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Azucena H Kakazu, Bokkyoo Jun, Eric J Knott, Nicolas G Bazan, Haydee E P Bazan; Stimulation of PEDF-R in Rabbit Corneal Epithelial Cells Induces a Rapid Release of DHA. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5501.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies have shown that treatment of rabbit corneas with pigment epithelial derived factor (PEDF) and the ω-3 fatty acid docosahexaenoic acid (DHA) increases neuroprotectin D1 (NPD1) synthesis and promotes nerve regeneration after experimental surgery (Cortina MS et al., IOVS, 2010). In tissues, NPD1 synthesis requires the release of DHA by activation of a non-defined phospholipase A2 (PLA2). In retinal cells, a PEDF receptor (PEDF-R) has been identified that has a sequence homology similar to PLA enzymes (Notari L et al., J Biol Chem, 2006). Here we investigate the action of PEDF on the release of DHA in rabbit corneas and demonstrate the expression of the PEDF-R and independent PLA2 (iPLA2) in the corneal epithelium.
Rabbit eyes were obtained from Pel Freez Biologicals (Rogers, AR). Organ cultured corneas were incubated with 40 ng/ml PEDF for 5, 10, 15, 30, and 60 minutes at 37°C. At the end of each time point, medium and epithelium were collected and their lipids extracted and analyzed by liquid-chromatography-tandem mass spectrometry. The expression of PEDF-R was evaluated by Western blot and immunofluorescence staining with specific antibodies on rabbit corneal epithelial cells (RCEC) in culture. iPLA2 was identified by Western blot in rabbit epithelia from cells in culture and organ culture.
A 56 Kda band corresponding to the PEDF-R was expressed in RCEC. The band was confirmed by a blocking peptide that abolished the band. PEDF-R was localized by immunofluorescence in the membrane and around the nuclei. A strong 80 KDa band corresponding to iPLA2 was expressed in the corneal epithelium. Stimulation of corneas in organ culture with PEDF induced a rapid release of DHA. Five minutes after stimulation, the release of DHA increased 4 times with respect to controls in the epithelium and 8 times in the media. By 30 minutes, the values were similar to controls.
Stimulation of the corneal epithelium by PEDF rapidly releases DHA from membrane phospholipids by activation of the PEDF-R linked to PLA2 activity.
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