Role of cytosolic phospholipase A(2) in retinal neovascularization.
OBJECTIVE
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.
Barnett
Joshua M
JM
Vanderbilt Eye Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8808, USA.
McCollum
Gary W
GW
Penn
John S
JS
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D000860:Hypoxia / Q000201:enzymology / Q000503:physiopathology; D007231:Infant, Newborn; D008297:Male; D010100:Oxygen / Q000633:toxicity; D054510:Phospholipases A2, Cytosolic / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015861:Retinal Neovascularization / Q000139:chemically induced / Q000201:enzymology* / Q000517:prevention & control; D055351:Retinal Neurons / Q000378:metabolism; D012178:Retinopathy of Prematurity / Q000139:chemically induced / Q000201:enzymology* / Q000517:prevention & control; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D048051:p38 Mitogen-Activated Protein Kinases; D054510:Phospholipases A2, Cytosolic; D015232:Dinoprostone; D010100:Oxygen