Role of cytosolic phospholipase A(2) in retinal neovascularization

Abstract

Purpose: To identify and characterize the role of cytosolic phospholipase A(2) (cPLA(2)) in retinal angiogenesis using relevant cell-based assays and a rodent model of retinopathy of prematurity.

Methods: The phosphorylation states of cPLA(2) and p38 MAP kinase and the expression of COX-2 were assessed by Western blot analysis in rat Müller cells. The activities of PLA(2) enzymes in rat retinal lysates were assessed using a commercially available assay. Prostaglandin E(2) (PGE(2)) and VEGF levels in Müller cell-conditioned medium and in retinal tissue samples were measured by ELISA. Rat retinal microvascular endothelial cell proliferation was measured using a BrdU assay. Efficacy of the cPLA(2) inhibitor CAY10502 was tested using the rat model of oxygen-induced retinopathy (OIR) in which neovascularization (NV) was assessed by computer-assisted image analysis.

Results: In Müller cells, hypoxia increased the phosphorylation of cPLA(2) and p38 MAP kinase by 4-fold and 3-fold respectively. The cPLA(2) inhibitor CAY10502 decreased hypoxia-induced PGE(2) and VEGF levels in Müller cell-conditioned medium by 68.6% (P < 0.001) and 46.6% (P < 0.001), respectively. Retinal cPLA(2) activity peaked 1 day after oxygen exposure in OIR rats. CAY10502 (250 nM) decreased OIR-induced retinal PGE(2) and VEGF levels by 69% (P < 0.001) and 40.2% (P < 0.01), respectively. Intravitreal injection of 100 nM CAY10502 decreased retinal NV by 53.1% (P < 0.0001).

Conclusions: cPLA(2) liberates arachidonic acid, the substrate for prostaglandin (PG) production by the cyclooxygenase enzymes. PGs can exert a proangiogenic influence by inducing VEGF production and by stimulating angiogenic behaviors in vascular endothelial cells. Inhibition of cPLA(2) inhibits the production of proangiogenic PGs. Thus, cPLA(2) inhibition has a significant influence on pathologic retinal angiogenesis.

Figure 1.

Retinal cPLA₂ activity ± SEM in OIR and RA rats between 14(0) and 14(6). *P < 0.01, and †P < 0.02; OIR versus RA.

Figure 2.

Relative PLA₂ activity in OIR retinas at 14(1). This graph demonstrates the contribution of cPLA₂ to total PLA₂ activity in OIR retinas. Most of the total PLA₂ activity in the retinal samples is derived from the cytosolic phospholipase family. Bromoenol lactone, iPLA₂ inhibitor; thioetheramide-PC, sPLA₂ inhibitor; MAFP (methylarachidonyl-fluorophosphonate), cPLA₂ inhibitor with some iPLA₂ inhibition; CAY10502, cPLA_2α-specific inhibitor. *P < 0.05, and †P < 0.001; inhibitor versus untreated.

Figure 3.

The effect of hypoxia on cPLA2 activation and related proteins in rat Müller cells. Hypoxia increases the phosphorylation of cPLA2 and p38 and the total protein levels of COX-2.

Figure 4.

The effect of cPLA₂ inhibition on PGE₂ in normoxic and hypoxic rat Müller cells. Cells were pretreated in normoxia for 24 hours and then were treated with normoxia or hypoxia plus drug for 12 hours. The cPLA₂ inhibitor CAY10502 led to a dose-dependent reduction in PGE₂ production. *P < 0.05, and †P < 0.001 relative to vehicle (0.1% DMSO).

Figure 5.

The effect of cPLA₂ inhibition on retinal VEGF and PGE₂ in OIR rats 24 hours after removal from oxygen treatment. cPLA₂ inhibition by CAY10502 demonstrates a dose-responsive reduction on both retinal VEGF (dark bars with axis on the left) and PGE₂ (light bars with axis on the right). *P < 0.05, †P < 0.01, and ‡P< 0.001 relative to vehicle.

Figure 6.

The effect of cPLA₂ inhibition on NV area in rat OIR. The cPLA₂ inhibitor CAY10502 led to a dose-dependent inhibition in NV area. *P < 0.0001 relative to vehicle.

Figure 7.

Comparison of two representative retinal quadrants from eyes treated with 0.01% DMSO vehicle (A) or the cPLA₂ inhibitor CAY10502 at 100 nM (B).