Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase.

OBJECTIVE: To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS). METHODS & RESULTS: A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. CONCLUSIONS: Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Dexamethasone upregulates Nox1 expression in vascular smooth muscle cells.

BACKGROUND/AIM: It has been demonstrated that dexamethasone-induced hypertension can be prevented by the NADPH oxidase inhibitor apocynin. The effect of dexamethasone on NADPH oxidase, however, is unknown. The present study was conducted to investigate the effect of dexamethasone on the gene expression of Nox1, the major NADPH oxidase isoform in vascular smooth muscle cells. RESULTS: Oral treatment of Wistar-Kyoto rats with dexamethasone (0.03 mg/kg/day) for 12 days led to an upregulation of Nox1 mRNA expression in the aorta. In cultured A7r5 rat aortic smooth muscle cells, dexamethasone increased Nox1 mRNA expression in a concentration- and time-dependent manner. The upregulation of Nox1 mRNA expression was completely prevented by the glucocorticoid receptor antagonist mifepristone. The effect of dexamethasone on Nox1 expression was likely to be indirect as it could be largely blocked by cycloheximide, an inhibitor of protein biosynthesis. Dexamethasone increased Nox1 mRNA stability as well as Nox1 transcription. The dexamethasone-induced Nox1 expression was completely prevented by scriptaid, a pan-inhibitor of histone deacetylases (HDAC), indicating a crucial role for HDAC enzymes. In total, A7r5 cells expressed 8 HDAC isoforms, with HDAC1, 5, 6 and 7 being the most abundant ones. Knockdown of these 4 individual HDAC enzymes did not prevent the effect of dexamethasone on Nox1 expression, although HDAC5 knockdown markedly reduced basal Nox1 expression. CONCLUSION: Dexamethasone upregulates Nox1 expression in vascular smooth muscle cells. This effect involves the glucocorticoid receptor and HDAC enzymes.

Glutathione peroxidase-1 deficiency potentiates dysregulatory modifications of endothelial nitric oxide synthase and vascular dysfunction in aging.

Recently, we demonstrated that gene ablation of mitochondrial manganese superoxide dismutase and aldehyde dehydrogenase-2 markedly contributed to age-related vascular dysfunction and mitochondrial oxidative stress. The present study has sought to investigate the extent of vascular dysfunction and oxidant formation in glutathione peroxidase-1-deficient (GPx-1(-/-)) mice during the aging process with special emphasis on dysregulation (uncoupling) of the endothelial NO synthase. GPx-1(-/-) mice on a C57 black 6 (C57BL/6) background at 2, 6, and 12 months of age were used. Vascular function was significantly impaired in 12-month-old GPx-1(-/-) -mice as compared with age-matched controls. Oxidant formation, detected by 3-nitrotyrosine staining and dihydroethidine-based fluorescence microtopography, was increased in the aged GPx-1(-/-) mice. Aging per se caused a substantial protein kinase C- and protein tyrosine kinase-dependent phosphorylation as well as S-glutathionylation of endothelial NO synthase associated with uncoupling, a phenomenon that was more pronounced in aged GPx-1(-/-) mice. GPx-1 ablation increased adhesion of leukocytes to cultured endothelial cells and CD68 and F4/80 staining in cardiac tissue. Aged GPx-1(-/-) mice displayed increased oxidant formation as compared with their wild-type littermates, triggering redox-signaling pathways associated with endothelial NO synthase dysfunction and uncoupling. Thus, our data demonstrate that aging leads to decreased NO bioavailability because of endothelial NO synthase dysfunction and uncoupling of the enzyme leading to endothelial dysfunction, vascular remodeling, and promotion of adhesion and infiltration of leukocytes into cardiovascular tissue, all of which was more prominent in aged GPx-1(-/-) mice.

Expression of NO synthases and redox enzymes in umbilical arteries from newborns born small, appropriate, and large for gestational age.

BACKGROUND: Modified expression of nitric oxide synthases (NOSs) and an imbalance between the pro-oxidative and the antioxidative system accompany endothelial dysfunction, the first stage of atherosclerosis. Humans born small (SGA) or large (LGA) for gestational age are at higher risk of developing atherosclerosis later in life than humans born appropriate for gestational age (AGA). We hypothesized that indicators of endothelial dysfunction could be detectable at birth. The purpose of this study was to find out whether the expression patterns of NO synthases (endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS)), pro-oxidative enzymes (components of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, NADPH oxidase 1 (NOX1), NOX2, NOX4, p22phox, and p47phox), and antioxidative enzymes (superoxide dismutase 1-3 (SOD1-3), glutathione peroxidase 1 (GPX1), and catalase) in umbilical arteries differ among SGA, LGA, and AGA newborns. METHODS: Thirty-six umbilical cords were obtained from healthy, normal, full-term SGA, AGA, and LGA newborns. The umbilical arteries were dissected and homogenized. mRNA expression was analyzed with quantitative real-time PCR. Western blotting was performed to determine protein expression. RESULTS: mRNA and protein expression of NO synthases, pro-oxidative enzymes, and antioxidative enzymes did not differ in the umbilical arteries from newborns of the three groups. CONCLUSION: Indicators of endothelial dysfunction in terms of differences in enzyme expression in SGA or LGA newborns vs. AGA newborns were not present at birth.

AVE3085, an enhancer of endothelial nitric oxide synthase, restores endothelial function and reduces blood pressure in spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Nitric oxide (NO) plays an important role in endothelial function, and impaired NO production is involved in hypertension. Therefore, compounds that regulate endothelial NO synthase (eNOS) may be of therapeutic benefit. A novel, low molecular weight compound AVE3085 is a recently developed compound with the ability to enhance eNOS transcription. The present study investigated the effects of AVE3085 in endothelial dysfunction associated with hypertension. EXPERIMENTAL APPROACH: Spontaneously hypertensive rats (SHRs) were treated with AVE 3085 (10 mg·kg·day(-1) , orally) for 4 weeks. Isometric force measurement was performed on rings of isolated aortae in organ baths. Protein expression of eNOS, phosphorylated-eNOS and nitrotyrosine in the aortae were examined by Western blotting. mRNA for eNOS in rat aortae were examined by reverse-transcriptase polymerase chain reaction (RT-PCR). KEY RESULTS: AVE3085 greatly improved endothelium-dependent relaxations in the aortae of SHRs. This functional change was accompanied by up-regulated expression of eNOS protein and mRNA, enhanced eNOS phosphorylation and decreased formation of nitrotyrosine. Furthermore, AVE3085 treatment reduced the blood pressure in SHR without affecting that of hypertensive eNOS(-/-) mice. CONCLUSIONS AND IMPLICATIONS: The eNOS-transcription enhancer AVE3085 restored impaired endothelial function in a hypertensive model. The present study provides a solid basis for the potential development of eNOS-targeting drugs to restore down-regulated eNOS, as a new strategy in hypertension.