scholarly journals Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfishCyanea capillata

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3172 ◽  
Author(s):  
Beilei Wang ◽  
Dan Liu ◽  
Chao Wang ◽  
Qianqian Wang ◽  
Hui Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Critical Role ◽  
No Production ◽  
Enos Activity ◽  
Enos Phosphorylation ◽  
Isolated Aorta ◽  
Umbilical Vein Endothelial Cells

Our previous study demonstrated that tentacle extract (TE) from the jellyfishCyanea capillata(C. capillata) could cause a weak relaxation response mediated by nitric oxide (NO) using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca2+signaling pathways in human umbilical vein endothelial cells (HUVECs). Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin) significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca2+concentration ([Ca2+]i), which was significantly attenuated by either IP3receptor blocker (Heparin) or PKC inhibitor (PKC 412). In contrast, extracellular Ca2+-free, L-type calcium channel blocker (Nifedipine), or PKA inhibitor (H89) had no influence on the [Ca2+]ielevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca2+in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca2+]ielevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca2+-dependent pathways.

scholarly journals The role of HYAL2 in LSS-induced glycocalyx impairment and the PKA-mediated decrease in eNOS–Ser-633 phosphorylation and nitric oxide production

2016 ◽  
Vol 27 (25) ◽  
pp. 3972-3979 ◽  
Author(s):  
Xiangquan Kong ◽  
Liang Chen ◽  
Peng Ye ◽  
Zhimei Wang ◽  
Junjie Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Shear Stress ◽  
Umbilical Vein ◽  
Endothelial Glycocalyx ◽  
No Production ◽  
Enos Phosphorylation ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Hyaluronan (HA) in the endothelial glycocalyx serves as a mechanotransducer for high-shear-stress–stimulated endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. Low shear stress (LSS) has been shown to contribute to endothelial inflammation and atherosclerosis by impairing the barrier and mechanotransduction properties of the glycocalyx. Here we focus on the possible role of hyaluronidase 2 (HYAL2) in LSS-induced glycocalyx impairment and the resulting alterations in eNOS phosphorylation and NO production in human umbilical vein endothelial cells (HUVECs). We show that LSS strongly activates HYAL2 to degrade HA in the glycocalyx. The dephosphorylation of eNOS–Ser-633 under LSS was triggered after HA degradation by hyaluronidase and prevented by repairing the glycocalyx with high–molecular weight hyaluronan. Knocking down HYAL2 in HUVECs protected against HA degradation in the glycocalyx by inhibiting the expression and activity of HYAL2 and further blocked the dephosphorylation of eNOS–Ser-633 and the decrease in NO production in response to LSS. The LSS-induced dephosphorylation of PKA was completely abrogated in HYAL2 siRNA–transfected HUVECs. The LSS-induced dephosphorylation of eNOS–Ser-633 was also reversed by the PKA activator 8-Br-cAMP. We thus suggest that LSS inhibits eNOS–Ser-633 phosphorylation and, at least partially, NO production by activating HYAL2 to degrade HA in the glycocalyx.

Protective effect of neferine on endothelial cell nitric oxide production induced by lysophosphatidylcholine: the role of the DDAH–ADMA pathway

2011 ◽  
Vol 89 (4) ◽  
pp. 289-294 ◽  
Author(s):  
Zhen-Yu Peng ◽  
Sai-Dan Zhang ◽  
Shao Liu ◽  
Bai-Mei He
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Protective Effect ◽  
Asymmetric Dimethylarginine ◽  
Umbilical Vein ◽  
Antioxidant Properties ◽  
No Production ◽  
Seed Embryo ◽  
Umbilical Vein Endothelial Cells

Neferine, extracted from the seed embryo of Nelumbo nucifera Gaertn., has multiple cardiovascular pharmacological effects. The dimethylarginine dimethylaminohydrolase (DDAH) – asymmetric dimethylarginine (ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of neferine on endothelial NO production was related to the DDAH–ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were first exposed to neferine (0.1, 1.0, or 10.0 μmol/L) for 1 h, and then incubated with lysophosphatidylcholine (LPC; 10 μg/mL) in the presence of neferine for 24 h. The medium was collected for measuring the levels of NO, maleic dialdehyde (MDA), as well as ADMA. The endothelial cells were collected for measuring DDAH activity and the level of reactive oxygen species (ROS). LPC significantly decreased NO concentration and DDAH activity and increased the levels of ADMA, ROS, and MDA. Neferine could partially counteract the changes induced by LPC. These findings suggested that neferine could modulate the DDAH–ADMA pathway via its antioxidant properties, which was involved in its beneficial effect on endothelial NO production.

scholarly journals Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10287
Author(s):  
Chih-Hsien Wu ◽  
Yi-Lin Chiu ◽  
Chung-Yueh Hsieh ◽  
Guo-Shiang Tsung ◽  
Lian-Shan Wu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Umbilical Vein ◽  
Sirtuin 1 ◽  
No Production ◽  
Beneficial Effects ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

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.

Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Vinicius P Garcia ◽  
Jamie G Hijmans ◽  
Kelly A Stockelman ◽  
Madden Brewster ◽  
Hannah Fandl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
No Production ◽  
Cell Dysfunction ◽  
Enos Activity ◽  
Umbilical Vein Endothelial Cells ◽  
And Function ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

Endothelial nitric oxide production during in vitro simulation of external limb compression

2002 ◽  
Vol 282 (6) ◽  
pp. H2066-H2075 ◽  
Author(s):  
Guohao Dai ◽  
Olga Tsukurov ◽  
Michael Chen ◽  
Jonathan P. Gertler ◽  
Roger D. Kamm
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Umbilical Vein ◽  
Control Group ◽  
No Production ◽  
Vein Thrombosis ◽  
Enos Mrna ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Nitric Oxide

External pneumatic compression (EPC) is effective in preventing deep vein thrombosis (DVT) and is thought to alter endothelial thromboresistant properties. We investigated the effect of EPC on changes in nitric oxide (NO), a critical mediator in the regulation of vasomotor and platelet function. An in vitro cell culture system was developed to simulate flow and vessel collapse conditions under EPC. Human umbilical vein endothelial cells were cultured and subjected to tube compression (C), pulsatile flow (F), or a combination of the two (FC). NO production and endothelial nitric oxide synthase (eNOS) mRNA expression were measured. The data demonstrate that in the F and FC groups, there is a rapid release of NO followed by a sustained increase. NO production levels in the F and FC groups were almost identical, whereas the C group produced the same low amount of NO as the control group. Conditions F and FC also upregulate eNOS mRNA expression by a factor of 2.08 ± 0.25 and 2.11 ± 0.21, respectively, at 6 h. Experiments with different modes of EPC show that NO production and eNOS mRNA expression respond to different time cycles of compression. These results implicate enhanced NO release as a potentially important factor in the prevention of DVT.

Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

2007 ◽  
Vol 293 (1) ◽  
pp. C458-C467 ◽  
Author(s):  
Jian-Zhong Sheng ◽  
Andrew P. Braun
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Early Event ◽  
No Production ◽  
Primary Role ◽  
Membrane Hyperpolarization ◽  
Intracellular Ca ◽  
Umbilical Vein Endothelial Cells

The contribution of small-conductance (SKCa) and intermediate-conductance Ca2+-activated K+ (IKCa) channels to the generation of nitric oxide (NO) by Ca2+-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca2+ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca2+, as judged by 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl2 (50 μmol/l) + ouabain (100 μmol/l), depleting intracellular Ca2+ stores by thapsigargin or removing external Ca2+ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SKCa and IKCa channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca2+ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SKCa and IKCa channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SKCa and IKCa channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca2+ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca2+ stores is to trigger the opening of both KCa channels along with Ca2+ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SKCa and IKCa channels are essential for NO-mediated vasorelaxation.

scholarly journals Programmed cell death 5 suppresses AKT-mediated cytoprotection of endothelium

2018 ◽  
Vol 115 (18) ◽  
pp. 4672-4677 ◽  
Author(s):  
Seung-Hyun Lee ◽  
Jaesung Seo ◽  
Soo-Yeon Park ◽  
Mi-Hyeon Jeong ◽  
Hyo-Kyoung Choi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Umbilical Vein ◽  
Density Lipoprotein ◽  
No Production ◽  
Dependent Signal ◽  
Programmed Cell Death 5 ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Nitric Oxide

Programmed cell death 5 (PDCD5) has been associated with human cancers as a regulator of cell death; however, the role of PDCD5 in the endothelium has not been revealed. Thus, we investigated whether PDCD5 regulates protein kinase B (PKB/AKT)-endothelial nitric oxide synthase (eNOS)–dependent signal transduction in the endothelium and affects atherosclerosis. Endothelial-specific PDCD5 knockout mice showed significantly reduced vascular remodeling compared with wild-type (WT) mice after partial carotid ligation. WT PDCD5 competitively inhibited interaction between histone deacetylase 3 (HDAC3) and AKT, but PDCD5L6R, an HDAC3-binding–deficient mutant, did not. Knockdown of PDCD5 accelerated HDAC3–AKT interaction, AKT and eNOS phosphorylation, and nitric oxide (NO) production in human umbilical vein endothelial cells. Moreover, we found that serum PDCD5 levels reflect endothelial NO production and are correlated with diabetes mellitus, high-density lipoprotein cholesterol, and coronary calcium in human samples obtained from the cardiovascular high-risk cohort. Therefore, we conclude that PDCD5 is associated with endothelial dysfunction and may be a novel therapeutic target in atherosclerosis.

VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells

1998 ◽  
Vol 274 (3) ◽  
pp. H1054-H1058 ◽  
Author(s):  
John D. Hood ◽  
Cynthia J. Meininger ◽  
Marina Ziche ◽  
Harris J. Granger
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Northern Blotting ◽  
No Production ◽  
Vascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Growth Factor ◽  
Stimulation Of

Vascular endothelial growth factor (VEGF) is an endothelium-specific secreted protein that potently stimulates vasodilation, microvascular hyperpermeability, and angiogenesis. Nitric oxide (NO) is also reported to modulate vascular tone, permeability, and capillary growth. Therefore, we hypothesized that VEGF might regulate endothelial production of NO. The production of nitrogen oxides by human umbilical vein endothelial cells (HUVECs) was measured after 1, 12, 24, and 48 h of incubation with VEGF. VEGF treatment resulted in both an acute (1 h) and chronic (>24 h) stimulation of NO production. Furthermore, Western and Northern blotting revealed a VEGF-elicited, dose-dependent increase in the cellular content of endothelial cell nitric oxide synthase (ecNOS) message and protein that may account for the chronic upregulation of NO production elicited by VEGF. Finally, endothelial cells pretreated with VEGF for 24 h and subsequently exposed to A-23187 for 1 h produced NO at approximately twice the rate of cells that were not pretreated with VEGF. We conclude that VEGF upregulates ecNOS enzyme and elicits a biphasic stimulation of endothelial NO production.

scholarly journals DHA-phospholipids control membrane fusion and transcellular tunnel dynamics

2021 ◽  
Author(s):  
Meng-Chen Tsai ◽  
Lucile Fleuriot ◽  
Sébastien Janel ◽  
David Gonzalez-Rodriguez ◽  
Camille Morel ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Umbilical Vein ◽  
Critical Role ◽  
Biomechanical Properties ◽  
Cell Cortex ◽  
And Shifts ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact ◽  
Cell Thickness

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses show that human umbilical vein endothelial cells (HUVECs) subjected to DHA-diet undergo a 6-fold enrichment in DHA-PLs at plasma membrane (PM) at the expense of monounsaturated OA-PLs. Consequently, DHA-PLs enrichment at the PM induces a reduction of cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM leads to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PLs levels in membranes affect cell biomechanical properties.

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