Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells

Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEG F synthesis is not clear. Accordingly, the effect of endogenous and exogeno us NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was inv estigated. Two in vitro models were used: (1) VSMCs stimulated to produce N O by treatment with interleukin (IL)-1 beta (10 ng/mL) and (2) VSMCs lipotr ansfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N-omega-nitro-L -arginine methyl ester (L-NAME, 2 to 5 mmol/L) or diaminohydroxypyrimidine (DAHP, 2.5 to 5 mmol/L), inhibitors of NOS and GTP cyclohydrolase I, respec tively. Some cells treated with L-NAME or DAMP were supplemented with L-arg inine (10 mmol/L) or tetrahydrobiopterin (BH4; 100 mu mol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 10 0 mu mol/L) and chemically related compounds, potassium ferrocyanide and fe rricyanide, on VEGF generation. IL-1 beta induced iNOS expression and NO ge neration and significantly upregulated VEGF mRNA expression and protein syn thesis. L-NAME and DAHP totally inhibited NO generation and decreased the I L-1 beta-upregulated VEGF synthesis by 30% to 40%. Supplementation with L-a rginine or BH4 increased NO generation by L-NAME- or DAMP-treated cells, an d VEGF synthesis was augmented by addition of BH4. The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF th an cells transfected with control plasmids. Inhibition of NO generation by L-NAME decreased VEGF synthesis. In contrast to the effect of endogenous NO , we observed the inhibition of VEGF synthesis in the presence of high (10 or 100 mu mol/L) concentrations of SNP. This effect was mimicked by chemica lly related ferricyanide and ferrocyanide compounds, suggesting that the in hibitory effect of sodium nitroprusside may be mediated by an NO-independen t mechanism. The results indicate that endogenous NO enhances VEGF synthesi s. The positive interaction between endogenous NO and VEGF may have implica tions for endothelial regeneration after balloon angioplasty and for angiog enesis.