Cellular Mechanisms of Hydrogen Peroxide (H
            2
            O
            2
            )‐induced Permeability Increases in Intact Venules: Role of H
            2
            O
            2
            ‐induced Ca
            2+
            ‐independent Nitric Oxide (NO) Production in Endothelial Cells

Эндотелиальные клетки, выстилающие стенки сосудов, преобразовывают деформацию собственных структур, вызванную током крови, в химические сигналы, одним из которых является важный регулятор просвета сосуда - оксид азота (NO). К настоящему моменту накоплен большой объём данных о клеточных механизмах активации продукции NO, однако сведений о динамике генерации оксида азота эндотелиальными клетками в зависимости от гидродинамических условий недостаточно. В этой связи разработка микрофлюидных систем in vitro, имитирующих кровеносное русло, и изучение в них эндотелия в сложных гидродинамических условиях является актуальной задачей. В данной работе для создания контролируемых гидродинамических условий для монослоя эндотелиоцитоподобных клеток EA.hy926 была спроектирована и разработана микрофлюидная система, имитирующая линейные участки микрососудистого русла. Методом непрямого определения содержания оксида азота (II) NO с использованием флуоресцентного зонда 4,5-диаминофлуоресцеина DAF-2 впервые получены данные об увеличении продукции NO клетками EA.hy926 при механическом стрессе, создаваемом потоком ростовой среды. Представлены расчетные гидродинамические характеристики микрофлюидной системы, а также методика измерения продукции NO. Возможность исследования функциональной активности эндотелия позволяет использовать разработанную микрофлюидную модельную систему как для изучения клеточно-автономных регуляторных свойств эндотелия при действии ряда вазоактивных фармакологических препаратов и других методов воздействия на эндотелий, так и при моделируемой дисфункции эндотелия. Endothelial cells lining vascular walls transform the flow-induced deformation of their own structures into chemical signals, one of which, nitric oxide (NO), is an important regulator of the vascular lumen diameter. By present, a large amount of data on cellular mechanisms for activation of NO production has been accumulated. However, there is insufficient information on changes in endothelial NO generation under different hydrodynamic conditions. Therefore, development of microfluidic systems that model blood vessels in vitro and using them to study the endothelium under complex hydrodynamic conditions are relevant tasks. In this study, a microfluidic system was developed to create controlled hydrodynamic conditions for a monolayer of endotheliocyte-like cells EAhy.926. This system simulates linear sections of the microvasculature. By indirect measurement of NO (II) content with a fluorescent 4,5-diaminofluorescein (DAF-2) probe, we showed an increase in the NO production by EAhy.926 cells under mechanical stress generated by the medium flow. The article presents the method for measuring NO production and the calculated hydrodynamic characteristics of the microfluidic system. The results showed that the developed microfluidic model system is promising for studying cell-autonomous regulatory properties of the endothelium both under the action of vasoactive agents and in simulated endothelial dysfunction.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.3.c753 ◽

1994 ◽

Vol 267 (3) ◽

pp. C753-C758 ◽

Cited By ~ 170

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.

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20-HETE-induced nitric oxide production in pulmonary artery endothelial cells is mediated by NADPH oxidase, H2O2, and PI3-kinase/Akt

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00298.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. L564-L574 ◽

Cited By ~ 22

Author(s):

Sreedhar Bodiga ◽

Stephanie K. Gruenloh ◽

Ying Gao ◽

Vijay L. Manthati ◽

Narsimhaswamy Dubasi ◽

...

Keyword(s):

Nitric Oxide ◽

Hydrogen Peroxide ◽

Endothelial Cells ◽

Pulmonary Artery ◽

Nadph Oxidase ◽

Pi3 Kinase ◽

No Production ◽

Akt Inhibitor ◽

Akt Phosphorylation ◽

No Release

We have shown that 20-hydroxyeicosatetraenoic acid (20-HETE) increases both superoxide and nitric oxide (NO) production in bovine pulmonary artery endothelial cells (BPAECs). The current study was designed to determine mechanisms underlying 20-HETE-stimulated NO release, and particularly the role of NADPH oxidase, reactive oxygen species, and PI3-kinase in stimulated NO release. Intracellular hydrogen peroxide (H2O2) and NO production were detected by dichlorofluorescein or dihydrorhodamine and diaminofluorescein fluorescence, respectively. Activation of endothelial nitric oxide synthase (eNOS) (Ser1179) and Akt (Ser473) was assessed by comparing the ratio of phosphorylated to total protein expression by Western blotting. Addition of 20-HETE to BPAECs caused an increase in superoxide and hydrogen peroxide, but not peroxynitrite. 20-HETE-evoked activation of Akt and eNOS, as well as enhanced NO release, are dependent on H2O2 as opposed to superoxide in that these endpoints are blocked by PEG-catalase and not PEG-superoxide dismutase. Similarly, 20-HETE-stimulated NO production in BPAECs is blocked by NADPH oxidase inhibitors apocynin or gp91 blocking peptide, and by PI3-kinase/Akt blockers wortmannin, LY-294002, or Akt inhibitor, implicating NADPH oxidase, PI3-kinase, and Akt signaling pathways, respectively, in this process. Together, these data suggest the following scheme: 20-HETE stimulates NADPH oxidase-dependent formation of superoxide. Superoxide is rapidly dismutated to hydrogen peroxide, which then mediates activation of PI3-kinase/Akt, phosphorylation of eNOS, and enhanced release of NO from eNOS in response to 20-HETE in BPAECs.

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Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00316.2010 ◽

2011 ◽

Vol 301 (3) ◽

pp. L334-L345 ◽

Cited By ~ 28

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.

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Nitric Oxide and Hydrogen Peroxide Signaling in Extractive Shiraia Fermentation by Triton X-100 for Hypocrellin A Production

International Journal of Molecular Sciences ◽

10.3390/ijms21030882 ◽

2020 ◽

Vol 21 (3) ◽

pp. 882

Author(s):

Xin Ping Li ◽

Yue Wang ◽

Yan Jun Ma ◽

Jian Wen Wang ◽

Li Ping Zheng

Keyword(s):

Nitric Oxide ◽

Hydrogen Peroxide ◽

H2o2 Production ◽

No Production ◽

Extractive Fermentation ◽

Hypocrellin A ◽

Cell Membrane Permeabilization ◽

Rapid Generation ◽

Triton X

Shiraia mycelial culture is a promising biotechnological alternative for the production of hypocrellin A (HA), a new photosensitizer for anticancer photodynamic therapy (PDT). The extractive fermentation of intracellular HA in the nonionic surfactant Triton X-100 (TX100) aqueous solution was studied in the present work. The addition of 25 g/L TX100 at 36 h of the fermentation not only enhanced HA exudation to the broth by 15.6-fold, but stimulated HA content in mycelia by 5.1-fold, leading to the higher production 206.2 mg/L, a 5.4-fold of the control on day 9. After the induced cell membrane permeabilization by TX100 addition, a rapid generation of nitric oxide (NO) and hydrogen peroxide (H2O2) was observed. The increase of NO level was suppressed by the scavenger vitamin C (VC) of reactive oxygen species (ROS), whereas the induced H2O2 production could not be prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), suggesting that NO production may occur downstream of ROS in the extractive fermentation. Both NO and H2O2 were proved to be involved in the expressions of HA biosynthetic genes (Mono, PKS and Omef) and HA production. NO was found to be able to up-regulate the expression of transporter genes (MFS and ABC) for HA exudation. Our results indicated the integrated role of NO and ROS in the extractive fermentation and provided a practical biotechnological process for HA production.

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Role of cytoskeleton in shear stress-induced endothelial nitric oxide production

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.1.h347 ◽

1997 ◽

Vol 273 (1) ◽

pp. H347-H355 ◽

Cited By ~ 23

Author(s):

H. L. Knudsen ◽

J. A. Frangos

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

Plasma Membrane ◽

Cytochalasin D ◽

Induced Response ◽

No Production ◽

Dependent Manner ◽

Mechanochemical Transduction

To study the role of the cytoskeleton in mechanochemical transduction, human umbilical vein endothelial cells were exposed to cytoskeleton-disrupting or -stabilizing agents, and the flow-induced production of nitric oxide (NO) as monitored by intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) was examined. A shear stress of 20 dyn/cm2 elevated cGMP levels approximately twofold relative to basal (stationary) levels (1.9 +/- 0.1 pmol cGMP in stationary controls; P < 0.01). Treatment with 1 microM phalloidin and 0.5 microM cytochalasin D did not significantly affect the flow-induced response (1.77 +/- 0.23 and 2.89 +/- 0.18 pmol cGMP in stationary controls, respectively), whereas disruption of microtubules with 0.5 microM colchicine significantly elevated the response (3.64 +/- 0.18 pmol cGMP in stationary controls; P < 0.01). The NO synthase inhibitor NG-amino-L-arginine abrogated all flow-induced elevations of cGMP, indicating that increased cGMP levels were mediated by NO. Cytoskeletal disruption with 0.2 microM cytochalasin D or 0.5 microM colchicine did not alter cGMP levels in response to 10 nM bradykinin. The role of the plasma membrane in mechanochemical transduction was examined by treatment with cholesteryl hemisuccinate, which attenuated the flow-induced response in a dose-dependent manner. In conclusion, the pathways of flow- and bradykinin-mediated NO production in endothelial cells did not require actin filament turnover or intact actin or microtubule cytoskeletons, and cholesterol, possibly by stiffening the plasma membrane, attenuated the flow response.

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Role of nitric oxide in autocrine control of growth and apoptosis of endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.272.2.h760 ◽

1997 ◽

Vol 272 (2) ◽

pp. H760-H768 ◽

Cited By ~ 5

Author(s):

A. Lopez-Farre ◽

L. Sanchez de Miguel ◽

C. Caramelo ◽

J. Gomez-Macias ◽

R. Garcia ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Endothelial Cell ◽

Growth Inhibitor ◽

No Production ◽

Alternative Mechanism ◽

Competitive Antagonist ◽

Autocrine Control ◽

Bovine Endothelial Cells

Nitric oxide (NO) is a growth inhibitor for diverse cellular types. In the present study, we have found that the inhibition of NO production in bovine endothelial cells by an L-arginine competitive antagonist induces DNA replication and promotes the transition from prereplicative to replicative phases of the endothelial cell cycle and an increase in c-myc and c-fos oncogene-encoded protein expression. The inhibition of NO generation had, however, a markedly different outcome depending on the state of confluence of the cells, i.e., proliferation was found in subconfluent cells, whereas apoptosis occurred in confluent cells. Moreover, Western blot analysis revealed differences in the constitutive NO synthase expression in proliferating compared with growth-arrested cells. In conclusion, these results disclose an alternative mechanism of endothelial cell apoptosis at the confluent state, which is related to NO inhibition. Moreover, the fact that the apoptotic phenomenon occurred in the presence of growth factors indicates the existence of apoptotic mechanisms that do not require growth factor deprivation.

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Protective Role of Nitric Oxide in a Model of Thrombotic Microangiopathy in Rats

Journal of the American Society of Nephrology ◽

10.1681/asn.v12102088 ◽

2001 ◽

Vol 12 (10) ◽

pp. 2088-2097

Author(s):

JING SHAO ◽

TOSHIO MIYATA ◽

KOEI YAMADA ◽

NORIO HANAFUSA ◽

TAKEHIKO WADA ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Thrombotic Microangiopathy ◽

Periodic Acid ◽

Minor Role ◽

Protective Mechanism ◽

No Production ◽

Nos Inhibitor ◽

Inducible Nos

Abstract. A new model of thrombotic microangiopathy (TMA) was previously developed, and it was demonstrated that endothelial nitric oxide (NO) synthase (NOS) is upregulated in glomeruli in this model. It was hypothesized that the synthesis of NO, a potent vasodilator and platelet inhibitory factor, is induced as a defense mechanism. The goal of this study was to clarify the role of NO in this model.Ex vivoexperiments using Western blotting and functional assays demonstrated upregulation of endothelial NOS in isolated glomeruli from TMA rats. Inin vivoexperiments, five groups of rats were studied, including rats with TMA treated with vehicle,NG-nitro-L-arginine methyl ester (L-NAME) (a NOS inhibitor), or L-N6-(1-iminoethyl)lysine (L-NIL) (a specific inducible NOS inhibitor) and normal control rats treated with vehicle or L-NAME. Blood urea nitrogen levels, BP, urinary nitrate/nitrite excretion, and proteinuria were measured. Histologic assessments using periodic acid-Schiff staining and immunohistologic studies with markers for endothelium, platelets, fibrin, cell proliferation, and apoptosis were also performed. L-NAME inhibition of NO synthesis in rats with TMA resulted in more severe glomerular and tubulointerstitial injury, which was accompanied by thrombus formation and a marked loss of endothelial cells, with more apoptotic cells. These changes were associated with severe renal function deterioration. In contrast, these features were less pronounced in the vehicle- or L-NIL-treated rats with TMA and were absent in the control animals. In conclusion, inhibition of NO production in this model of TMA markedly exacerbated renal injury. The absence of effects with L-NIL treatment suggests a minor role for inducible NOS in this model. These results suggest that production of NO, most likely by endothelial cells, is an important protective mechanism in TMA.

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1140-170 Hydrogen peroxide-induced superoxide production in endothelial cells: Role of nitric oxide synthase

Journal of the American College of Cardiology ◽

10.1016/s0735-1097(04)92149-3 ◽

2004 ◽

Vol 43 (5) ◽

pp. A508

Author(s):

Christian H Coyle ◽

Neal L Weintraub ◽

Khalid N Kader

Keyword(s):

Nitric Oxide ◽

Hydrogen Peroxide ◽

Endothelial Cells ◽

Nitric Oxide Synthase ◽

Superoxide Production ◽

Oxide Synthase

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

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y11-021 ◽

2011 ◽

Vol 89 (4) ◽

pp. 289-294 ◽

Cited By ~ 11

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.

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