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N.G. Bazan, V.L. Marcheselli, J. Hu, J. Finley, D. Bok, B. Chandamuri; Pigment Epithelium–Derived Growth Factor (PEDF) Selectively Up–Regulates NPD1 Synthesis and Release Through the Apical Side of Human RPE Cells in Primary Cultures . Invest. Ophthalmol. Vis. Sci. 2005;46(13):167.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Neuroprotectin D1 (NPD1) is derived from docosahexaenoic acid (DHA) by enzyme–mediated oxygenation, synthesized in RPE cells, and displays potent cytoprotective activity against oxidative stress (PNAS 101:8491–8496; 2004). When RPE cells are confronted with oxidative stress, NPD1 down–regulates cytokine–mediated pro–inflammatory gene expression and pro–apoptotic Bcl–2, Bax, and Bad proteins. In addition, NPD1 up–regulates Bcl–2 and Bcl–xl proteins. As a result, NPD1 inhibits oxidative stress–mediated apoptosis. Because of the pro–survival bioactivity of NPD1, we explored here several growth factors as potential modulators of NPD1 synthesis in RPE cells. We also aimed to define whether release of NPD1 was via the apical or basal cell surface. Methods: Human RPE cells were seeded onto Millicell–HA culture plates (Millipore, Bedford, MA); after reaching confluence, cultures were used that displayed transepithelial resistance of at least 500 Ω × cm–2. Cells were grown in plastic culture flasks using a chemically defined culture medium. After adding growth factors, apical and basal media were separately collected and measured by LC–PDA–ESI–MS–MS–based lipidomic analysis. Results: We incubated cells in the presence of GDNF, rCNTF, rhNGF, hFGF, rhBDNF, or PEDF at concentrations from 50–200 pM over 48 to 72 hr. PEDF elicited concentration–dependent synthesis of NPD1, which was released mostly through the apical cell surface. This was overwhelmingly seen when PEDF was added to the apical medium, and not to the basal medium. CNTF was the second most potent of the growth factors studied to elicit NPD1 synthesis but its activity was about 15% that of PEDF. All other growth factors also triggered NPD1 synthesis, but at much lower than 15% of that elicited by PEDF. Dose–dependent NPD1 synthesis occurred when cells were treated on the apical side with DHA, but when co–cultured with PEDF, a 3.8–fold increase was measured. Conclusions: PEDF is a potent activator of the synthesis and release of NPD1 from human RPE cells. The concentration–dependency of this effect (50–200 pM of the growth factor) and the specificity of the apical cell surface suggest that growth factor–mediated regulation of NPD1 synthesis may be either autocrine or paracrine, and that the NPD1 release to the interphotoreceptor matrix may exert survival actions in other cells (photoreceptors?). Moreover, the mediator NPD1 may also be engaged in anti–angiogenic actions of PEDF. NIH EY05121
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