Abstract
12(R)-Hydroxy-5,8,14-eicosatrienoic acid (HETrE) is a potent inflammatory and angiogenic eicosanoid in ocular and dermal tissues. Previous studies suggested that 12(R)-HETrE activates microvessel endothelial cells via a high affinity binding site; however, the cellular mechanisms underlying 12(R)-HETrE angiogenic activity are unexplored. Because the synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its interactions with vascular endothelial growth factor (VEGF) in rabbit limbal microvessel endothelial cells. Addition of 12(R)-HETrE (0.1 nm) to the cells increased VEGF mRNA levels with maximum 5-fold increase at 45 min. The increase in VEGF mRNA was followed by an increase in immunoreactive VEGF protein. 12(R)-HETrE (0.1 nm) rapidly activated the extracellular signal-regulated kinases (ERKs) ERK1 and ERK2. Moreover, preincubation of cells with PD98059, a selective inhibitor of MEK-1, inhibited 12(R)-HETrE-induced VEGF mRNA. Addition of VEGF antibody to cells grown in Matrigel-coated culture plates inhibited 12(R)-HETrE-induced capillary tube-like formation, suggesting that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation of ERK1/2 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 after hypoxic injury, their interaction may be an important determinant in the development of neovascularized tissues.
Highlights
A wide variety of disorders of the cornea evoke a vasculogenic response; a considerable amount of information has accumulated about the circumstances under which newly formed blood vessels sprout and extend centripetally into the cornea
Because the synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its interactions with vascular endothelial growth factor (VEGF) in rabbit limbal microvessel endothelial cells
The current study examines whether VEGF is a component of the angiogenic activity of 12(R)-HETrE and characterizes mechanisms underlying the effect of 12(R)-HETrE on VEGF expression
Summary
Materials—12(R)-HETrE and 12(S)-HETrE were synthesized and purified as described previously [16]. Cells were cultured in Dulbecco’s modified Eagle’s medium (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (Cellgro), 1% antibiotic-antimycotic (Cellgro), and endothelial cell growth supplements (ECGS; Sigma). Cells were washed twice in phosphate-buffered saline, pH 7.6, and quiesced in antibiotic- and growth factor-free Dulbecco’s modified Eagle’s medium containing 0.5% fetal bovine serum for 24 –36 h. Membranes were cross-linked (1200 mkJ/cm2), dried at 80 °C for 2 h, and incubated overnight in hybridization buffer containing the 32P-end-labeled mouse VEGF cDNA probe at 65 °C. Aliquots of cell-free homogenates (150 g) were denatured in Laemmli loading buffer (2 min at 95 °C), resolved on 11% or 14% SDS-polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (BioRad). The length of the tube-like was structures was quantified using Image Pro-Express Software (Cyber Media)
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