scholarly journals Nitric oxide degradation of heparin and heparan sulphate

1997 ◽  
Vol 324 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Rolando E. VILAR ◽  
Dineshchandra GHAEL ◽  
Min LI ◽  
Devan D. BHAGAT ◽  
Lisa M. ARRIGO ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Mass ◽  
Inflammatory Responses ◽  
Degradation Products ◽  
Bone Development ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
No Synthase ◽  
Strong Anion Exchange

NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.

scholarly journals Endothelial NO Synthase-Dependent S-Nitrosylation of β-Catenin Prevents Its Association with TCF4 and Inhibits Proliferation of Endothelial Cells Stimulated by Wnt3a

2017 ◽  
Vol 37 (12) ◽  
Author(s):  
Ying Zhang ◽  
Rony Chidiac ◽  
Chantal Delisle ◽  
Jean-Philippe Gratton
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Transcriptional Activity ◽  
No Synthase ◽  
Endothelial Cell Proliferation ◽  
Dependent Manner ◽  
Endothelial No Synthase ◽  
Binding Interface ◽  
Critical Residue

ABSTRACT Nitric oxide (NO) produced by endothelial NO synthase (eNOS) modulates many functions in endothelial cells. S-nitrosylation (SNO) of cysteine residues on β-catenin by eNOS-derived NO has been shown to influence intercellular contacts between endothelial cells. However, the implication of SNO in the regulation of β-catenin transcriptional activity is ill defined. Here, we report that NO inhibits the transcriptional activity of β-catenin and endothelial cell proliferation induced by activation of Wnt/β-catenin signaling. Interestingly, induction by Wnt3a of β-catenin target genes, such as the axin2 gene, is repressed in an eNOS-dependent manner by vascular endothelial growth factor (VEGF). We identified Cys466 of β-catenin as a target for SNO by eNOS-derived NO and as the critical residue for the repressive effects of NO on β-catenin transcriptional activity. Furthermore, we observed that Cys466 of β-catenin, located at the binding interface of the β-catenin–TCF4 transcriptional complex, is essential for disruption of this complex by NO. Importantly, Cys466 of β-catenin is necessary for the inhibitory effects of NO on Wnt3a-stimulated proliferation of endothelial cells. Thus, our data define the mechanism responsible for the repressive effects of NO on the transcriptional activity of β-catenin and link eNOS-derived NO to the modulation by VEGF of Wnt/β-catenin-induced endothelial cell proliferation.

Decreased Expression of KLF2 and KLF4 Induced by Antiphospholipid Antibodies Promotes NF-Kb-Mediated Endothelial Cell Activation and Is Modulated by CBP/p300

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4315-4315
Author(s):  
Kristi L Allen ◽  
Mukesh K Jain ◽  
Keith R McCrae
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Antiphospholipid Antibodies ◽  
Transcriptional Activity ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Cell Phenotype ◽  
Endothelial Cell Activation

Abstract Abstract 4315 Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of the core proteins in proteoglycans synthesized by vascular endothelial cells

1989 ◽  
Vol 261 (1) ◽  
pp. 145-153 ◽  
Author(s):  
A Lindblom ◽  
I Carlstedt ◽  
L Å Fransson
Keyword(s):  
Endothelial Cells ◽  
Molecular Mass ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Buoyant Density ◽  
Cell Extract ◽  
Apparent Molecular Mass ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Proteoglycans, metabolically labelled with [3H]leucine and 35SO4(2-), were isolated from the spent media and from guanidinium chloride extracts of cultured human umbilical-vein endothelial cells by using isopycnic density-gradient centrifugation, gel filtration and ion-exchange h.p.l.c. The major proteoglycan species were subjected to SDS/polyacrylamide-gel electrophoresis before and after enzymic degradation of the polysaccharide chains. The cell extract contained mainly a heparan sulphate proteoglycan that has a buoyant density of 1.31 g/ml and a protein core with apparent molecular mass 300 kDa. The latter was heterogeneous and migrated as one major and one minor band. After reduction, the apparent molecular mass of the major band increased to approx. 350 kDa, indicating the presence of intrachain disulphide bonds. The proteoglycan binds to octyl-Sepharose and its polysaccharide chains are extensively degraded by heparan sulphate lyase. The proteoglycans of the medium contained 90% of all the incorporated 35SO4(2-). Here the predominant heparan sulphate proteoglycan was similar to that of the cell extract, but was more heterogeneous and contained an additional core protein with apparent molecular mass 210 kDa. Furthermore, two different chondroitin sulphate proteoglycans were found: one 200 kDa species with a high buoyant density (approx. 1.45 g/ml) and one 100 kDa species with low buoyant density (approx. 1.3 g/ml). Both these proteoglycans have a core protein of molecular mass approx. 47 kDa.

scholarly journals C-Type Natriuretic Peptide (CNP) Inhibition of Interferon-γ-Mediated Gene Expression in Human Endothelial Cells In Vitro

Biosensors ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 86
Author(s):  
Amy Day ◽  
Zoe Jameson ◽  
Carolyn Hyde ◽  
Bigboy Simbi ◽  
Robert Fowkes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Vascular Inflammation ◽  
Western World ◽  
Human Endothelial Cells ◽  
Ifn Γ

Cardiovascular diseases, including atherosclerosis, now account for more deaths in the Western world than from any other cause. Atherosclerosis has a chronic inflammatory component involving Th1 pro-inflammatory cytokines such as IFN-γ, which is known to induce endothelial cell inflammatory responses. On the other hand CNP, which acts via its receptors to elevate intracellular cGMP, is produced by endothelium and endocardium and is upregulated in atherosclerosis. It is believed to be protective, however its role in vascular inflammation is not well understood. The aim of this study was to investigate the effects of CNP on human endothelial cell inflammatory responses following IFN-γ stimulation. Human umbilical vein endothelial cells were treated with either IFN-γ (10 ng/mL) or CNP (100 nm), or both in combination, followed by analysis by flow cytometry for expression of MHC class I and ICAM-1. IFN-γ significantly increased expression of both molecules, which was significantly inhibited by CNP or the cGMP donor 8-Bromoguanosine 3’,5’-cyclic monophosphate (1 µm). CNP also reduced IFN-γ mediated kynurenine generation by the IFN-γ regulated enzyme indoleamine-2,3-deoxygenase (IDO). We conclude that CNP downmodulates IFN-γ induced pro-inflammatory gene expression in human endothelial cells via a cGMP-mediated pathway. Thus, CNP may have a protective role in vascular inflammation and novel therapeutic strategies for CVD based on upregulation of endothelial CNP expression could reduce chronic EC inflammation.

scholarly journals Distribution of iduronate 2-sulphate residues in heparan sulphate. Evidence for an ordered polymeric structure

1991 ◽  
Vol 273 (3) ◽  
pp. 553-559 ◽  
Author(s):  
J E Turnbull ◽  
J T Gallagher
Keyword(s):  
Molecular Mass ◽  
Average Length ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Degree Of Polymerization ◽  
Central Position ◽  
Human Skin Fibroblast ◽  
Cleavage Sites ◽  
Average Molecular Mass ◽  
Polymeric Structure

The structure of human skin fibroblast heparan sulphate has been examined by depolymerization with heparinase, which specifically cleaves highly sulphated disaccharides of structure GlcNSO3 (+/-6S)-alpha 1,4IdoA(2S) [N-sulphated glucosamine (6-sulphate)-alpha 1,4-iduronic acid 2-sulphate]. Heparan sulphate contained only a small proportion (approximately 10%) of linkages susceptible to this enzyme. The major products of depolymerization with heparinase were large oligosaccharides with an average molecular mass of 10 kDa (dp approximately 40, where dp is degree of polymerization; for disaccharides, dp = 2 etc.) as assessed by gel filtration on Sepharose CL-6B, compared with a molecular mass of 45 kDa (dp approximately 200) for the intact chains. The large heparinase-resistant oligosaccharides were highly susceptible to depolymerization with the enzyme heparitinase, which cleaves heparan sulphate in areas of low sulphation, where N-acetylated disaccharides [GlcNAc-alpha 1,4GlcA (N-acetylglucosaminyl-alpha 1,4-glucuronic acid)] are the predominant structural unit. Further analysis of the location of the heparinase cleavage sites indicated that they were predominantly found in a central position in GlcNSO3-alpha 1,4IdoA repeat sequences of average length four to seven disaccharides (dp 8-14). These results indicate that heparinase cleaves heparan sulphate in approximately four or five N-sulphated domains, each domain containing a cluster of two or three susceptible disaccharides; the domains are separated by long N-acetyl-rich sequences that are markedly deficient in sulphate groups. On the basis of these findings a model is proposed which depicts heparan sulphate as an ordered polymeric structure composed of an alternate arrangement of sulphate-rich and sulphate-poor regions. The sulphate-rich regions are likely to be flexible areas of the chain because of their high content of the conformationally versatile IdoA and IdoA(2S) residues. The model has important implications for the biosynthesis and functions of heparan sulphate.

scholarly journals Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties

1990 ◽  
Vol 271 (1) ◽  
pp. 75-86 ◽  
Author(s):  
J Bielicki ◽  
C Freeman ◽  
P R Clements ◽  
J J Hopwood
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Human Kidney ◽  
Reduced Form ◽  
Dermatan Sulphate ◽  
Native Molecular Mass ◽  
Phosphate Ions

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

Impaired EDRF production by endothelial cells exposed to fibrin monomer and FDP

1995 ◽  
Vol 268 (2) ◽  
pp. C520-C526 ◽  
Author(s):  
J. E. Freedman ◽  
A. Fabian ◽  
J. Loscalzo
Keyword(s):  
Endothelial Cells ◽  
Degradation Products ◽  
Vascular Dysfunction ◽  
No Synthase ◽  
Northern Analysis ◽  
Aortic Endothelial Cells ◽  
Relaxing Factor ◽  
Bovine Aortic Endothelial Cells ◽  
Fibrin Monomer ◽  
Endothelium Derived Relaxing Factor

Fibrin and fibrinogen and their proteolytic degradation products, found within the atheroma, may contribute to vascular dysfunction. We monitored the production of endothelium-derived relaxing factor (EDRF) by bovine aortic endothelial cells (BAEC) after exposure to fibrinogen, fibrin monomer (FM), and fibrin and fibrinogen degradation products (FDP). Cells incubated with FM and FDP, compared with cells incubated with fibrinogen, were less able to inhibit platelet aggregation, and this effect correlated with a concentration-dependent decrease in EDRF production: BAEC incubated with FM or FDP, but not fibrinogen, produced significantly less nitric oxide (NO), as determined using a photolysis-chemiluminescence system. Northern analysis of BAEC incubated with fibrinogen, FM, or FDP and probed for constitutive bovine endothelial NO synthase mRNA demonstrated decreased expression in cells exposed to FDP or FM. These data show that FM and FDP reduce EDRF produced by BAEC and attenuate constitutive NO synthase expression. These effects may represent a mechanism by which thrombotic determinants adversely affect endothelial function and thereby potentially impair vasomotor responses and contribute to atherothrombogenesis.

Rhodiola crenulata Suppresses High Glucose-Induced Matrix Metalloproteinase Expression and Inflammatory Responses by Inhibiting ROS-Related HMGB1-TLR4 Signaling in Endothelial Cells

2020 ◽  
Vol 48 (01) ◽  
pp. 91-105
Author(s):  
Li-Yen Huang ◽  
I-Chuan Yen ◽  
Wei-Cheng Tsai ◽  
Shih-Yu Lee
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
High Glucose ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Mechanisms Of Action ◽  
Primary Response ◽  
Protective Effects ◽  
Rhodiola Crenulata

Rhodiola crenulata, a popular folk medicine for anti-altitude sickness in Tibet, has been shown to have protective effects against high glucose (HG)-induced endothelial cell dysfunction in human umbilical vein endothelial cells (HUVECs). However, its mechanisms of action are unclear. Here, we aimed to examine the effects and the mechanisms of action of Rhodiola crenulata extract (RCE) on matrix metalloproteinases (MMPs) and inflammatory responses under HG conditions. HUVECs were pretreated with RCE or untreated and then exposed to 33[Formula: see text]mM glucose medium for 24[Formula: see text]h. The levels of oxidative stress markers, MMPs, endogenous tissue inhibitors of MMPs (TIMPs), and adhesion molecules were determined. Zymography assays were also carried out. We found that RCE significantly decreased HG-induced increases in reactive oxygen species (ROS) and activation of MAPK and NF-[Formula: see text]B pathways. In addition, RCE not only significantly reduced the expression and activities of MMPs but also upregulated TIMP protein levels. Consistently, HG-induced activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response protein (MyD88) signaling pathway, intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and high mobility group box 1 (HMGB1) as well as endothelial cell apoptosis was inhibited by RCE treatment. RCE exerts protective effects on endothelial cells against HG insult, partially by suppressing the HMGB1/TLR4 axis. These findings indicate that Rhodiola crenulata may be a potential therapeutic agent for diabetes-associated vascular diseases.

Podokinesis in endothelial cell migration: role of nitric oxide

1998 ◽  
Vol 274 (1) ◽  
pp. C236-C244 ◽  
Author(s):  
Eisei Noiri ◽  
Eugene Lee ◽  
Jacqueline Testa ◽  
James Quigley ◽  
David Colflesh ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
No Synthase ◽  
Endothelial Cell Migration ◽  
Guidance Cues ◽  
Cell Matrix ◽  
Cell Matrix Adhesion

Previously, we demonstrated the role of nitric oxide (NO) in transforming epithelial cells from a stationary to locomoting phenotype [E. Noiri, T. Peresleni, N. Srivastava, P. Weber, W. F. Bahou, N. Peunova, and M. S. Goligorsky. Am. J. Physiol. 270 ( Cell Physiol. 39): C794–C802, 1996] and its permissive function in endothelin-1-stimulated endothelial cell migration (E. Noiri, Y. Hu, W. F. Bahou, C. Keese, I. Giaever, and M. S. Goligorsky. J. Biol. Chem. 272: 1747–1753, 1997). In the present study, the role of functional NO synthase in executing the vascular endothelial growth factor (VEGF)-guided program of endothelial cell migration and angiogenesis was studied in two independent experimental settings. First, VEGF, shown to stimulate NO release from simian virus 40-immortalized microvascular endothelial cells, induced endothelial cell transwell migration, whereas N G-nitro-l-arginine methyl ester (l-NAME) or antisense oligonucleotides to endothelial NO synthase suppressed this effect of VEGF. Second, in a series of experiments on endothelial cell wound healing, the rate of VEGF-stimulated cell migration was significantly blunted by the inhibition of NO synthesis. To gain insight into the possible mode of NO action, we next addressed the possibility that NO modulates cell matrix adhesion by performing impedance analysis of endothelial cell monolayers subjected to NO. The data showed the presence of spontaneous fluctuations of the resistance in ostensibly stationary endothelial cells. Spontaneous oscillations were induced by NO, which also inhibited cell matrix adhesion. This process we propose to term “podokinesis” to emphasize a scalar form of micromotion that, in the presence of guidance cues, e.g., VEGF, is transformed to a vectorial movement. In conclusion, execution of the program for directional endothelial cell migration requires two coexisting messages: NO-induced podokinesis (scalar motion) and guidance cues, e.g., VEGF, which imparts a vectorial component to the movement. Such a requirement for the dual signaling may explain a mismatch in the demand and supply with newly formed vessels in different pathological states accompanied by the inhibition of NO synthase.

Fibrinogen degradation product fragment D increases endothelial monolayer permeability

1991 ◽  
Vol 261 (4) ◽  
pp. L283-L289 ◽  
Author(s):  
M. Ge ◽  
T. J. Ryan ◽  
H. Lum ◽  
A. B. Malik
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Clearance Rate ◽  
Degradation Products ◽  
Specific Binding ◽  
Gel Filtration ◽  
Fold Increase ◽  
Gamma Chain ◽  
Fibrinogen Degradation Product ◽  
Albumin Clearance

We assessed the effects of the two primary high-molecular-weight fibrinogen degradation products (FDP), fragments D and E, on the pulmonary vascular endothelial barrier function. Fragments D and E were purified to homogeneity by QAE Sephadex chromatography followed by gel filtration. Incubation of bovine pulmonary artery endothelial monolayers with 0.5–2.0 microM fragment D for 2 h caused a doubling of transendothelial 125I-albumin clearance rate (a measure of 125I-albumin permeability). Fragment E only produced a 0.6-fold increase in 125I-albumin clearance rate at concentration of 4.0 microM. Both FDP remained active in incubating media with serum. The permeability-increasing effect of fragment D was reversible and was not due to cell detachment or lysis. The fragment-D effect was time dependent and was associated with redistribution of endothelial F-actin microfilaments. The effect was independent of the carboxy-terminal sequence on gamma-chain of fragment D. Fragments D and E binding to pulmonary artery endothelial cells was specific and reversible, but fragment D binding was three-fold greater than fragment E, which may account for the greater permeability increase mediated by fragment D. The results indicate that FDP, especially fragment D, increase endothelial permeability to albumin. The response involves specific binding of fragment D to endothelial cells and redistribution of intracellular actin.

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