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Sietse Braakman, Kristin Perkumas, Darryl Overby, David Woodward, W Daniel Stamer; Sphingosine-1-Phosphate signaling in cultured human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1995.
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© ARVO (1962-2015); The Authors (2016-present)
Sphingosine-1-Phosphate (S1P) is a bioactive lipid that decreases conventional outflow facility in human, porcine and mouse eyes, likely by decreasing the permeability of Schlemm’s canal endothelium (SCE). Located upstream of SCE, the trabecular meshwork (TM) is a prime candidate for S1P synthesis. In this study, we test the hypothesis that human TM cells synthesize S1P as a possible mechanism to regulate facility. We also measure S1P levels in human aqueous humor (AH).
TM cell lines were isolated from non-glaucomatous human donors and differentiated by allowing confluent cultures to mature for 1 week in DMEM containing 10% fetal bovine serum (FBS) followed by 1 week in 1% FBS. After differentiation, medium was refreshed (1% FBS) and conditioned by the cells for 2 hours, before being sampled. AH samples were obtained from 5 non-glaucomatous patients during cataract surgery. Conditioned medium, fresh medium containing 1% FBS, and pooled AH were analyzed for S1P and prostaglandin E2 (PGE2) using LC-MS/MS. Cell viability was measured by lactate dehydrogenase (LDH) activity using a colorimetric NAD assay. Cell lysates were analyzed by western blot for sphingosine-kinase 1 (SphK1).
S1P in fresh medium (1% FBS) was measured to be 3.5 nM, which decreased to 1.4±0.3nM (mean±SD, n=29) after 2h of cell conditioning. In contrast, PGE2 levels remained constant (65±22nM vs. 60nM, n=29, p=0.6). Ceramides and sphingosine, substrates for S1P synthesis, were present in conditioned media (26±9.7 and 5.0±1.4nM, n=29). In AH, S1P was below the detection limit, while PGE2 was measured to be 28.8nM. There were no signs of cell death, as measured by LDH (6.3-14.3 mIU/mL vs. 1139mIU/mL in positive control). SphK1 was detected by western blot in both TM and SC cells.
S1P was undetectable in AH and conditioned media from TM, however both TM and SC cells expressed the enzymes and substrates necessary for S1P generation and receptors for signaling. Since S1P agonists/antagonists alter outflow facility, endogenous S1P generation must be local (autocrine/paracrine), stimulated and/or short lived. Acknowledgements: Lipidomics Core Facility MUSC
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