May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
PEDF Promotes Biosynthesis and Efflux of NPD1 in ARPE-19 Cells
Author Affiliations & Notes
  • M. A. Reinoso
    Ophthalmology, LSU/Ochsner, New Orleans, Louisiana
  • V. L. Marcheselli
    Neuroscience Center,
    LSU, New Orleans, Louisiana
  • P. K. Mukherjee
    Neuroscience Center,
    LSU, New Orleans, Louisiana
  • Y. Lu
    Neuroscience Center,
    LSU, New Orleans, Louisiana
  • F. R. Jackson
    Neuroscience Center,
    LSU, New Orleans, Louisiana
  • D. R. Bergsma
    Ophthalmology, LSU/Ochsner, New Orleans, Louisiana
  • N. G. Bazan
    Ophthalmology and Neuroscience Center,
    LSU, New Orleans, Louisiana
  • Footnotes
    Commercial Relationships M.A. Reinoso, None; V.L. Marcheselli, None; P.K. Mukherjee, None; Y. Lu, None; F.R. Jackson, None; D.R. Bergsma, None; N.G. Bazan, None.
  • Footnotes
    Support NEI EY05121, American Health Assistance Foundation, NCRR, R21 COBRE and Research to Prevent Blindness, NY (LSU Eye Center).
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2959. doi:
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      M. A. Reinoso, V. L. Marcheselli, P. K. Mukherjee, Y. Lu, F. R. Jackson, D. R. Bergsma, N. G. Bazan; PEDF Promotes Biosynthesis and Efflux of NPD1 in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2959.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: NPD1 is a stereospecific cytoprotective messenger synthetized from docosahexaenoic acid in RPE cells challenged by oxidative stress, cytokines or calcium ionophore (TINS 29:263-271, 2006). A key regulatory step in NPD1 synthesis is to define how growth factors may modulate the formation of this lipid mediator and its bioavailabity. Here we have explored the action of PEDF, a pleiotropic neurotrophin made in RPE, on the synthesis and cellular release of NPD1.

Methods:: ARPE-19 cells, three days after plating, were serum starved and exposed to TNFα/H2O2 in the presence and absence of PEDF (10 mg/ml). Cells and incubation media were collected as a function of time up to 12 hrs. Then we used LC-PDA-MS-MS - based lipidomic analysis to identify and quantitate NPD1. In addition, we have used 2H5-22:6, n-3 to trace the DHA conversion into docosanoids as well as to further explore the effect of PEDF. This non-radioactive approach has several advantages; deuterium is on specific methylene carbons not metabolically altered and its heavier molecular mass is separated and identified by mass spectrometry. Therefore analysis of endogenous as well as of added DHA derivatives are detected and quantified.

Results:: Oxidative stress promotes 10 fold increases in NPD-1 levels in ARPE-19 cells after 6 hrs incubation. Time course of induction of NPD-1 levels when ARPE cells were treated with 2H5-DHA and oxidative stress shows a rapid increase up to 6 hrs of incubation of 12 folds measured on cell pellets. Cell media, on the other hand, shows time dependent accumulation of NPD1 up to 55 fold after 12 hrs incubation. Treatment with 50 nM PEDF increased such profile by at least two fold. Deuterated DHA was incorporated to cell membranes, and readily converted into NPD-1, it constituted about 10% of total mass, and profile kinetics are comparable to endogenous DHA.

Conclusions:: Here we demonstrate that PEDF is an activator of NPD1 synthesis in ARPE-19 cells exposed to oxidative stress. Since 70% of the synthetized NPD1 is released from the cells we postulate that the neurotrophin-stimulated NPD1 synthesis exerts at least part of its bioactivity through autocrine mechanisms. A major action of PEDF-stimulated NPD1 synthesis shown here is RPE cytoprotection.

Keywords: second messengers • apoptosis/cell death • neuroprotection 
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