Secretome analysis of chondroitin sulfate-treated chondrocytes reveals anti-angiogenic, anti-inflammatory and anti-catabolic propertiesReport as inadecuate

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Arthritis Research and Therapy

, 14:R202

First Online: 02 October 2012Received: 02 June 2012Revised: 18 July 2012Accepted: 02 October 2012


IntroductionChondroitin sulfate CS is a symptomatic slow-acting drug for osteoarthritis OA widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli IL-1β is regulated by CS, employing a novel quantitative proteomic approach.

MethodsHuman articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1β 5 ng-ml with or without CS pretreatment 200 µg-ml. Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results.

ResultsWe could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance six increased and 12 decreased. In normal chondrocytes stimulated with IL-1β, CS reduces inflammation directly by decreasing the presence of several complement components CFAB, C1S, CO3, and C1R and also indirectly by increasing proteins such as TNFα-induced protein TSG6. TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation observed in MMP1 and MMP3 levels. Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1.

ConclusionWe have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1β. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.

AbbreviationsCSchondroitin sulfate

Ctthreshold cycle

DMEMDulbecco-s modified Eagle-s medium

ECMextracellular matrix

GSglucosamine sulfate

HAChuman articular chondrocyte


LCliquid chromatography

mAbmonoclonal antibody

MALDImatrix-assisted laser desorption-ionization

MSmass spectrometry


PCRpolymerase chain reaction

SILACstable isotope labeling with amino acids in cell culture

TOFtime of flight

TFATrifluoroacetic acid

TNFtumor necrosis factor

TSG6TNFα-induced protein


Electronic supplementary materialThe online version of this article doi:10.1186-ar4040 contains supplementary material, which is available to authorized users.

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Author: Valentina Calamia - Lucía Lourido - Patricia Fernández-Puente - Jesús Mateos - Beatriz Rocha - Eulalia Montell - Josep V


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