In vitro and in vivo Studies of cPLA2 Involvement in VEGF Mediated Retinal Neovascularization

Abstract

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