May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
In vitro and in vivo Studies of cPLA2 Involvement in VEGF Mediated Retinal Neovascularization
Author Affiliations & Notes
  • J.M. Barnett
    Ophth, Vanderbilt, Nashville, TN
  • J.A. Fowler
    Ophth, Vanderbilt, Nashville, TN
  • J.S. Penn
    Ophth, Vanderbilt, Nashville, TN
  • Footnotes
    Commercial Relationships  J.M. Barnett, None; J.A. Fowler, None; J.S. Penn, None.
  • Footnotes
    Support  NIH Grant EY08126, EY07533, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 2600. doi:
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      J.M. Barnett, J.A. Fowler, J.S. Penn; In vitro and in vivo Studies of cPLA2 Involvement in VEGF Mediated Retinal Neovascularization . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2600.

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

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Purpose: : Cytosolic Phospholipase A2 (cPLA2) promotes VEGF induction through catalyzing the hydrolysis of phospholipids directly releasing arachidonic acid, the precursor of prostaglandins. In order to better understand the role of cPLA2 in retinal VEGF regulation, the temporal distribution and level of retinal cytosolic Phospholipase A2 involvement was examined using in vitro and in vivo models.

Methods: : Primary retinal Müller cells were isolated from 14 day old Long Evans rat pups and exposed to hypoxia for 24 hours after treatment with the cPLA2 inhibitor, cPLA2 protein and mRNA were then collected from the cells and used in western blots and microarray analysis. Growth medium from the cells was also collected and assessed for VEGF levels. Sprauge–Dawley rat litters were exposed to alternating 50% and 10% oxygen atmospheres from birth through P14. Upon removal from the exposure chamber, some rats received an intraocular injection of a cPLA2 inhibitor, N–{(2S, 4R)–4–Biphenyl–2–ylmethyl–isobutyl–amino)–1–[2–(2,4–difluorobenzoyl)–benzoyl]–pyrrolidin–2–ylmethyl}–3–[4–(2,4–dioxothiazolidin–5–ylidenemethyl)–phenyl]acrylamide, HCl. These animals were sacrificed at 6 days later, and their retinas were ADPase–stained and evaluated for neovascularization (NV) and avascular area. Other animals were sacrificed at 1, 3, and 6 days post oxygen exposure, and retinal cPLA2 levels were measured by western blot analysis and gene microarray.

Results: : Primary retinal Müller cells exposed to hypoxia for 24 hours exhibited a 3–fold increase in VEGF secretion, while only a 1.1–fold increase was observed in samples treated with a cPLA2 inhibitor. Additionally, these cells demonstrated a 4.2–fold increase in cPLA2 mRNA and a 3– to 5–fold induction of phosphorylated cPLA2 protein levels. A 5.7–fold increase in retinal cPLA2 mRNA and a 3.2–fold increase in phosphorylated retinal cPLA2 protein was observed in oxygen exposed animals compared to room air raised controls at 1 day post oxygen exposure. The evaluation of the retinas from animals treated with an intraocular injection of a cPLA2 inhibitor revealed a 30% (p<0.05) decrease in NV and a 26% (p<0.05) decrease in avascular area compared to control retinas.

Conclusions: : Increased cPLA2 mRNA and protein levels were associated with increased VEGF levels and both increased retinal NV and avascular area, which where both significantly decreased with the inhibition of cPLA2. Continued research into the induction of VEGF through arachidonic acid derivatives may identify additional and novel therapeutic targets.

Keywords: Muller cells • retinal neovascularization • enzymes/enzyme inhibitors 

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