Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

2021 ◽  
Author(s):  
Sarah Basehore ◽  
Samantha Bohlman ◽  
Callie Weber ◽  
Swathi Swaminathan ◽  
Yuji Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
No Production ◽  
Disturbed Flow ◽  
Enos Activity ◽  
Enos Phosphorylation ◽  
Steady Laminar ◽  
In Cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

scholarly journals MAGI1 Mediates eNOS Activation and NO Production in Endothelial Cells in Response to Fluid Shear Stress

Cells ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 388 ◽  
Author(s):  
Kedar Ghimire ◽  
Jelena Zaric ◽  
Begoña Alday-Parejo ◽  
Jochen Seebach ◽  
Mélanie Bousquenaud ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Protein Kinase ◽  
Fluid Shear Stress ◽  
Adaptor Protein ◽  
No Production ◽  
Fluid Shear ◽  
Enos Phosphorylation

Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE-cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1-mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress-induced and PKA/AMPK dependent eNOS activation and NO production.

scholarly journals Impairment of autophagy in endothelial cells prevents shear-stress-induced increases in nitric oxide bioavailability

2014 ◽  
Vol 92 (7) ◽  
pp. 605-612 ◽  
Author(s):  
Leena P. Bharath ◽  
Robert Mueller ◽  
Youyou Li ◽  
Ting Ruan ◽  
David Kunz ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Oxidant Stress ◽  
Critical Role ◽  
No Production ◽  
Enos Phosphorylation ◽  
Autophagy Pathway ◽  
No Bioavailability ◽  
Static Conditions

Autophagy is a lysosomal catabolic process by which cells degrade or recycle their contents to maintain cellular homeostasis, adapt to stress, and respond to disease. Impairment of autophagy in endothelial cells studied under static conditions results in oxidant stress and impaired nitric oxide (NO) bioavailability. We tested the hypothesis that vascular autophagy is also important for induction of NO production caused by exposure of endothelial cells to shear stress (i.e., 3 h × ≈20 dyn/cm2). Atg3 is a requisite autophagy pathway mediator. Control cells treated with non-targeting control siRNA showed increased autophagy, reactive oxygen species (ROS) production, endothelial NO synthase (eNOS) phosphorylation, and NO production upon exposure to shear stress (p < 0.05 for all). In contrast, cells with >85% knockdown of Atg3 protein expression (via Atg3 siRNA) exhibited a profound impairment of eNOS phosphorylation, and were incapable of increasing NO in response to shear stress. Moreover, ROS accumulation and inflammatory cytokine production (MCP-1 and IL-8) were exaggerated (all p < 0.05) in response to shear stress. These findings reveal that autophagy not only plays a critical role in maintaining NO bioavailability, but may also be a key regulator of oxidant–antioxidant balance and inflammatory–anti-inflammatory balance that ultimately regulate endothelial cell responses to shear stress.

scholarly journals VE-PTP inhibition elicits eNOS phosphorylation to blunt endothelial dysfunction and hypertension in diabetes

2020 ◽  
Author(s):  
Mauro Siragusa ◽  
Alberto Fernando Oliveira Justo ◽  
Pedro Felipe Malacarne ◽  
Anna Strano ◽  
Akshay Buch ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Dysfunction ◽  
Vascular Reactivity ◽  
Therapeutic Option ◽  
Diabetic Patients ◽  
No Production ◽  
Vascular Endothelial ◽  
Enos Activity

Abstract Aims Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity. Methods and results In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium-intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases, i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and down-regulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition. Conclusions VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension.

scholarly journals Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase

2008 ◽  
Vol 86 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Syamantak Majumder ◽  
Ajit Muley ◽  
Gopi Krishna Kolluru ◽  
Samir Saurabh ◽  
K. P. Tamilarasan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Egg Yolk ◽  
Cellular Migration ◽  
No Production ◽  
Enos Phosphorylation ◽  
Low Levels ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.

scholarly journals Androgen Receptor-Dependent Activation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells: Role of Phosphatidylinositol 3-Kinase/Akt Pathway

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1822-1828 ◽  
Author(s):  
Jing Yu ◽  
Masahiro Akishita ◽  
Masato Eto ◽  
Sumito Ogawa ◽  
Bo-Kyung Son ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Androgen Receptor ◽  
Pi3 Kinase ◽  
Maximal Response ◽  
No Production ◽  
Dependent Manner ◽  
Small Interference Rna ◽  
Enos Phosphorylation ◽  
Interference Rna

The mechanisms of testosterone-induced vasodilatation are not fully understood. This study investigated the effect of testosterone on nitric oxide (NO) synthesis and its molecular mechanism using human aortic endothelial cells (HAEC). Testosterone at physiological concentrations (1–100 nm) induced a rapid (15–30 min) increase in NO production, which was associated with phosphorylation and activation of endothelial NO synthase (eNOS). Then, the involvement of the androgen receptor (AR), which is abundantly expressed in HAEC, was examined. The effect of testosterone on eNOS activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA. In contrast, testosterone-induced eNOS phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERα small interference RNA. 5α-Dihydrotestosterone, a nonaromatizable androgen, also stimulated eNOS phosphorylation. Next, the signaling cascade that leads to eNOS phosphorylation was explored. Testosterone stimulated rapid phosphorylation of Akt in a time- and dose-dependent manner, with maximal response at 15–60 min. The rapid phosphorylation of eNOS or NO production induced by testosterone was inhibited by Akt inhibitor SH-5 or by phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p85α subunit of PI3-kinase. In conclusion, testosterone rapidly induces NO production via AR-dependent activation of eNOS in HAEC. Activation of PI3-kinase/Akt signaling and the direct interaction of AR with p85α are involved, at least in part, in eNOS phosphorylation.

Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. C753-C758 ◽  
Author(s):  
M. J. Kuchan ◽  
H. Jo ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
G Protein ◽  
G Proteins ◽  
No Production ◽  
Dependent Manner ◽  
Protein Activation ◽  
Synthase Inhibitor

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.

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.

Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow

2002 ◽  
Vol 9 (1) ◽  
pp. 27-41 ◽  
Author(s):  
Alan R. Brooks ◽  
Peter I. Lelkes ◽  
Gabor M. Rubanyi
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Laminar Flow ◽  
Mrna Levels ◽  
Monocyte Adhesion ◽  
Disturbed Flow ◽  
Aortic Endothelial Cells ◽  
Human Aortic Endothelial Cells ◽  
Steady Laminar

Subtraction cloning and cDNA arrays were used to compare steady-state mRNA levels in cultured human aortic endothelial cells (HAEC) exposed for up to 24 h to either high-shear (13 dyn/cm2) steady laminar flow (LF), an established representation of “atheroprotective” flow conditions, or low-shear (<1 dyn/cm2), pulsatile, nonsteady, non-unidirectional flow (disturbed flow, DF) that simulates conditions in the atherosclerosis-prone areas of the arterial circulation. More than 100 genes not previously known to be flow regulated were identified. Analysis of selected genes by quantitative RT-PCR confirmed the results obtained from the microarrays. These data demonstrate that DF is not simply the absence of LF but in fact represents a distinct biomechanical stimulus that has a profound impact upon the gene expression profile of HAEC in culture. In line with previous studies, many of the changes in mRNA levels induced by LF are atheroprotective. In contrast, DF upregulated the mRNA levels of a plethora of proatherosclerotic genes including proinflammatory, proapoptotic, and procoagulant molecules. For some of the genes whose expression was altered by DF and LF, corresponding changes in EC function (proliferation and monocyte adhesion) could be demonstrated. Specifically, the sustained upregulation of VCAM-1 and increased monocyte adhesion to EC exposed to DF was similar to that found in EC in vivo at atherosclerosis-prone regions, confirming the relevance of our model system for in vivo conditions. Distinct differences in the cellular response induced by TNFα and DF suggest that the effects of DF are not mediated entirely by the same signaling pathways that activate NF-κB. These studies demonstrate extensive and pathophysiologically relevant changes in sustained gene expression patterns in aortic EC exposed to DF compared with LF which are predicted to induce a proatherogenic EC phenotype.

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.

Deferiprone increases endothelial nitric oxide synthase phosphorylation and nitric oxide production

2018 ◽  
Vol 96 (9) ◽  
pp. 879-885 ◽  
Author(s):  
Thanaporn Sriwantana ◽  
Pornpun Vivithanaporn ◽  
Kittiphong Paiboonsukwong ◽  
Krit Rattanawonsakul ◽  
Sirada Srihirun ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Endothelial Cells ◽  
Endothelial Function ◽  
Healthy Subjects ◽  
Iron Chelation ◽  
No Production ◽  
Hemoglobin E ◽  
Enos Phosphorylation

Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/β-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS (Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells.

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