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(Investigative Ophthalmology and Visual Science. 2004;45:2915-2921.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0128
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Platelet-Activating Factor (PAF) Induces Corneal Neovascularization and Upregulates VEGF Expression in Endothelial Cells

Xiang Ma,1,2 Paulo Ottino,1 Haydee E. P. Bazan,1 and Nicolas G. Bazan1

1From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center School of Medicine in New Orleans, New Orleans, Louisiana.

PURPOSE. Platelet-activating factor (PAF) is a potent proinflammatory mediator that accumulates in the cornea after injury and induces the expression of genes related to inflammation and wound healing. The current study was conducted to investigate the direct effect of PAF on corneal neovascularization and on the expression of angiogenic growth factors in vascular endothelial cells.

METHODS. Pellets containing carbamyl-PAF (cPAF) were implanted in corneas of wild-type or PAF-receptor (PAF-R)-knockout mice, and the progression of angiogenesis was monitored by microscope. In some experiments, mice were treated with a daily intraperitoneal injection of the PAF-R antagonist LAU8080. Migration assays of human umbilical cord vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs) were performed in a Boyden chamber after addition of various concentrations of cPAF or bovine fibroblast growth factor (FGF-2). Cell proliferation was assessed by fluorescence-binding assay in the presence of cPAF or FGF-2 for 8 days. Vascular endothelial growth factor (VEGF) and FGF-2 expression was studied by RT-PCR and Northern- and Western-blot analyses in cells stimulated with cPAF at different concentrations and for different times.

RESULTS. Six days after cPAF pellet implantation, there were new vessels growing from the limbus to the center of the cornea. The PAF-induced neovascularization was significantly reduced in PAF-R-knockout mice and in mice treated with the PAF antagonist. cPAF added to the lower well of the Boyden chamber produced a dose-dependent migration of HUVECs and HMVECs that was inhibited in cells preincubated with LAU8080 or with a VEGF-blocking antibody. In contrast, cPAF did not stimulate proliferation of endothelial cells. cPAF induced VEGF mRNA and protein expression but not FGF-2 expression in HUVECs and HMVECs.

CONCLUSIONS. PAF stimulates corneal neovascularization by a receptor-mediated mechanism. Induction of VEGF expression and stimulation of vascular endothelial cell migration are initial events in PAF-promoted corneal angiogenesis.




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