Simvastatin prevents articular chondrocyte dedifferentiation induced by nitric oxide by inhibiting the expression of matrix metalloproteinases 1 and 13

In this study, we investigated whether simvastatin (SVT), a statin commonly prescribed to decrease cholesterol levels, might have a therapeutic effect in OA. Primary rabbit chondrocytes were pre-treated with SVT (50 μM), then treated with sodium nitroprusside (SNP; 1 mM), a donor of nitric oxide (NO) known as a pro-inflammatory mediator, and analyzed for the expression levels of type II collagen, SOX-9, aggrecan, matrix metalloproteinases (MMPs) 1, and 13. SNP increased NO generation in a dose-dependent manner, causing a loss of type II collagen and aggrecan indicative of chondrocyte dedifferentiation, which was inhibited by SVT. SVT also reversed the increase in MMP-1 and -13 and inhibited NO production and NO synthase expression induced by SNP in articular chondrocytes. Given that MMP-1 and -13 knockdown by siRNA increased the level of type II collagen in SNP-treated cells, our results show that SVT prevented NO-induced chondrocyte damage and dedifferentiation through downregulation of MMP expression. This study showed that SVT could attenuate the degradation of articular cartilage components, which is characteristic for OA, through inhibition of MMPs in NO-treated chondrocytes, suggesting that SVT may be a novel candidate therapeutic agent for the prevention and/or treatment of OA. Impact statement Dedifferentiation of chondrocytes is the main character of cartilage degradation. Therefore the understanding of chondrocytes dedifferentiation is essential for arthritis therapy. However, the molecular mechanism of cartilage destroy is mostly unknown. In this work we show that simvastatin (SVT) inhibits dedifferentiation by nitric oxide by blocking the expression of matrix metalloproteinases 1 and 13. These effects of SVT on dedifferentiation suggest that SVT may be used as a drug for the cure of arthritis.

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Asiatic Acid Attenuates Osteoarthritis by Regulating NF-κB Signaling Pathway in Chondrocytes

10.21203/rs.3.rs-31316/v1 ◽

2020 ◽

Author(s):

Zhengmeng Yang ◽

Lu Feng ◽

Xiaoting Zhang ◽

Weiping Lin ◽

Bin Wang ◽

...

Keyword(s):

Nitric Oxide ◽

Signaling Pathway ◽

Articular Chondrocytes ◽

Cartilage Damage ◽

Luciferase Assay ◽

No Production ◽

Asiatic Acid ◽

Dependent Manner ◽

Cox 2 ◽

Dose Dependent

Abstract Background: Natural small molecules have become more attractive as alternatives to non-steroidal anti-inflammatory drugs in osteoarthritis (OA) treatments. This study aims to investigate the effects of Asiatic acid (AA) on OA in chondrocytes and the surgery-induced OA animal model. Methods: Cytotoxicity of AA in primary rat articular chondrocytes was determined. Chondrocytes were pretreated with AA at the safe concentrations and subsequently treated with IL-1β. The production of inflammatory mediators including nitric oxide (NO), nitric oxide synthase (iNOS), as well as cyclooxygenase (COX)-2, and the expression of chondrogenic and hypertrophic markers including Sox 9, Aggrecan, Col 2a1, and matrix metalloproteinase-13 (MMP13) in the cells were measured. The effect of AA on nuclear factor-kappa B (NF-κB) signaling pathway was further determined by dual luciferase assay and western blot. The surgery-induced OA animals were treated with AA or saline for 6 weeks. The pathological changes in the affected joints were measured by micro-CT and histological analysis. Results: We found a broad safety spectrum of AA from 0 to 25 μM. A dose-dependent inhibitory effect of AA on NO production, as well as iNOS and COX-2 expression were found. Meanwhile, AA promoted chondrogenesis and inhibited hypertrophy in chondrocyte treated with IL-1β. In addition, AA inhibited NF-κB signaling pathway with a dose-dependent manner. Furthermore, results from animal study revealed that AA prevented articular cartilage damage as well as subchondral bone remodeling in the surgery-induced OA animal.

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P83 CONTRIBUTION OF MATRIX METALLOPROTEINASES (MMPS) AND CYSTEINE PROTEASES IN OSTEOARTHRITIS (OA) CARTILAGE DEGRADATION: AN EX-VIVOHUMAN CARTILAGE EXPLANT APPROACH USING THE TYPE II COLLAGEN FRAGMENTS HELIX-II AND CTX-II

Osteoarthritis and Cartilage ◽

10.1016/s1063-4584(07)60535-4 ◽

2006 ◽

Vol 14 ◽

pp. S56-S57

Author(s):

A.B.C. Jensen ◽

N. Charni ◽

J.M. Delaissé ◽

P. Garnero

Keyword(s):

Matrix Metalloproteinases ◽

Type Ii Collagen ◽

Cysteine Proteases ◽

Cartilage Degradation ◽

Type Ii ◽

Cartilage Explant

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Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.1.c245 ◽

1998 ◽

Vol 274 (1) ◽

pp. C245-C252 ◽

Cited By ~ 27

Author(s):

Junsuke Igarashi ◽

Masashi Nishida ◽

Shiro Hoshida ◽

Nobushige Yamashita ◽

Hiroaki Kosaka ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Cardiac Myocytes ◽

Myocardial Injury ◽

No Production ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

No Donor ◽

Oxide Synthase ◽

Gpx Activity

The effects of nitric oxide (NO) produced by cardiac inducible NO synthase (iNOS) on myocardial injury after oxidative stress were examined. Interleukin-1β induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS,l-arginine enhanced NO production in a concentration-dependent manner. Glutathione peroxidase (GPX) activity in myocytes was attenuated by elevated iNOS activity and by an NO donor, S-nitroso- N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition of H2O2(0.1 mM, 1 h). Inhibition of iNOS with Nω-nitro-l-arginine methyl ester ameliorated the effects of NO-enhancing treatments on myocardial injury and GPX activity. SNAP augmented the myocardial injury induced by H2O2. Inhibition of GPX activity with antisense oligodeoxyribonucleotide for GPX mRNA increased myocardial injury by H2O2. Results suggest that the induction of cardiac iNOS promotes myocardial injury due to oxidative stress via inactivation of the intrinsic antioxidant enzyme, GPX.

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Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

Journal of Biological Chemistry ◽

10.1074/jbc.m110.175448 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19215-19228 ◽

Cited By ~ 13

Author(s):

Frederic Cailotto ◽

Pascal Reboul ◽

Sylvie Sebillaud ◽

Patrick Netter ◽

Jean-Yves Jouzeau ◽

...

Keyword(s):

Growth Factor ◽

Promoter Activity ◽

Transforming Growth Factor ◽

Articular Chondrocytes ◽

Specific Protein ◽

Dependent Manner ◽

Articular Chondrocyte ◽

Experimental Conditions ◽

Inorganic Pyrophosphate ◽

Tgf Β1

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca2+-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa2+) or cytosolic Ca2+ (cCa2+) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa2+ input (fluorescent probe). Voltage-operated Ca2+-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa2+ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa2+ dose-dependent manner. TGF-β1 effects were suppressed by cCa2+ chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca2+. SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa2+ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.

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Nitric oxide activates matrix metalloproteinases indirectly in human articular chondrocytes

International Journal of Experimental Pathology ◽

10.1046/j.1365-2613.2000.0145s.x ◽

2008 ◽

Vol 81 (1) ◽

pp. A17-A17 ◽

Cited By ~ 1

Author(s):

Acey ◽

Grabowski ◽

Skerry

Keyword(s):

Nitric Oxide ◽

Matrix Metalloproteinases ◽

Articular Chondrocytes ◽

Human Articular Chondrocytes

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Adenosine enhances nitric oxide production by vascular endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.2.c519 ◽

1995 ◽

Vol 269 (2) ◽

pp. C519-C523 ◽

Cited By ~ 104

Author(s):

J. M. Li ◽

R. A. Fenton ◽

B. S. Cutler ◽

J. G. Dobson

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Vascular Tone ◽

Vascular Endothelial Cells ◽

Competitive Inhibitor ◽

No Production ◽

Dependent Manner ◽

Bathing Medium ◽

Adenosine Receptor Antagonist ◽

Potent Vasodilator

Adenosine per se is a potent vasodilator of vascular smooth muscle. Endothelial cells modulate vascular tone via the release of nitric oxide (NO), which also elicits vasodilation. This study was undertaken to determine whether adenosine could directly stimulate endothelial cells to enhance NO production, which could subsequently reduce vascular tone. NO production was evaluated in porcine carotid artery endothelial cells (PCAEC) and human saphenous vein endothelial cells (HSVEC) seeded on multiwell plates, grown to confluence, and treated with adenosine for 1 h. The bathing medium was collected, and the NO production was determined as reflected by the formation of NO2- and NO3-. NO production by PCAEC was significantly increased by adenosine in a dose-dependent manner, whereas there was only an insignificant tendency for an increase by HSVEC. The addition of the NO synthase competitive inhibitor, NG-monomethyl-L-arginine (NMMA), or the adenosine receptor antagonist, theophylline, prevented the increase in NO production by adenosine. The results suggest that adenosine stimulates, by a receptor-mediated mechanism, the production of NO by arterial, but not by venous, endothelial cells.

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Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1819 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1819-C1824 ◽

Cited By ~ 37

Author(s):

Yao Song ◽

Jay L. Zweier ◽

Yong Xia

Keyword(s):

Nitric Oxide ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Heat Shock Protein 90 ◽

Tryptophan Fluorescence ◽

Hsp90 Inhibitor ◽

Resonance Spectroscopy ◽

No Production ◽

Dependent Manner ◽

Intact Cells

Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.

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l-Arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense against Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.00578-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4305-4315 ◽

Cited By ~ 74

Author(s):

Rupesh Chaturvedi ◽

Mohammad Asim ◽

Nuruddeen D. Lewis ◽

Holly M. Scott Algood ◽

Timothy L. Cover ◽

...

Keyword(s):

Nitric Oxide ◽

Helicobacter Pylori ◽

Protein Expression ◽

Translation Initiation Factor ◽

No Production ◽

Dependent Manner ◽

Inos Protein ◽

Inos Protein Expression ◽

H Pylori ◽

Inducible Nitric Oxide

ABSTRACT Helicobacter pylori infection of the stomach causes an active immune response that includes stimulation of inducible nitric oxide (NO) synthase (iNOS) expression. Although NO can kill H. pylori, the bacterium persists indefinitely, suggesting that NO production is inadequate. We determined if the NO derived from iNOS in macrophages was dependent on the availability of its substrate, l-arginine (l-Arg). Production of NO by H. pylori-stimulated RAW 264.7 cells was dependent on the l-Arg concentration in the culture medium, and the 50% effective dose for l-Arg was 220 μM, which is above reported plasma l-Arg levels. While iNOS mRNA induction was l-Arg independent, iNOS protein increased in an l-Arg-dependent manner that did not involve changes in iNOS protein degradation. l-Lysine, an inhibitor of l-Arg uptake, attenuated H. pylori-stimulated iNOS protein expression, translation, NO levels, and killing of H. pylori. While l-Arg starvation suppressed global protein translation, at concentrations of l-Arg at which iNOS protein was only minimally expressed in response to H. pylori, global translation was fully restored and eukaryotic translation initiation factor α was dephosphorylated. H. pylori lacking the gene rocF, which codes for a bacterial arginase, induced higher levels of NO production by increasing iNOS protein levels. When murine gastric macrophages were activated with H. pylori, supraphysiologic levels of l-Arg were required to permit iNOS protein expression and NO production. These findings indicate that l-Arg is rate limiting for iNOS translation and suggest that the levels of l-Arg that occur in vivo do not permit sufficient NO generation by the host to kill H. pylori.

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