Abstract
Purpose: :
Endogenous bioactive mediators synthesized in RPE cells in response to enhanced oxidative stress are important for photoreceptor function and integrity (IOVS:48,4866-81,2007). DHA is concentrated and avidly retained in photoreceptors. Also DHA traffics daily through RPE cells. Thus, following the discovery of neuroprotectin D1 (PNAS:101,8491-6,2004), we now report the induction of a 14S-hydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid pathway from DHA in RPE cells. Addition of deuterium-labeled DHA in the presence of oxidative stress led to the synthesis of these novel docosanoids - the maresins - reported in activated human macrophages (J.Exp.Med.:206,15-23, 2009). We found divergent induction of the 14S- (maresins) and 17S- (NPD1) pathways by enhanced oxidative stress in RPE cells.
Methods: :
Human ARPE-19 cells were incubated in the medium with DHA-d5 (4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic-21,21,22,22,22-d5 acid) supplemented for 6h, and the medium was replaced with DHA-d5 free medium. The cells were under oxidative stress with H2O2 (50, 400, 500 and 600μM) and TNF-α. Lipids were extracted and loaded onto an Eclipse Pluse C18 column (Agilent Technologies, Santa Clara, CA) and run gradient for 12min by changing solvent A (100:0.01 MeOH:CH3COOH) from 70% to 100% while decreasing solvent B (100:0.01 H2O: CH3COOH), 6min of 100% B before changing to A:B=70:30 for 5min. TSQ Quantum Ultra (Thermo-Finnigan, San Jose, CA) triple quadrupole tandem mass spectrometer (with HESI probe) was used for the analysis. Selected parent and daughter ion pairs of maresin and maresin-d5 were 359/250 m/z and 364/250 m/z, respectively.
Results: :
Endogenous maresin from DHA incorporated in the cellular membrane was found 2 ~ 5 times smaller than maresin-d5 from supplemented DHA-d5, depending on the amount of oxidative stress. One of the maresin isomers, 7S-maresin, was also increased from supplemented DHA. The maresin synthesis decreased as oxidative stress was enhanced, which is the opposite of what occurs for NPD1 synthesis.
Conclusions: :
We show here for the first time the ability of RPE cells to activate a 14S-pathaway for the synthesis of maresins. Maresin synthesis from DHA is activated by low oxidative stress conditions (50 μM H2O2). These new RPE docosanoids may contribute to cope with inflammatory resolution. The significance of the presence of maresins in macrophages also is being explored in RPE cells for their potential involvement in outer segment phagocytosis.
Keywords: lipids • neuroprotection • apoptosis/cell death