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.

Abstract 3635: Inhibition of Inhibitor Kappa B Kinase Beta Augments Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production in Aortic Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Sumathy Mohan ◽  
Ryzard Konopinski ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark 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 kappa B kinases α, β and γ) in non-vascular 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 (4.02 ± 0.81-folds) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.04 ± 0.06-folds). Another important and novel finding is that both IKKβ and eNOS could be co-immunoprecipitated with Hsp-90 (Figures A & B ) thus indicating the possible existence of a complex of IKKβ and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2μM) or Radicicol (20μM) mitigated (0.45 ± 0.04 -fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.416-fold). Blocking of IKKβ expression by IKK inhibitor II (15μM wedelolactone) or 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.

Abstract 345: Inhibitor Kappa B Kinase Regulates Nitric Oxide Synthase Activity in Vascular Endothelial Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Mohan Natarajan ◽  
Manikcam Krishnan ◽  
Ryzad Konopinski ◽  
Colleen Gaudette ◽  
Preethi Janardhanan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Binding Site ◽  
High Glucose ◽  
No Production ◽  
Aortic Endothelial Cells ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase

The generation of nitric oxide (NO) in endothelial cells is predominantly regulated by endothelial nitric oxide synthase (eNOS) activity. The role of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been established. Earlier, we demonstrated an increased interaction of Hsp-90 with inhibitor kappa B kinase β (IKKβ) in aortic endothelial cells (ECs) under conditions of high glucose (HG). These studies implicated the possibility of a site-specific competition between eNOS and IKKβ to bind to Hsp-90. In this study, the binding site of IKKβ on Hsp-90β is demonstrated by generating 5’deletional mutants of Flag tagged Hsp-90 and full length enhanced yellow fluorescent protein tagged IKKβ (EYFP-IKKβ). Forceful co-expression of the Hsp-90 deletion constructs with EYFP-IKKβ plasmid followed by immunoprecipitation/immunoblotting revealed the binding site of IKKβ to be between 307-711aa on Hsp-90, the middle domain (440-600 aa) where eNOS has been shown to bind to Hsp-90. Co-immunoprecipitation experiments performed in lysates of human aortic endothelial cells incubated with 25mM high glucose exhibited enhanced IKK beta interaction with both Hsp-90 α and β isoforms. Independently blocking Hsp-90α or β expression by siRNA transfection significantly blocked VEGF-induced NO production implicating the possible mechanism that IKK beta regulates NO production by competing with eNOS for both the isoforms of Hsp-90. These results confirm the previously unknown participation of IKKβ in the downregulation of eNOS activity under the influence of HG and provide a novel therapeutic strategy of using IKKβ as an effective target to improve NO generation in diabetic arteries.

scholarly journals Estrogen Stimulates Heat Shock Protein 90 Binding to Endothelial Nitric Oxide Synthase in Human Vascular Endothelial Cells

2000 ◽  
Vol 275 (7) ◽  
pp. 5026-5030 ◽  
Author(s):  
Kerry Strong Russell ◽  
M. Page Haynes ◽  
Teresa Caulin-Glaser ◽  
James Rosneck ◽  
William C. Sessa ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
Heat Shock Protein 90 ◽  
Vascular Endothelial ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

scholarly journals Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells

2015 ◽  
Vol 308 (8) ◽  
pp. C673-C683 ◽  
Author(s):  
Mohan Natarajan ◽  
Ryszard Konopinski ◽  
Manickam Krishnan ◽  
Linda Roman ◽  
Alakesh Bera ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
High Glucose ◽  
Vascular Complications ◽  
Critical Event ◽  
In Vivo Model ◽  
No Production ◽  
Vascular Endothelial ◽  
Enos Activity ◽  
Endothelial Nitric Oxide

Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-κB kinase-β (IKKβ)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKKβ on Hsp90. Interestingly, IKKβ binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKKβ to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKKβ. The pathophysiological relevance of the IKKβ-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2Akita in vivo model. Our study further defines the preferential involvement of α- vs. β-isoforms of Hsp90 in the IKKβ-eNOS-Hsp90 interaction, even though both Hsp90α and Hsp90β stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKKβ within the cell system that regulates NO production, but they also confirm that the IKKβ-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications 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 Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

2011 ◽  
Vol 301 (3) ◽  
pp. L334-L345 ◽  
Author(s):  
Ru-Jeng Teng ◽  
Jianhai Du ◽  
Hao Xu ◽  
Ivane Bakhutashvili ◽  
Annie Eis ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Pulmonary Artery ◽  
Ex Vivo ◽  
No Production ◽  
Cellular Mechanisms ◽  
Dimer Formation ◽  
Enos Activity ◽  
Endothelial Nitric Oxide

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.

Microtubule-active agents modify nitric oxide production in pulmonary artery endothelial cells

2002 ◽  
Vol 282 (6) ◽  
pp. L1183-L1189 ◽  
Author(s):  
Yunchao Su ◽  
Sergei I. Zharikov ◽  
Edward R. Block
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Culture Medium ◽  
Hsp90 Inhibitor ◽  
No Production ◽  
Enos Activity ◽  
Tubulin Protein ◽  
Hsp 90 ◽  
Enos Protein

The effects of specific microtubule-active agents on nitric oxide (NO) production were examined in pulmonary artery endothelial cells (PAEC). PAEC were incubated with taxol, which stabilizes microtubules, or nocodazole, which disrupts microtubules, or both for 2–4 h. We then examined NO production, endothelial NO synthase (eNOS) activity, and eNOS association with heat shock protein (HSP) 90. Incubation of PAEC with taxol (15 μM) for 2–4 h resulted in an increase in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. Incubation of PAEC with nocodazole (50 μM) for 2–4 h induced a decrease in NO production, eNOS activity, and the amount of HSP90 binding to eNOS. The presence of taxol in the culture medium prevented the effects of nocodazole on NO production and eNOS activity in PAEC. Geldanamycin, a HSP90 inhibitor, prevented the taxol-induced increase in eNOS activity. Taxol and nocodazole did not affect eNOS, HSP90, and tubulin protein contents in PAEC, as detected using Western blot analysis. These results indicate that the polymerization state of the microtubule cytoskeleton regulates NO production and eNOS activity in PAEC. The changes in eNOS activity induced by modification of microtubules are due, at least in part, to the altered binding of HSP90 to eNOS protein.

Effects of simulated hyperglycemia, insulin, and glucagon on endothelial nitric oxide synthase expression

2000 ◽  
Vol 279 (1) ◽  
pp. E11-E17 ◽  
Author(s):  
Yaoxian Ding ◽  
Nosratola D. Vaziri ◽  
Richard Coulson ◽  
Vaijinath S. Kamanna ◽  
Daeyoung D. Roh
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
High Glucose ◽  
Glucose Concentration ◽  
No Production ◽  
High Glucose Concentration ◽  
Enos Expression ◽  
Cultured Endothelial Cells ◽  
Increased Risk

Diabetes is associated with endothelial dysfunction and increased risk of hypertension, cardiovascular disease, and renal complications. Earlier studies have revealed that hyperglycemia impairs nitric oxide (NO) production and diabetes causes endothelial dysfunction in humans and experimental animals. This study was designed to test the effects of altered concentrations of glucose, insulin, and glucagon, the principal variables in types I and II diabetes, on NO production and endothelial NO synthase (eNOS) expression in cultured human coronary endothelial cells. Cultured endothelial cells were incubated in the presence of glucose at either normal (5.6 mM) or high (25 mM) concentrations for 7 days. The rates of basal and bradykinin-stimulated NO production (nitrate + nitrite) and eNOS protein expression (Western blot) were then determined at the basal condition and in the presence of insulin (10−8 and 10−7 M), glucagon (10−8 and 10−7 M), or both. Incubation with a high-glucose concentration for 7 days significantly downregulated, whereas insulin significantly upregulated, basal and bradykinin-stimulated NO production and eNOS expression in cultured endothelial cells. The stimulatory action of insulin was mitigated by high-glucose concentration and abolished by cotreatment of cells with glucagon. Thus hyperglycemia, insulinopenia, and hyperglucagonemia, which frequently coexist in diabetes, can work in concert to suppress NO production by human coronary artery endothelial cells.

scholarly journals Far-Infrared-Emitting Sericite Board Upregulates Endothelial Nitric Oxide Synthase Activity through Increasing Biosynthesis of Tetrahydrobiopterin in Endothelial Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Seonhee Kim ◽  
Ikjun Lee ◽  
Hee-Jung Song ◽  
Su-jeong Choi ◽  
Harsha Nagar ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Function ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Diseases ◽  
Far Infrared ◽  
No Production ◽  
Sprague Dawley ◽  
Endothelial Nitric Oxide

Far-infrared ray (FIR) therapy has been reported to exert beneficial effects on cardiovascular function by elevating endothelial nitric oxide synthesis (eNOS) activity and nitric oxide (NO) production. Tetrahydrobiopterin (BH4) is a key determinant of eNOS-dependent NO synthesis in vascular endothelial cells. However, whether BH4 synthesis is associated with the effects of FIR on eNOS/NO production has not yet been investigated. In this study, we investigated the effects of FIR on BH4-dependent eNOS/NO production and vascular function. We used FIR-emitting sericite boards as an experimental material and placed human umbilical vein endothelial cells (HUVECs) and Sprague–Dawley rats on the boards with or without FIR irradiation and then evaluated vascular relaxation by detecting NO generation, BH4 synthesis, and Akt/eNOS activation. Our results showed that FIR radiation significantly enhanced Akt/eNOS phosphorylation and NO production in human endothelial cells and aorta tissues. FIR can also induce BH4 storage by elevating levels of enzymes (e.g., guanosine triphosphate cyclohydrolase-1, 6-pyruvoyl tetrahydrobiopterin synthase, sepiapterin reductase, and dihydrofolate reductase), which ultimately results in NO production. These results indicate that FIR upregulated eNOS-dependent NO generation via BH4 synthesis and Akt phosphorylation, which contributes to the regulation of vascular function. This might develop potential clinical application of FIR to treat vascular diseases by augmenting the BH4/NO pathway.

scholarly journals Genistein Acutely Stimulates Nitric Oxide Synthesis in Vascular Endothelial Cells by a Cyclic Adenosine 5′-Monophosphate-Dependent Mechanism

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5532-5539 ◽  
Author(s):  
Dongmin Liu ◽  
Laurie L. Homan ◽  
Joseph S. Dillon
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Function ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Endothelial ◽  
Protective Effects ◽  
Nitric Oxide Synthesis ◽  
Enos Activity ◽  
Endothelial Nitric Oxide

Abstract Genistein may improve vascular function, but the mechanism of this effect is unclear. We tested the hypothesis that genistein directly regulates vascular function through stimulation of endothelial nitric oxide synthesis. Genistein activated endothelial nitric oxide synthase (eNOS) in intact bovine aortic endothelial cells and human umbilical vein endothelial cells over an incubation period of 10 min. The maximal eNOS activity was at 1 μm genistein. Consistent with this activation pattern, 1 μm genistein maximally stimulated the phosphorylation of eNOS at serine 1179 at 10 min of incubation. The rapid activation of eNOS by genistein was not dependent on RNA transcription or new protein synthesis and was not blocked by a specific estrogen receptor antagonist. In addition, inhibition of MAPK or phosphatidylinositol 3-OH kinase/Akt kinase had no affect on eNOS activation by genistein. Furthermore, the genistein effect on eNOS was also independent of tyrosine kinase inhibition. However, inhibition of cAMP-dependent kinase [protein kinase A (PKA)] by H89 completely abolished the genistein-stimulated eNOS activation and phosphorylation, suggesting that genistein acted through a PKA-dependent pathway. These findings demonstrated that genistein had direct nongenomic effects on eNOS activity in vascular endothelial cells, leading to eNOS activation and nitric oxide synthesis. These effects were mediated by PKA and were unrelated to an estrogenic effect. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for soy phytoestrogens.

Sign in / Sign up

Export Citation Format

Share Document