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Azucena H Kakazu, Thang Luong PHAM, Jiucheng He, Bokkyoo Jun, Nicolas G Bazan, Haydee E P Bazan; A novel resolvin D6 (RvD6) isomer released in tears stimulates corneal innervation and wound healing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5678. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recently, we demonstrated that treatment of injured mouse corneas with pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) stimulated nerve regeneration and the synthesis of a new DHA lipid mediator released in tears. The fragmentation pattern and UV spectrum of the docosanoid matched with RvD6, chemically named 4S,17S-dihydroxy-DHA (2018 ARVO). However, the retention time (RT) of the new lipid was different from the RvD6 standard RT, suggesting the presence of a different compound. Here we elucidated the structure and biological properties of this novel lipid mediator.
The corneas from anesthetized CD-1 mice were injured by rotating a 2mm trephine in order to cut the subbasal nerves. After 16 hours, the wounded corneas were excised and cultured in medium containing PEDF + DHA for 4h. In some experiments, the DHA was replaced with deuterated form (DHA-d5). To elucidate the structure of the novel docosanoid, enzyme inhibitors [100 nM Fluvoxamine, inhibitor of cytochrome P450 (CYP1A2); 50uM 17-ODYA, inhibitor of cytochrome P450 (CYP4F3); 200nM ML351, inhibitor of 15-lipoxygenase; 60uM Zileuton or 100uM MK591 both inhibitors of 5-lipoxygenase] were added to the medium. Lipids were extracted and analyzed by LC-UV-MS/MS. To study the biological activity of the new docosanoid, the compound was isolated by HPLC and topically applied to injured corneas. The corneal nerve regeneration was measured by immunostaining with PGP9.5 antibody.
The docosanoid isolated from the medium was identified as RvD6 by its fragmentation patterns that matched at least 6 fragments from RvD6 standard and by the UV spectrum with a peak at 238 nm, matching the UV spectrum from RvD6 standard. When our compound was co-injected with RvD6 standard, it showed different RT, suggesting the presence of a RvD6 stereoisomer. In experiments using DHA-d5, the RT matched the RT observed when DHA was used, demonstrating that the RvD6 isomer is synthesized from exogenous DHA. Fluvoxamine, an inhibitor of cytochrome P450 (CYP1A2), significantly inhibited the synthesis of RvD6 isomer.
PEDF+DHA induces the synthesis of a RvD6 isomer that is more powerful than its precursors on corneal wound healing and nerve regeneration. Inhibition of its synthesis by Fluvoxamine suggests that the structure of the RvD6 isomer could be 4S,17R-dihydroxy-DHA, also known as aspirin-triggered RvD6.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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