Abstract
Purpose :
We have previously demonstrated that treatment of corneas with PEDF or with the 44-mer PEDF peptide plus DHA increases the synthesis of neuroprotection D1 (NPD1; 10R,17S¬dihydroxy-docosa-4Z,7Z,11E,15E,19Z hexaenoic acid) and stimulates nerve regeneration after experimental refractive surgery (Cortina MS et al, IOVS, 2010 ). NPD1 synthesis requires the release of DHA by the activation of a phospholipase A2 (PLA2) (Bazan N, IOVS, 2007). Here we investigate if PEDF or 44 mer-PEDF plus DHA activates the synthesis of NPD1 and other docosanoids in rabbit corneas and if the synthesis is inhibited by the iPLA2 inhibitor bromoenol lactone (BEL).
Methods :
Rabbit corneas were pre-incubated with 5µM DHA for 2h at 37°C, washed and then stimulated with 40ng/ml PEDF or 5ng/ml 44-mer for 3, 16, or 24h at 37°C. In some experiments, 50µM BEL was added 30 minutes before stimulation. Corneas and media were collected and lipids extracted using solid-phase extraction and followed by LC-MS/MS lipid analysis.
Results :
Stimulation of corneas with PEDF or the 44-mer PEDF for 5 minutes enhanced the unesterified DHA pool size. Since this effect was inhibited by the specific inhibitor of i-PLA2, BEL, we suggest an i-PLA-mediated event. When the corneas were incubated with PEDF or 44-mer PEDF, NPD1 synthesis took place. In addition, we found several DHA-dihydroxylated derivatives that currently are being characterized. They include peak 0 (retention time 3.28 min), peak 1 (3.37 min), peak 2 (3.63 min), peak 3 (3.74 min), and peak 4 (3.96 min). These peaks displayed a fragmentation ion 206.12, which was also detected in NPD1. Peak 0 was stimulated by 44-mer PEDF in corneal tissue at 16 and 24h after incubation. Peaks 1, 3, an 4 were increased in the media, where peak 1 showed a strong increase at 3h with PEDF and 44-mer. Peak 2 was increased in cells and media with 44-mer PEDF at 3 and 16h after incubation.
Conclusions :
Activation of PEDF-R induces a rapid release of DHA from phospholipids of corneas by the stimulation of i-PLA2. In addition to NPD1, PEDF and the neurotrophic peptide 44-mer induce the synthesis of additional docosanoids that may display neurotrophic activity. It is of interest that there is a differential release of docosanoids to the incubation media, suggesting that they may display autocrine or paracrine bioactivity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.