Synthesis and identification of two docosanoids in mouse corneas and tears stimulated with pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA)

Abstract

Purpose : We have recently demonstrated that treatment of injured mouse corneas with PEDF+DHA stimulated nerve regeneration and the synthesis of 17- hydroxyDHA (17-HDHA) and 14-HDHA and these synthesis was inhibited by the specific inhibitor for PEDF-R, Atglistatin (Pham TL et al. J. Biol. Chem. 2017; 292: 18486). These two HDHA are side products in the synthesis of a family of specialized pro-resolving mediators (SPMs). Here we identify two new DHA mediators activated by PEDF that are involved in corneal wound healing and nerve regeneration

Methods : Eight to ten week-old male CD-1 mice were used. Corneas from anesthetized mice were injured by rotating a 2 mm trephine to cut the subbasal nerves and then treated with PEDF+DHA or vehicle. In some experiments 500uM Zileuton, inhibitor of 5-lipoxygenease (5-LOX) or 200uM ML351 inhibitor of 15-LOX were added 30 minutes before stimulation. Tears were collected at 4 and 16h after injury. The wounded corneas were excised and cultured in media containing PEDF+DHA, PEDF+DHA with inhibitors, or vehicle for 4 hour at 37^o C. Lipids were extracted from the tears, media and corneas and analyzed by LC-UV-MS/MS. To study the effect of the docosanoid resolvin D6 (RvD6) on corneal wound healing and nerve regeneration, the corneal epithelium was removed and then treated topically with 10ng/eye RvD6, 3 times/day. The wound closure was measured after 20 h.

Results : Two DHA-dihydroxylated derivatives were detected in tears, media and corneas, with retention times (RT) 8.06 and 8.28 min. The peaks were identify on its RT, fragmentation pattern and UV spectrum. The peak with RT 8.06 min matched with the 4,14-diHDHA , belonging to the maresin family; the peak with a RT of 8.28 min matched with RvD6. Both peaks showed strong activation with PEDF+DHA and were significantly inhibited in presence of inhibitors of 5- and 15-LOX. RvD6 significantly stimulated corneal wound healing after 20h of treatment.

Conclusions : PEDF+DHA induce the synthesis of RvD6 that stimulated wound healing and of the maresin 4,14-diHDHA Both SPMs are released in tears and in the media of incubated corneas suggesting that they may display autocrine and/or paracrine bioactivity that enhance nerve regeneration.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.