In vitro capillary formation assay using Matrigel was used to assess the functional relationship between 12(R)-HETrE and VEGF. Results: 12(R)-HETrE increased VEGF mRNA and protein in time and concentration dependent manners. Both transcriptional post-transcriptional mechanisms accounted for the increase in VEGF mRNA. 12(R)-HETrE activated ERK 1/2 as indicated by rapid increases in the levels of phosphorylated ERK 1/2 and activity measured as phosphorylated Elk. 12(R)-HETrE-stimulated ERK 1/2 activity as well as 12(R)-HETrE-induced VEGF expression was inhibited by the MEK 1 selective inhibitor PD98059. Addition of VEGF antibody to RLMVE cells grown in Matrigel-coated plates inhibited 12(R)-HETrE-induced capillary formation. Conclusions: The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation of ERK 1/2 and that VEGF mediates at least in part the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea following hypoxic injury, their interaction may be an important determinant in the development of neovascularized cornea. CR:None. Support: NIH grant EY06513
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