Abstract-Vascular endothelial growth factor (VEGF) is known to induce the release of nitric oxide (NO) from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. Accordingly, the effect of endogenous and exogenous NO on VEGF synthesis by rat vascular smooth muscle cells (VSMCs) was investigated. Two in vitro models were used:(1) VSMCs stimulated to produce NO by treatment with interleukin (IL)-1 (10 ng/mL) and (2) VSMCs lipotransfected with pKecNOS plasmid, containing the endothelial constitutive NO synthase (ecNOS) cDNA. The synthesis of NO was inhibited by N -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, respectively. Some cells treated with L-NAME or DAHP were supplemented with L-arginine (10 mmol/L) or tetrahydrobiopterin (BH 4 ; 100 mol/L), respectively. In addition, we studied the effect of sodium nitroprusside (SNP; 10 and 100 mol/L) and chemically related compounds, potassium ferrocyanide and ferricyanide, on VEGF generation. IL-1 induced iNOS expression and NO generation and significantly upregulated VEGF mRNA expression and protein synthesis. L-NAME and DAHP totally inhibited NO generation and decreased the IL-1-upregulated VEGF synthesis by 30% to 40%. Supplementation with L-arginine or BH 4 increased NO generation by L-NAME-or DAHP-treated cells, and VEGF synthesis was augmented by addition of BH 4 . The cells generating NO after pKecNOS transfection released significantly higher amounts of VEGF than 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 mol/L) concentrations of SNP. This effect was mimicked by chemically related ferricyanide and ferrocyanide compounds, suggesting that the inhibitory effect of sodium nitroprusside may be mediated by an NO-independent mechanism. The results indicate that endogenous NO enhances VEGF synthesis. The positive interaction between endogenous NO and VEGF may have implications for endothelial regeneration after balloon angioplasty and for angiogenesis.
These studies reveal that NO derived from NO-donors or generated by NOS within the cells, upregulates the synthesis of VEGF in vascular smooth muscle cells. Administration of NO donors, or augmentation of endogenous NO synthesis, may be an alternative approach in therapeutic angiogenesis.
Objective-Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. Methods and Results-On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT Յ23), medium (moderately active, GTϭ24 to 28), and long (least active, GT Ն29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J 2 , hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor-induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1, interleukin-6, and soluble intercellular adhesion molecule-1. Conclusion-The
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