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Sachidananda Kenchegowda, C ZANG, Nicolas G. BAZAN, Haydee E. BAZAN; Increased Release Of Pigment Epithelial-derived Factor After Corneal Injury Stimulates Neuroprotectin D1 Synthesis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1824.
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Treatment with a combination of pigment epithelial-derived factor (PEDF) and docosahexaenoic acid (DHA) stimulates corneal nerve regeneration after damage by experimental surgery in rabbits, accelerating wound healing and returning corneal sensitivity to non-injured levels (Cortina et al, Arch Ophthalmology, 2011). PEDF in the presence of DHA induces NPD1 synthesis in vivo, suggesting the docosanoid is a mediator in the action of PEDF plus DHA (Cortina et al, IOVS, 2010). Here, we investigated if PEDF is released from corneal epithelial cells after injury and if the cells can synthesize NPD1.
Rabbit corneas were injured with a 7 mm epithelial debridement and cultured for 48 h. Media was collected and PEDF expression was analyzed by western blot. . In a parallel experiment, non-injured and injured corneas were fixed and sectioned, and immunofluorescence was performed using anti-PEDF antibodies. Human corneal epithelial cells were incubated with DHA Na+ solution (5µM), for 2 h to promote incorporation of the fatty acid and then stimulated with 40 ng/ml PEDF for 24 and 48 h. In some experiments, epithelial cells were treated with CDC (10 µM) (12/15 lipoxygenease inhibitor) or PD146176 (10 µM) (15-lipoxygenease-1 inhibitor) in the presence of PEDF. NPD1 synthesis was analyzed in cells and media by LC-ESI-MS/MS.
There was a drastic increase of PEDF expression in the media of injured cornea compared to non-injured ones. Immunofluorescence showed strong PEDF expression in the basal layer of epithelial cells in normal corneas, while in the injured group PEDF expression was only observed in the surface of the epithelium. A significant increase in NPD1 synthesis was found after 24 and at 48 h of PEDF stimulation. NPD1 synthesis was completely blocked by CDC and PD146176. An interesting finding was the release of NPD1 to the media after PEDF stimulation.
After injury, PEDF is secreted by corneal epithelial cells and by an autocrine mechanism activate its receptor to stimulate NPD1 synthesis. On the other hand, the release of NPD1 to the media suggests autocrine/paracrine actions of this docosanoid.
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