Suppression of hypoxia-associated vascular endothelial growth factor gene expression by nitric oxide via cGMP

PURPOSE. TO investigate the suppressive effect of nitric oxide (NO) on vasc ular endothelial growth factor (VEGF) gene expression and to elucidate its mechanism of action. METHODS. Immortalized human retinal epithelial (RPE) cells, H-ras-transfect ed murine capillary endothelial cells, and nuclear factor-kappa B (NF-kappa B) RelA knockout 3T3 fibroblasts had VEGF gene expression stimulated by hy poxia, TPA (phorbol eater 12-O-tetradecanoylphorbol-13 acetate), and ms-tra nsfection. The dose response and time course of inhibition of VEGF gene exp ression by NO were characterized by northern blot analysis, ribonuclease pr otection assay, and enzyme-linked immununosorbent assay. The effects of NF- kappa B and cGMP in the NO-induced suppression of VEGF gene expression were quantitated. cGMP production was inhibited by LY 83583 (6-anilino-5,8-quin olinedione), a specific inhibitor of guanylate cyclase production, and cGMP accumulation was quantitated by immunoassay. RelA knockout 3T3 fibroblasts were used to assess the contribution of NF-KB to the downregulation of VEG F by NO. RESULTS. The NO donor sodium nitroprusside (SNP) decreased hypoxia-induced VEGF gene expression in a dose- and time-dependent manner. One hundred fift y micromolar SNP completely suppressed hypoxia-induced VEGF mRNA levels for at least 24 hours. Constitutive VEGF expression was not altered by SNP. Th e SNP-mediated decreases in VEGF expression were associated with increases in intracellular cGMP and were blocked by LY 83583. Sodium nitroprusside wa s able to decrease hypoxia-induced VEGF mRNA increases in fibroblasts defic ient in the RelA subunit of NF-kappa B. Nitric oxide was also effective at suppressing increased VEGF expression secondary to mutant ras and TPA. CONCLUSIONS. These data indicate that NO decreases hypoxia-induced VEGF via a cGMP-dependent mechanism and suggest that NO may serve as an endogenous inhibitor of both hypoxia- and non-hypoxia-enhanced VEGF expression in vivo .