Role of cytosolic phospholipase A2 in retinal neovascularization.

Angiogenesis, the formation of new capillaries from existing blood vessels, occurs during physiological processes such as reproduction, growth and development, and wound healing.1–6 Conversely, diseases such as arthritis, tumor growth, and retinopathies are characterized by pathologic, persistent angiogenesis.6–8 In the context of the retina, pathologic, persistent angiogenesis is often referred to as retinal neovascularization (NV). Age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity are potentially blinding conditions characterized by choroidal or retinal NV. Retinal NV is often caused by tissue hypoxia.9–11 Hypoxia stimulates the activation of various intracellular signaling pathways, which lead to the production of growth factors and cytokines that stimulate quiescent endothelial cells to develop a neovascular phenotype.12–17 Of the vasoactive factors identified to date, there is considerable evidence that vascular endothelial growth factor (VEGF) is most consistently and dramatically upregulated by retinal hypoxia.18 Hypoxia induces VEGF synthesis in a number of retinal cell types, including endothelial cells, astrocytes, retinal pigment epithelial cells, Muller cells, and ganglion cells.19–23 Muller cells have been shown to be the principal source of VEGF in animal models of retinal NV.21–23 Previous studies suggest that cyclooxygenase (COX)/prostaglandin (PG)-dependent signaling mechanisms contribute to retinal VEGF production and neovascular disease.24–27 The initial step in PG biosynthesis is the liberation of arachidonic acid (AA) from membrane phospholipids by phospholipase A2 (PLA2) enzymes. There are at least 19 groups of PLA2s that are generally classified as cytosolic (cPLA2), secretory (sPLA2), or calcium-independent (iPLA2). PLA2 is activated in response to a number of stimuli including ischemia, oxidative stress, and cell signaling molecules.28 cPLA2 is activated when serines 505 and 727 are phosphorylated by p38 and p42/44 MAP kinases.29 Active cPLA2 then catalyzes the hydrolysis of membrane phospholipids at the sn-2 position, releasing AA directly into the cytoplasm.30 Free AA either diffuses out of the cell, is reincorporated into phospholipids, or is metabolized by the COX, lipoxygenase, or cytochrome P450 enzymes.30–32 There are two well-characterized COX enzymes. COX-1, a constitutive isoform, and COX-2, which is responsive to growth factors, cytokines, and environmental stimuli, catalyze the reaction between two molecules of oxygen (O2) and AA to produce prostaglandin H2 (PGH2). Cell-specific synthases catalyze isomerization, oxidation, and reduction of PGH2 to yield the prostaglandins E (PGE), F (PGF), and D (PGD).33–35 PGs may exert a proangiogenic influence by inducing the upregulation of VEGF.36–39 The following lines of evidence suggest a COX/PG-dependent component to retinal VEGF induction and subsequent NV: (1) hypoxia stimulates the upregulation of COX-2 (as well as VEGF) in Muller cells40; (2) hypoxia stimulates an approximate 3-fold increase in Muller cell PGE2 synthase (McCollum GW, et al. IOVS 2005;46:ARVO E-Abstract 2974); (3) PGE2 induces the upregulation of VEGF and basic fibroblast growth factor (bFGF; a potent angiogenesis inducer) in Muller cells39; (4) in vitro data show that amfenac, a nonsteroidal anti-inflammatory drug (NSAID), dose dependently inhibits hypoxia-induced VEGF production in Muller cells41; (5) cPLA2, COX, and VEGF are coordinately upregulated during the post-oxygen treatment phase (retinal hypoxia) in the rat model of oxygen-induced retinopathy (OIR) (Lukiw JW, et al. IOVS 2002;46:ARVO E-Abstract 2974) and in retinal endothelial cells exposed to hypoxia42; and (6) NSAIDs that inhibit COX and, consequently, PG synthesis, reduce the NV response in rodent models of OIR.24–27 In these studies, cPLA2-dependent mechanisms of retinal angiogenesis were investigated. In vitro experiments used Muller and endothelial cells as models of the primary VEGF-producing cell type and the proliferating cell type of neovascular lesions, respectively. Consequently, cPLA2 activity, VEGF levels, and PGE2 levels were measured in the Muller cells, and proliferation was measured in endothelial cells in response to inhibiting cPLA2. In vivo experiments using the rat OIR model were structured to complement and build on the in vitro studies; to that end, we measured the relative contribution of PLA2 isoforms, cPLA2 activity, VEGF levels, PGE2 levels, and neovascular areas with cPLA2 inhibition.