Abstract 1497: Differential Modulation of Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathways by Tetrahydrobiopterin Synthesis vs. Salvage

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Toru Sugiyama ◽  
Ruqin Kou ◽  
Thomas Michel
Keyword(s):  
Endothelial Dysfunction ◽  
Signaling Pathways ◽  
De Novo ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Akt Phosphorylation ◽  
Disease States ◽  
Redox Active ◽  
Enzymatic Reduction ◽  
Endothelial Nitric Oxide

Tetrahydrobiopterin (BH4) is an essential redox-active cofactor for eNOS that can be both synthesized de novo or salvaged by enzymatic reduction of the oxidized compound. The endothelial dysfunction associated with diabetes is accompanied a decrease in the abundance of bioactive BH4. De novo biosynthesis of BH4 is catalyzed by GTP cyclohydrolase-1 (GTPCH1); recycling of BH4 is catalyzed by dihydrofolate reductase (DHFR). The relative roles of de novo BH4 synthesis and BH4 redox recycling in regulation of eNOS bioactivity remain incompletely defined. In the present study, we have used siRNA methods to investigate the effects of BH4 “knockdown” on eNOS regulation and endothelial signal transduction pathways in bovine aortic endothelial cells. We transfected duplex siRNA constructs designed to specifically target DHFR and GTPCH1, and suppressed levels of these proteins by ~90% (n = 37) relative to control siRNA-transfected cells. Transfection of siRNA constructs targeting DHFR or GTPCH1 suppressed VEGF-induced eNOS activity (using [ 3 H]-citrulline assay) or NO production (using an electrochemical NO sensor) by 90 ± 9% (n = 8, p < 0.01). siRNA-mediated knockdown of either DHFR or GTPCH1 had no effect on the abundance of stability of eNOS dimers, assessed using low-temperature SDS-PAGE (n = 4). DHFR knockdown completely blocked VEGF-induced eNOS dephosphorylation at the inhibitory phosphoserine residue 116 (n = 4, p < 0.01), but had no effect on agonist-modulated eNOS phosphorylation at the activating phosphoserine residue 1179. GTPCH1 knockdown had no effect either on phosphorylation or dephosphorylation of eNOS at these residues. Phosphorylation of Akt was decreased by 85 ± 4% by DHFR knockdown (p < 0.001, n = 4) but Akt phosphorylation was unaffected by GTPCH1 knockdown. These studies demonstrate for the first time a striking contrast in the consequences for eNOS signaling pathways from the suppression of BH4 salvage/reduction vs. de novo BH4 synthetic pathways. The abrogation of VEGF-mediated Akt activation by siRNA-mediated DHFR knockdown indicates that alterations in BH4 recycling may have broad effects on cell signaling pathways, with important consequences for the development of endothelial dysfunction in vascular disease states.

scholarly journals Angiotensin II Impairs Endothelial Nitric-oxide Synthase Bioavailability under Free Cholesterol-enriched Conditions via Intracellular Free Cholesterol-rich Membrane Microdomains

2013 ◽  
Vol 288 (20) ◽  
pp. 14497-14509 ◽  
Author(s):  
Eisuke Amiya ◽  
Masafumi Watanabe ◽  
Norihiko Takeda ◽  
Tetsuya Saito ◽  
Taro Shiga ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Endothelial Dysfunction ◽  
Free Cholesterol ◽  
Small Gtpase ◽  
Membrane Microdomains ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Modified Ldl ◽  
Rich Domain ◽  
Endothelial Nitric Oxide

Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS (eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.

Abstract 1446: Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in eNOS-Mediated Vascular Function and Angiogenic Response -Unmasked by PYK2 Knockout Mice-

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Akihiro Matsui ◽  
Mitsuhiko Okigaki ◽  
Asako Katsume ◽  
Shinsaku Matsunaga ◽  
Hiroyuki Yamada ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Vascular Function ◽  
Tube Formation ◽  
No Production ◽  
Wild Type ◽  
Aortic Endothelial Cells ◽  
Akt Phosphorylation ◽  
Angiogenic Response ◽  
Calcium Dependent ◽  
Endothelial Nitric Oxide

Background: Endothelial nitric oxide synthase (eNOS) is activated by PI3-kinase/Akt or Ca 2+ -dependent CaM kinases, whereas the involvement of Ca 2+ -dependent tyrosine kinase PYK2 remains to be determined. Methods and Results: We newly generated the PYK2-deficient (−/−) mice and investigated the effects of PYK2 on NO-mediated vascular functions and angiogenic response. The PYK2−/− mice exhibited a significant reduction in the basal eNOS phosphorylation and VEGF- or Acetylcholin-mediated response in the aorta and aortic endothelial cells (ECs) compared with the wild-type mice, and blood flow recovery and neovessel formation after hindlimb ischemia were reduced with affecting mobilization of endothelial progenitors. Acetylcholin-mediated aortic vasorelaxation and cGMP production were decreased (54% and 44%, P<0.01, respectively) and blood pressure in the PYK2−/− mice is higher than the wild-type mice (mean 97 vs. 86 mmHg, P<0.05). In the PYK2−/− mice, VEGF-mediated Akt phosphorylation, NO production and cytoplasmic Ca 2+ mobilization were markedly decreased. And also the phosphorylation of c-Src and PLCγ1, interaction of c-Src/PLCγ1 were clearly reduced. VEGF-dependent migration, tube formation and actin cytoskeletal reorganization were markedly inhibited in PYK2−/− ECs, while addition of L-arginine or cGMP-dependent kinase activator reversed the attenuated response. Conclusion: These findings demonstrate that PYK2 is involved in the receptor-activated signaling events leading to the phosphorylation of eNOS and thus regulates eNOS-mediated vasoactive function and angiogenesis.

scholarly journals Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization

2019 ◽  
Vol 316 (1) ◽  
pp. H80-H88 ◽  
Author(s):  
Fumin Chang ◽  
Sheila Flavahan ◽  
Nicholas A. Flavahan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Normal Activity ◽  
Aortic Endothelial Cells ◽  
Relative Expression ◽  
Fischer 344 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Homodimer formation is essential for the normal activity of endothelial nitric oxide synthase (eNOS). Structural uncoupling of eNOS, with generation of enzyme monomers, is thought to contribute to endothelial dysfunction in several vascular disorders, including aging. However, low-temperature SDS-PAGE of healthy arteries has revealed considerable variation between studies in the relative expression of eNOS dimers and monomers. While assessing structural uncoupling of eNOS in aging arteries, we identified methodological pitfalls that might contribute to such variation. Therefore, using human cultured aortic endothelial cells and aortas from young and aged Fischer-344 rats, we investigated optimal approaches for analyzing the expression of eNOS monomers and dimers. The results demonstrated that published differences in treatment of cell lysates can significantly impact the relative expression of several eNOS species, including denatured monomers, partially folded monomers, dimers, and higher-order oligomers. In aortas, experiments initially confirmed a large increase in eNOS monomers in aging arteries, consistent with structural uncoupling. However, these monomers were actually endogenous IgG, which, under these conditions, has mobility similar to eNOS monomers. Increased IgG levels in aged aortas likely reflect the aging-induced disruption of endothelial junctions and increased arterial penetration of IgG. After removal of the IgG signal, there were low levels of eNOS monomers in young arteries, which were not significantly different in aged arteries. Therefore, structural uncoupling of eNOS is not a prominent feature in young healthy arteries, and the process is not increased by aging. The study also identifies optimal approaches to analyze eNOS dimers and monomers. NEW & NOTEWORTHY Structural uncoupling of endothelial nitric oxide synthase (eNOS) is considered central to endothelial dysfunction. However, reported levels of eNOS dimers and monomers vary widely, even in healthy arteries. We demonstrate that sample processing can alter relative levels of eNOS species. Moreover, endothelial dysfunction in aging aortas results in IgG accumulation, which, because of similar mobility to eNOS monomers, could be misinterpreted as structural uncoupling. Indeed, enzyme monomerization is not prominent in young or aging arteries.

Anthocyanin increases adiponectin secretion and protects against diabetes-related endothelial dysfunction

2014 ◽  
Vol 306 (8) ◽  
pp. E975-E988 ◽  
Author(s):  
Yan Liu ◽  
Dan Li ◽  
Yuhua Zhang ◽  
Ruifang Sun ◽  
Min Xia
Keyword(s):  
Endothelial Dysfunction ◽  
Normal Diet ◽  
Protective Effects ◽  
Aortic Endothelial Cells ◽  
Beneficial Effects ◽  
Forkhead Box ◽  
Endothelial Nitric Oxide ◽  
Human Aortic Endothelial Cells ◽  
Type Information

Adiponectin is an adipose tissue-secreted adipokine with beneficial effects on the cardiovascular system. In this study, we evaluated a potential role for adiponectin in the protective effects of anthocyanin on diabetes-related endothelial dysfunction. We treated db/db mice on a normal diet with anthocyanin cyanidin-3- O-β-glucoside (C3G; 2 g/kg diet) for 8 wk. Endothelium-dependent and -independent relaxations of the aorta were then evaluated. Adiponectin expression and secretion were also measured. C3G treatment restores endothelium-dependent relaxation of the aorta in db/db mice, whereas diabetic mice treated with an anti-adiponectin antibody do not respond. C3G treatment induces adiponectin expression and secretion in cultured 3T3 adipocytes through transcription factor forkhead box O1 (Foxo1). Silencing Foxo1 expression prevented C3G-stimulated induction of adiponectin expression. In contrast, overexpression of Foxo1-ADA promoted adiponectin expression in adipocytes. C3G activates Foxo1 by increasing its deacetylation via silent mating type information regulation 2 homolog 1 (Sirt1). Furthermore, purified anthocyanin supplementation significantly improved flow-mediated dilation (FMD) and increased serum adiponectin concentrations in patients with type 2 diabetes. Changes in adiponectin concentrations positively correlated with FMD in the anthocyanin group. Mechanistically, adiponectin activates cAMP-PKA-eNOS signaling pathways in human aortic endothelial cells, increasing endothelial nitric oxide bioavailability. These results demonstrate that adipocyte-derived adiponectin is required for anthocyanin C3G-mediated improvement of endothelial function in diabetes.

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway

Cardiology ◽  
2015 ◽  
Vol 132 (4) ◽  
pp. 252-260 ◽  
Author(s):  
Wen-Qi Han ◽  
Feng-Jun Chang ◽  
Qun-Rang Wang ◽  
Jun-Qiang Pan
Keyword(s):  
Nitric Oxide ◽  
Acute Coronary Syndrome ◽  
Endothelial Dysfunction ◽  
Heat Shock Protein 90 ◽  
No Production ◽  
Healthy Controls ◽  
Coronary Syndrome ◽  
Enos Phosphorylation ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Objectives: Endothelial dysfunction is involved in the development of the acute coronary syndrome (ACS). Plasma microparticles (MPs) from other diseases have been demonstrated to initiate coagulation and endothelial dysfunction. However, whether MPs from ACS patients impair vasodilatation and endothelial function remains unclear. Methods: Patients (n = 62) with ACS and healthy controls (n = 30) were recruited for MP isolation. Rat thoracic aortas were incubated with MPs from ACS patients or healthy controls to determine the effects of MPs on endothelial-dependent vasodilatation, the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), the interaction of eNOS with heat shock protein 90 (Hsp90), and nitric oxide (NO) and superoxide anion (O2-) production. The origin of MPs was assessed by flow cytometry. Results: MP concentrations were increased in patients with ACS compared with healthy controls. They were positively correlated with the degree of coronary artery stenosis. MPs from ACS patients impair endothelial-dependent vasodilatation, decrease both Akt and eNOS phosphorylation, decrease the interaction between eNOS and Hsp90, and decrease NO production but increase O2- generation in rat thoracic aortas. Endothelial-derived MPs and platelet-derived MPs made up nearly 75% of MPs. Conclusions: Our data indicate that MPs from ACS patients negatively affect endothelial-dependent vasodilatation via Akt/eNOS-Hsp90 pathways.

scholarly journals High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction

2009 ◽  
Vol 296 (1) ◽  
pp. C182-C192 ◽  
Author(s):  
Sumathy Mohan ◽  
Ryszard Konopinski ◽  
Bo Yan ◽  
Victoria E. Centonze ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
High Glucose ◽  
Vascular Endothelial Cells ◽  
Heat Shock Protein 90 ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor κB kinases (IKK)α, β, and γ] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 ± 0.65-fold) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.45 ± 0.4-fold). Both IKKβ and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 μM) or Radicicol (20 μM) mitigated (0.45 ± 0.04-fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.42-fold). Blocking of IKKβ expression by IKK inhibitor II (15 μM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

scholarly journals Superoxide-mediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers

2009 ◽  
Vol 296 (6) ◽  
pp. H1781-H1792 ◽  
Author(s):  
Gonzalo Peluffo ◽  
Pablo Calcerrada ◽  
Lucia Piacenza ◽  
Nelson Pizzano ◽  
Rafael Radi
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Tobacco Smoke ◽  
Endothelial Nitric Oxide Synthase ◽  
Flow Mediated Dilation ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Tobacco smoke is known to cause nitric oxide (·NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between·NO and superoxide (O2·−) radicals and the consequent impact on·NO bioavailability and nitroxidative stress in bovine aortic endothelial cells exposed to cigarette smoke extract (CSE) and in smokers. Bovine aortic endothelial cells in the presence of CSE triggered O2·−production as indicated by spin-trapping electron paramagnetic resonance experiments. O2·−was produced both extracellulary (3.4 vs. 1.0 nmol·h−1·mg−1; CSE vs. control; cytochrome c3+reduction assay) and intracellularly (40% inhibition of cytosolic aconitase). CSE also led to the production of peroxynitrite as evaluated by dihydrorhodamine oxidation and protein tyrosine nitration on cells. O2·−and peroxynitrite formation were decreased by ascorbate and α-tocopherol. Additionally, CSE led to the oxidation of endothelial nitric oxide synthase increasing the monomeric inactive form of endothelial nitric oxide synthase. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 IU all-rac-α-tocopherol every 12 h for 165 days. Smokers had endothelial dysfunction compared with control subjects (95% confidence interval: 2.5, 8.3 vs. 10.6, 14.2; P < 0.05) as assessed by flow-mediated dilation of the brachial artery, and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of flow-mediated dilation in smokers reverted after a long-term antioxidant supplementation (95% confidence interval: 13.9, 19.9; P < 0.05), reaching values comparable with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert·NO toward peroxynitrite by inducing O2·−production in vascular endothelial cells both in vitro and in vivo.

scholarly journals Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3190-3198 ◽  
Author(s):  
Hongwei Si ◽  
Jie Yu ◽  
Hongling Jiang ◽  
Hazel Lum ◽  
Dongmin Liu
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Enos Expression ◽  
Camp Response Element ◽  
Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Human Aortic Endothelial Cells

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

Tumor necrosis factor-α reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

2007 ◽  
Vol 293 (2) ◽  
pp. H1115-H1121 ◽  
Author(s):  
Bonnie L. Goodwin ◽  
Laura C. Pendleton ◽  
Monique M. Levy ◽  
Larry P. Solomonson ◽  
Duane C. Eichler
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Elevated Serum ◽  
Proximal Promoter ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Tnf Α ◽  
Argininosuccinate Synthase ◽  
Endothelial Nitric Oxide ◽  
Aortic Endothelial

Endothelial dysfunction associated with elevated serum levels of TNF-α observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-α (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-α with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-α suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-α may be mediated, in part, via the NF-κB signaling pathway. These findings demonstrate that TNF-α coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-α is an added insult to endothelial function because of its important role in NO production and in endothelial viability.

Impairment of endothelial nitric oxide production by acute glucose overload

2001 ◽  
Vol 280 (1) ◽  
pp. E171-E178 ◽  
Author(s):  
Chiwaka Kimura ◽  
Masahiro Oike ◽  
Tetsuya Koyama ◽  
Yushi Ito
Keyword(s):  
Nitric Oxide ◽  
No Production ◽  
Nitric Oxide Production ◽  
Superoxide Anions ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
A 23187 ◽  
Intracellular Ca ◽  
Endothelial Nitric Oxide ◽  
Cellular Productivity

We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM d-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677–685, 1998) that glucose overload impairs Ca2+ mobilization due to an accumulation of superoxide anions (O2 −) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to Ca2+ entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an O2 − donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular Ca2+ concentration increase induced by the Ca2+ ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and highd-glucose-treated cells in Ca2+-free solution. These results indicate that glucose overload impairs NO production by the O2 −-mediated attenuation of Ca2+entry.

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