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WJ Lukiw, WC Gordon, NG Bazan; Cytoplasmic Phospholipase A2 (cPLA2) and Inflammatory Gene Signaling During Neovascularization in a Model of Retinopathy of Prematurity . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2743.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The significance of inflammatory gene signaling during retinal neovascularizaton (NV) is not well understood. Here we have examined the expression of genes that encode (a) critical enzymes that synthesize inflammatory messengers (cPLA2, COX-1, COX-2), (b) angiogenic growth factor (VEGF) and (c) transcription factors (TFs) known to modulate inflammatory gene expression (AP1, AP2, HIF-1, NFkB and STAT1). Methods:A 2 week cycled oxygen protocol [10%-50% O2; 2 day/cycle] followed by 0-10 days normoxia (20% O2) was used to induce NV in neonatal Sprague-Dawley rat retina. Total RNA and retinal nuclear proteins were extracted from the same samples and cPLA2, COX-1, COX-2 and VEGF RNA messages were assayed using RT-PCR. AP1-, AP2-, HIF-1-, NFkB- and STAT1-DNA binding activities were analyzed using gel shift and gel supershift assay. cPLA2, COX-1, COX-2 and VEGF proteins were detected using Western immuno-dot blot analysis. Results:After the oxygen-cycling protocol and beginning at 2-to-5 days of normoxia peripheral retinal microangiopathy signified the triggering of de novo NV. At day 2 of normoxia, AP1-, AP2-, HIF-1-, NF-kB- and STAT1-DNA binding increased 1.1-, 1.2-, 2.7-, 2.5-, and 2.1-fold, respectively, over controls. By day 4 of normoxia cPLA2, COX-1, COX-2 and VEGF RNA message levels rose to 2.2-, 1.1-, 3.7- and 3.9-fold, respectively, over controls . By day 7 of normoxia, COX-1 and COX-2 protein levels rose 1.1- and 2.7-fold, respectively, over controls and cPLA2 and VEGF RNA levels rose 3.6- and 4.4-fold, respectively, over controls (p<0.05). Conclusion:A two week cycled oxygen protocol followed by normoxia activates a robust inflammatory gene signaling program in rat pup retina. These data suggest a strong temporal correlation between activation of the hypoxia sensitive TFs HIF-1-, NF-kB- and STAT1, their binding to target DNA in the cPLA2, COX-2 and VEGF gene promoters and enhanced expression from these inflammatory genes. These data also suggest increased production of arachidonic acid (AA) via up-regulated cPLA2 activity and enhanced COX-2 activity that uses AA as a substrate for prostaglandin (PG) synthesis. In turn, PGs are known potent stimulators of VEGF gene transcription. Unraveling these inflammatory signaling pathways will provide attractive genetic targets for pharmacological intervention to manage pathoangiogenesis.
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