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F. Seta, A. Mezentsev, M.W. Dunn, M. Laniado–Schwartzman; CORNEAL–DERIVED 12(R)–HYDROXYEICOSATRIENOIC ACID (12–HETrE), STIMULATES LIMBAL ENDOTHELIAL ANGIOGENESIS VIA ERK1/2 INDEPENDENT OF RAS/RAF ACTIVATION . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2343.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: 12(R)–HETrE is a corneal epithelial–derived angiogenic factor whose synthesis is induced in response to injury in vitro and in vivo; it acts in a paracrine manner on the limbal vessels to activate endothelial cells via a specific receptor/binding site and stimulates angiogenesis. We have identified the MAPK–ERK1/2 pathway as one mechanism mediating 12(R)–HETrE–induced VEGF production and angiogenesis. We further examined the cellular mechanisms upstream of ERK1/2 activation using endothelial cells derived from rabbit limbal microvessels (RLMVE cells). Methods: RLMVE cells (passages 3–10) were grown until 70% confluent and then quiesced for 36 hours. The cells were treated with 12(R)–HETrE (0.1–1 nM) in the presence and absence of Calphostin (a PKC inhibitor), Wortmannin (PI3K inhibitor), Gentistein (a tyrosine kinase inhibitor), PD98059 (a MEK inhibitor), and FTSA and Manumycin (inhibitors of Ras activation). RLMVE cells were transfected with plasmid expressing the dominant negative form of Ras or Raf proteins 24 h before treatment with 12(R)–HETrE; cells were harvested 5–30 min after 12(R)–HETrE addition and Western blot analysis was used to determine MAPK activation. In vitro capillary formation assay using Matrigel was used to assess the functional relationship between 12(R)–HETrE and ERK1/2 activation. Results: 12(R)–HETrE induced phosphorylation of ERK1/2 in a time– and concentration– dependent manner. The maximal response occurred within 5 min of addition of 0.1 nM 12(R)–HETrE. 12(R)–HETrE–induced ERK1/2 phosphorylation was inhibited by pretreatment of cells with Calphostin, Wortmannin, Gentistein and PD98059 but not with inhibitors of Ras activation. ERK1/2 activation in response to 12(R)–HETrE was not altered in cells expressing Ras or Raf dominant negative proteins. Inhibition of ERK1/2 activation attenuated 12(R)–HETrE–stimulated capillary–like tube formation in RLMVE cells. Conclusions: The results indicate that 12(R)–HETrE induces ERK1/2 phosphorylation in a mechanism that involves activation of PI3K, tyrosine kinase and PKC but not Ras and Raf. 12–(R)–HETrE is produced by the corneal epithelium in response to injury, displays potent inflammatory properties, is a mitogen for microvessel endothelial cells, and is angiogenic in vitro and in vivo. Identifying signaling molecules and processes linking its receptor binding to effect is critical for elucidating the pathophysiological ramification of this eicosanoid.
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