Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase expression in human chondrocytes

Accepted Manuscript Cannabinoid WIN-55,212-2 Mesylate Inhibits Interleukin-1β Induced Matrix Metalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase Expression in Human Chondrocytes S.L. Dunn, J.M. Wilkinson, A. Crawford, C.L. Le Maitre, R.A.D. Bunning PII:

S1063-4584(13)00999-0

DOI:

10.1016/j.joca.2013.10.016

Reference:

YJOCA 3011

To appear in:

Osteoarthritis and Cartilage

Received Date: 29 May 2013 Revised Date:

22 October 2013

Accepted Date: 26 October 2013

Please cite this article as: Dunn SL, Wilkinson JM, Crawford A, Le Maitre CL, Bunning R, Cannabinoid WIN-55,212-2 Mesylate Inhibits Interleukin-1β Induced Matrix Metalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase Expression in Human Chondrocytes, Osteoarthritis and Cartilage (2013), doi: 10.1016/j.joca.2013.10.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Cannabinoid WIN-55,212-2 Mesylate Inhibits Interleukin-1β Induced Matrix Metalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase Expression in Human Chondrocytes.

S L Dunn , J M Wilkinson , A Crawford , C L Le Maitre & RAD Bunning* †

‡

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†

Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam

University, Sheffield, S1 1WB, UK ‡

Academic Unit of Bone Metabolism, Department of Human Metabolism, University of

Sheffield, UK

Centre for Biomaterials & Tissue Engineering, University of Sheffield, School of

Clinical Dentistry, UK

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SL Dunn, e-mail: [email protected]

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†

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JM Wilkinson, e-mail: [email protected] A Crawford, e-mail: [email protected] CL Le Maitre, e-mail: [email protected]

*Author for correspondence: RAD Bunning,Tel: +44 114 225 3012 Fax: +44 114 225

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3066 E-mail: [email protected]

KEY WORDS: Cannabinoid, Cartilage degradation, Chondrocytes, Interleukin 1 (IL1), Matrix metalloproteinases (MMPs), Tissue Inhibitors of Matrix Metalloproteinases (TIMPs)

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Running title: Inhibition of MMP and TIMP production by WIN-55

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ABSTRACT Objective Interleukin-1β (IL-1β) is involved in the up-regulation of matrix

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metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are antiinflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2

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mesylate (WIN-55) may inhibit cartilage degradation.

Methods Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3

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and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time PCR was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and ELISA to measure the amount of MMP-3 and MMP-13 protein released into media.

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Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocytes cultures.

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Results Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte

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monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemisty demonstrated the expression of cannabinoid receptors in chondrocyte cultures. Conclusion Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal

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levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by

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which cannabinoids may act to prevent cartilage breakdown in arthritis.

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Cannabinoid WIN-55,212-2 Mesylate Inhibits Interleukin-1β Induced Matrix

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Metalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase Expression

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in Human Chondrocytes.

S L Dunn†, J M Wilkinson‡, A Crawford$, C L Le Maitre† & RAD Bunning*†

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†

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University, Sheffield, S1 1WB, UK

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‡

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Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam

Academic Unit of Bone Metabolism, Department of Human Metabolism, University

of Sheffield, UK

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$

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Clinical Dentistry, UK

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SL Dunn, e-mail: [email protected]

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JM Wilkinson, e-mail: [email protected]

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A Crawford, e-mail: [email protected]

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CL Le Maitre, e-mail: [email protected]

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*Author for correspondence: RAD Bunning,Tel: +44 114 225 3012 Fax: +44 114 225

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3066 E-mail: [email protected]

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Centre for Biomaterials & Tissue Engineering, University of Sheffield, School of

KEY WORDS: Cannabinoid, Cartilage degradation, Chondrocytes, Interleukin 1 (IL-

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1), Matrix metalloproteinases (MMPs), Tissue Inhibitors of Matrix Metalloproteinases

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(TIMPs)

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Running title: Inhibition of MMP and TIMP production by WIN-55

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24 ABSTRACT

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Objective Interleukin-1β (IL-1β) is involved in the up-regulation of matrix

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metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-

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inflammatory and reduce joint damage in animal models of arthritis. This study aimed

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to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2

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mesylate (WIN-55) may inhibit cartilage degradation.

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Methods Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3

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and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes

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were obtained from articular cartilage of patients undergoing total knee replacement.

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Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time

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PCR was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and

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ELISA to measure the amount of MMP-3 and MMP-13 protein released into media.

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Immunocytochemistry was used to investigate the expression of cannabinoid

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receptors in chondrocytes cultures.

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Results Treatment with WIN-55 alone or in combination with IL-1β, decreased or

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abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte

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monolayer and alginate bead cultures in both a concentration and time dependent

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manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3

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and -13 protein production by chondrocytes cultured in alginate beads.

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Immunocytochemisty demonstrated the expression of cannabinoid receptors in

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chondrocyte cultures.

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Conclusion Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene

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and protein expression of MMP-3 and -13. However WIN-55 also decreased basal

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levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by

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which cannabinoids may act to prevent cartilage breakdown in arthritis.

50 INTRODUCTION

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Osteoarthritis (OA) and rheumatoid arthritis (RA) are debilitating joint diseases, and

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although they have a different aetiology a key pathological feature of both is the loss

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of articular cartilage1. Cannabis-based medicine Sativex has been shown to have

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analgesic effects and to suppress disease activity in patients with RA

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Cannabinoids also have anti-inflammatory effects and reduce joint damage in animal

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models of arthritis 3-5. In vitro studies have shown that cannabinoids reduce cytokine

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production from RA fibroblasts and the release of matrix metalloproteinases (MMPs)

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from fibroblast-like synovial cells6-8. Cannabinoids also have direct effects on

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cartilage extracellular matrix (ECM) breakdown; reducing interleukin 1 (IL-1) induced

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proteoglycan and collagen degradation in bovine cartilage9. There is thus increasing

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evidence to suggest that cannabinoids have chondroprotective effects and may be of

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value in the treatment of arthritis10.

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During OA and RA there is a shift in the equilibrium between catabolic and anabolic

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activities1. As a result the breakdown of collagen and proteoglycans may exceed the

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rate of synthesis of new matrix molecules resulting in cartilage degradation. Another

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contributing factor in cartilage breakdown in OA and RA is an increase in

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inflammatory cytokines particularly IL-1 and tumour necrosis factor (TNF) produced

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by the articular chondrocytes or cells of the synovium11. This results in an increase in

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MMPs particularly MMP-3 and MMP-13, which are expressed in RA and OA

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cartilage and synovial tissue,12-20 without an increase in their tissue inhibitors of

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matrix metalloproteinases (TIMPs)21,

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possible mechanism to prevent breakdown of cartilage tissue in arthritis, provided

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the necessary functional specificity can be achieved 23.

. MMP inhibition has been proposed as a

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76 To investigate further the potential of cannabinoids to regulate cartilage breakdown

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we have studied the effects of synthetic cannabinoid WIN-55,212-2 mesylate (WIN-

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55) on basal and IL-1β stimulated MMP-3, -13, TIMP-1 and -2 expression in human

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articular chondrocytes from human OA cartilage tissue in monolayer and 3D alginate

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bead culture.

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To investigate further the potential of cannabinoids to regulate cartilage breakdown

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we have studied the effects of synthetic cannabinoid WIN-55,212-2 mesylate (WIN-

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55) on basal and IL-1β stimulated MMP-3, -13, TIMP-1 and -2 expression in human

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articular chondrocytes from human OA cartilage tissue in monolayer and 3D alginate

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bead culture. WIN-55 is an agonist at the classical cannabinoid receptors,

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cannabinoid receptor 1 and 2 (CB1 and 2), but also has been shown to activate

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other receptors including peroxisome proliferator activated receptors alpha and

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gamma (PPARα and γ)24-26. Thus the expression of these receptors in cultured OA

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chondrocytes was determined to identify potential targets of WIN-55.

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MATERIALS AND METHODS

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Human cartilage tissue Human chondrocytes were obtained from articular cartilage (n=9) removed from

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patients with symptomatic OA at the time of total knee replacement (Supplementary

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table 1). Cartilage was obtained under the National Research Ethics Service

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approval held by the Sheffield Musculoskeletal Biobank. All patients provided written,

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informed consent prior to participation. Cartilage blocks were taken from each

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anatomic compartment within the knee (n=6-7) (medial and lateral tibio-femoral and

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patello-femoral compartments). Cartilage tissue was graded macroscopically 0-4

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using the Outerbridge classification27. Chondrocytes were isolated from grade 0

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(n=3), grade 2 (n=6) and grade 3 (n=5) cartilage tissue as representative of non-

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degenerate, low degenerate and intermediate degenerate cartilage tissue. Cartilage

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from grade 4 severe degenerate tissue was not used in the study as the cell yield

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obtained was not sufficient.

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Isolation of human chondrocytes

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Cartilage tissue was digested in 0.25% trypsin (Sigma-Aldrich, Poole, UK) at 37°C

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for 30 minutes followed by digestion in 3 mg/ml collagenase type I (Sigma-Aldrich,

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Poole UK) in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) (Gibco,

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Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS)

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(Gibco, Paisley UK), 2mM glutamine (Gibco, Paisley, UK), 100 U/ml penicillin, 100

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µg/ml streptomycin (Gibco, Paisley UK), 2.5 µg/ml amphotericin B (Sigma-Aldrich,

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Poole, UK), and 50 µg/ml ascorbic acid (Sigma-Aldrich, Poole, UK) (complete media),

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at 37°C for 16 hours. Cells were passed through a 7 0 µM cell strainer and washed

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twice in DMEM/F12 supplemented with 2 mM glutamine, 100 U/ml penicillin, 100

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µg/ml, streptomycin, 2.5 µg/ml amphotericin B and 50 µg/ml ascorbic acid (serum

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free media). Cells were counted and the viability checked using trypan blue staining.

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Chondrocytes were cultured in monolayer in complete media in a humidified

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atmosphere of 5% CO2 at 37˚C and harvested at passage 2.

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127 Monolayer Culture

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Chondrocytes harvested at passage 2 were seeded at a cell density of 1x106

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cells/well in a 6 well plate and maintained in complete media in a humidified

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atmosphere of 5% CO2 at 37˚C for 24 hours prior to treatment to allow cells to

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adhere to the cell culture plate.

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Alginate Bead Culture

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When cultured in monolayer, chondrocytes dedifferentiate into fibroblast like cells, 28-

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native phenotype. Chondrocytes harvested at passage 2 were encapsulated in

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alginate beads at a cell density of 2x106 cells/ml, as previously described 31. Alginate

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beads were cultured in complete media in a humidified atmosphere of 5% CO2 at

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37°C and the media changed every other day. Chondro cytes were redifferentiated in

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alginate beads for 4 weeks prior to treatment.

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therefore alginate beads were used to redifferentiate the chondrocytes to their

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Cytotoxicity studies

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The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega,

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Southampton, UK) was used to determine the effects of 10µM WIN-55 on the cell

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viability of human chondrocytes according to the manufacturer’s instructions.

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Cannabinoid WIN-55 and IL-1β treatments

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Prior to stimulation complete media was removed cells washed with serum free

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media and serum free media containing 500 µg/ml bovine serum albumin (BSA)

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(Sigma-Aldrich, Poole, UK) added. Chondrocytes cultured in monolayer were

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unstimulated or stimulated with 10 ng/ml IL-1β (Peprotech, London, UK) with and

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without 1 µM, 2.5 µM, 5 µM, 7.5 µM and 10 µM WIN-55 (Sigma-Aldrich, Poole, UK)

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for 48 hours at 37°C. Chondrocytes in monolayer we re treated with 10 µM WIN-55

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for 3, 6, 24 and 48 hours at 37°C. Chondrocytes cul tured in alginate beads were

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unstimulated or stimulated with 10 ng/ml IL-1β with and without 10 µM WIN-55.

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Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Poole, UK) was used as a vehicle

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control at 0.1% (equivalent to that present in 10 µM WIN-55).

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MMP-3 and -13 Enzyme Linked Immunosorbent Assay (ELISA)

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ELISA (R&D Systems, Abingdon, UK) were used according to the manufacturer's

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instructions to measure pro and active MMP-3 protein expression (ng/ml) and pro

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MMP-13 protein expression (pg/ml) in alginate bead conditioned culture media

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following treatment with 10 µM WIN-55 with and without 10ng/ml IL-1β stimulation for

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48 hours.

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RNA extraction from monolayers

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For each of the 3 patient samples obtained from Outerbridge grade 0, 2 and 3 (n=9),

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patient samples were tested in triplicate. Following treatments, media was removed,

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cells washed in 1xPBS and RNA extracted in 1 ml of TRIzol reagent (Invitrogen,

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Paisley, UK) following the manufacturer's instructions. RNA was resuspended in 14

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µl sterile deionised water.

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RNA extraction from alginate beads

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For each of the 3 patient samples obtained from Outerbridge grade 0, 2 and 3 (n=9),

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patient samples were tested in triplicate RNA was extracted from alginate beads

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using 2 beads per extraction as described previously 31. The resulting RNA was then

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resuspended in 100µl of sterile deionised water and the RNA was purified using

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RNeasy clean up columns (Qiagen, Crawley, UK) according to the manufacturer's

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instructions and RNA eluted in 14 µl of sterile deionised water.

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Reverse transcription-real-time polymerase chain reaction (qRT-PCR)

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cDNA was formed as described previously31. Real-time PCR was used to investigate

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MMP-3, -13, TIMP-1, and -2 gene expression using Applied Biosystems Taqman

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Gene Expression Assays (Table 1). The reactions were performed for 40 cycles

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using Taqman Fast Universal PCR Master Mix (Applied Biosystems, Paisley, UK) on

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the ABI StepOnePlus real-time PCR machine (Applied Biosystems, Paisley, UK).

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The data were collected and the fold changes in gene expression analysed using the

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2-∆∆Ct method 32. Gene expression was normalised to housekeeping genes GAPDH

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and 18S together with untreated controls.

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Cannabinoid receptor expression in OA chondrocytes

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Chondrocytes isolated from 4 patient samples of grade 2-3 cartilage at passage 2

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were plated at a density of 1x105 cells in 8 well chamber slides and allowed to

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adhere overnight. Chondrocytes were fixed in 4% formalin and endogenous

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peroxidases were quenched. Following 0.01% chymotrypsin antigen retrieval at 37°C

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for 20 minutes cells were blocked in 25% v/v goat serum/BSA. Cells were incubated

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with rabbit polyclonal antibodies (Abcam) overnight at 4°C against CB1 (1/100), CB2

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(1/50), PPARα (1/150) and PPARγ (1/50). A rabbit isotype IgG antibody (Abcam)

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was used as negative control. Following washing cells were incubated with

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biotinylated goat anti-rabbit secondary antibody (1/300: Abcam) and binding

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detected using streptavidin-biotinylated horse radish peroxide complex (Vector

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Laboratories, Peterbrough, UK) with 3,3’-diaminobenzidine tetrahydrochloride

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substrate (Sigma-Aldrich). Cells were counterstained using Mayers Haematoxylin

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(Leica Microsystems, Milton Keynes, UK) dehydrated, cleared and mounted in

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Pertex (Leica Microsystems). Immunopositivity was visualised and images captured

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using

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(MediaCybernertic, Marlow, UK).

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Statistical Analysis

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Data was shown to be non-parametric via a Shapiro–Wilk test hence statistical

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significance between DMSO vehicle control and treatment groups was determined

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using the non-parametric Kruskall-Wallis multiple comparisons test and Conover-

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Inman post hoc test; p<0.05 was considered statistically significant for real-time PCR

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and ELISA analysis. All statistical analysis was performed using StatsDirect. All data

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analysis was performed using individual replicates for each treatment group.

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RESULTS

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Cytotoxicity studies

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Cytotoxicity studies showed that WIN-55 was not toxic to chondrocytes at the

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concentrations used (Data not shown).

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Time-course and concentration dependent effects of WIN-55 on MMP-3, MMP-

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13, TIMP-1 and TIMP-2 gene expression in monolayer

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Chondrocytes were treated with WIN-55 (10 µM) for 3, 6, 24 and 48 hours to

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determine the time point at which a maximum decrease in gene expression was

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observed. MMP-3, -13, TIMP-1 and -2 gene expression was decreased in a time-

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dependent manner, the largest decrease was observed at 48 hours (Supplementary

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Figure 1A-D); therefore this incubation time was used for all subsequent treatments.

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Gene expression of MMP-3, -13, TIMP-1 and -2 was decreased in a WIN-55

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concentration dependent manner both with and without IL-1β (10 ng/ml) stimulation

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(Supplementary Figure 2A-D). A significant decrease in gene expression was

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observed at concentrations ≥ 2.5 µM WIN-55 compared to DMSO (0.1%) vehicle

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controls (Supplementary Figure 2A-D). WIN-55 was used at 10 µM in all following

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treatments as this was the maximal inhibitory non-toxic concentration.

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Effects of DMSO in monolayer and alginate bead culture

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No differences in gene or protein expression were observed between untreated cells

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compared to cells treated with 0.1% DMSO and 10 ng/ml IL-1β treatment in

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combination with 0.1% DMSO compared to IL-1β treatment alone (Data not shown).

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Therefore 10 ng/ml IL-1β in combination with 0.1% DMSO and 0.1% DMSO controls

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were used for all real-time PCR and ELISA analysis.

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247 Effect of WIN-55 on IL-1β induced MMP-3 and MMP-13 gene expression in

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monolayer

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In order to determine the effects of WIN-55 on MMPs in the presence of the

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inflammatory cytokine IL-1β, real-time PCR was used to measure the gene

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expression of MMP-3 and -13 in chondrocytes obtained from macroscopically graded

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OA cartilage. For this purpose chondrocytes were stimulated with IL-1β to induce

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MMP expression and co-treated with WIN-55. The effects of WIN-55 alone were also

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investigated. IL-1β stimulation significantly induced MMP-3 gene expression in

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chondrocytes isolated from grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001)

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cartilage and MMP-13 in chondrocytes isolated from grade 0 (p<0.0001), grade 2

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(p=0.02) and grade 3 cartilage (p<0.0001) compared to DMSO vehicle control

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(Figure 1A-F). Treatment with WIN-55 in combination with IL-1β significantly reduced

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MMP-3 and -13 gene expression in chondrocytes derived from grade 0 (p<0.0001), 2

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(p<0.0001) and 3 (p<0.0001) cartilage compared to IL-1β stimulation alone (Figure

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1A-F). WIN-55 treatment in combination with IL-1β also significantly reduced MMP-3

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gene expression in chondrocytes isolated from grade 0 (p<0.0001), 2 (p<0.0001)

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and 3 (p<0.0001) cartilage and MMP-13 gene expression in chondrocytes isolated

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from grade 0 (p<0.0001), grade 2 (p=0.01) and grade 3 (p<0.0001) compared to

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DMSO vehicle control (Figure 1A-F). WIN-55 treatment alone significantly reduced

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MMP-3 gene expression in grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001)

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cartilage derived chondrocytes below basal levels compared to DMSO vehicle

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control

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13 gene expression in grade 0 (p=0.002) and 3 (p<0.0001) cartilage derived

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chondrocytes compared to DMSO control (Figure 1D, 2F). MMP-13 was only

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(Figure 1 A-C). WIN-55 treatment alone also significantly decreased MMP-

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expressed in two samples in chondrocytes isolated from grade 2 cartilage following

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WIN-55 treatment therefore statistical analysis could not be performed (Figure 1E).

274 Effects of WIN-55 on IL-1β induced MMP-3 and MMP-13 gene expression on

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chondrocytes cultured in alginate beads

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The effects of WIN-55 on IL-1β induced MMP-3 and -13 gene expression were

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determined in chondrocytes cultured in alginate beads in order to determine gene

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expression in chondrocytes following redifferentiation back to their native phenotype.

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IL-1β stimulation significantly induced MMP-3 gene expression in chondrocytes from

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grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage and MMP-13 gene

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expression in grade 0 (p=0.018), 2 (p<0.0001) and 3 (p<0.0001) cartilage derived

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chondrocytes compared to DMSO vehicle control (Figure 2A-F). Similarly WIN-55

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treatment in combination with IL-1β significantly reduced MMP-3 gene expression in

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grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage derived chondrocytes

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and MMP-13 gene expression in grade 0 (p<0.0001) and 3 (p<0.0001) cartilage

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derived chondrocytes compared to IL-1β stimulation alone (Figure 2A, 2B, 2C, 2D,

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2F). MMP-13 was not expressed in chondrocytes from grade 2 cartilage treated with

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IL-1β in combination with WIN-55 (Figure 2E). WIN-55 treatment in combination with

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IL-1β significantly reduced MMP-3 (p=0.03) and -13 (p=0.003) gene expression in

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chondrocytes from grade 0 chondrocytes compared to DMSO vehicle control (Figure

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2A-D). WIN-55 treatment in combination with IL-1β reduced MMP-3 gene expression

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in chondrocytes from grade 2 (p=0.11) cartilage and both MMP-3 (p=0.58) and -13

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(p=0.11) gene expression in chondrocytes from grade 3 cartilage compared to

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DMSO vehicle control; however this was not significant (Figure 2A, 2C, 2F). There

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was no significant difference from basal levels of MMP-3 gene expression in grade 0

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(p=0.052) and 3 (p=0.16) cartilage derived chondrocytes when treated with WIN-55

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alone (Figure 2A, 2C). MMP-3 was not expressed in grade 2 cartilage chondrocytes

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treated with WIN-55 alone (Figure 2B). MMP-13 was abolished in chondrocytes from

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grade 0, 2 and 3 cartilage treated with WIN-55 alone (Figure 2D-F).

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Effects of WIN-55 on IL-1β induced TIMP-1 and TIMP-2 gene expression in

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monolayer

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In order to determine the effects of WIN-55 on the inhibitors of MMP-3 and -13 the

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gene expression of TIMP-1 and -2 was investigated following WIN-55 treatment both

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alone and in combination with IL-1β. IL-1β stimulation had no significant effect on

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TIMP-1 gene expression in chondrocytes derived from grade 0 (p=0.75), 2 (p=0.60)

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and 3 (p=0.12) cartilage or TIMP-2 gene expression in chondrocytes isolated from

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grade 0 (p=0.77), 2 (p=0.49) and 3 (p=0.98) cartilage (Figure 3A-F). However, WIN-

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55 treatment in combination with IL-1β resulted in a significant decrease in TIMP-1

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gene expression compared to DMSO vehicle control and IL-1β stimulated

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chondrocytes derived from grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p=0.001)

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cartilage (Figure 3A-C). WIN-55 alone also significantly reduced TIMP-1 gene

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expression in chondrocytes derived from grade 0 (p<0.0001) and 2 (p<0.0001)

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cartilage below basal levels (Figure 3A, 3B). TIMP-1 gene expression was

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decreased in chondrocytes derived from grade 3 cartilage following WIN-55

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treatment however this was not significant (p=0.11) (Figure 3C). TIMP-2 gene

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expression was significantly reduced following WIN-55 treatment in combination with

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IL-1β compared to DMSO vehicle control and IL-1β stimulation in chondrocytes

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derived from grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage

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3D-F). WIN-55 alone significantly reduced the gene expression of TIMP-2 below

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322

basal levels in chondrocytes isolated from grade 0 (p<0.0001), 2 (p<0.0001) and 3

323

(p<0.0001) cartilage (Figure 3D-F).

324 Effects of WIN-55 on IL-1β induced TIMP-1 and TIMP-2 gene expression on

326

chondrocytes cultured in alginate beads

327

TIMP-1 gene expression was significantly increased in grade 2 (p<0.0009) and 3

328

cartilage derived chondrocytes (p=0.02) following IL-1β stimulation (Figure 4B-C) but

329

not in grade 0 (p=0.24) cartilage chondrocytes compared to DMSO vehicle control

330

(Figure 4A). In contrast TIMP-2 gene expression was significantly decreased

331

following IL-1β treatment in chondrocytes extracted from grade 0 (p=0.009), 2

332

(p=0.001) and 3 (p<0.0001) cartilage compared to DMSO vehicle control (Figure 4D-

333

F). WIN-55 treatment in combination with IL-1β resulted in a significant decrease in

334

TIMP-1 gene expression in chondrocytes derived from grades 0 (p<0.0001), 2

335

(p<0.0001), and 3 (p<0.0001), cartilage and TIMP-2 gene expression in

336

chondrocytes isolated from grades 0 (p<0.0001), 2 (p<0.0001), and 3 (p=0.0007),

337

cartilage compared to IL-1β stimulation (Figure 4A-F). WIN-55 treatment in

338

combination with IL-1β also resulted in a significant decrease in TIMP-1 gene

339

expression in chondrocytes derived from grades 0 (p<0.0001), 2 (p<0.0001), and 3

340

(p<0.0001), cartilage and TIMP-2 gene expression in chondrocytes isolated from

341

grades 0 (p<0.0001), 2 (p<0.0001), and 3 (p<0.0001) cartilage compared to DMSO

342

control (Figure 4A-F). WIN-55 treatment alone significantly reduced both TIMP-1

343

gene expression in chondrocytes derived from grade 0 (p=0.0005), 2 (p<0.0001) and

344

3 (p=0.0003) cartilage below basal levels (Figure 4A-C). WIN-55 treatment alone

345

significantly reduced both TIMP-2 gene expression in chondrocytes derived from

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346

grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage below basal levels

347

(Figure 4D-F).

348 The effects of WIN-55 on MMP-3 and -13 protein expression

350

In order to determine whether WIN-55 also inhibited MMP-3 and -13 expression at

351

the protein level, pro and active MMP-3 and pro MMP-13 were measured in culture

352

media using ELISA. Culture media obtained from chondrocytes isolated from grade 3

353

cartilage which had been cultured in alginate beads were used, as these

354

demonstrated clear gene expression effects. Following stimulation of chondrocytes

355

with IL-1β there was a significant increase in MMP-3 (p<0.0001) and -13 (p=0.046)

356

protein release into the media (Table 2). Treatment of chondrocytes with WIN-55 in

357

combination with IL-1β significantly reduced both MMP-3 (p=0.0007) and -13

358

(p=0.0005) protein compared to IL-1β treatment alone (Table 2). WIN-55 treatment

359

alone significantly reduced MMP-3 protein release to below basal levels (p=0.04)

360

and MMP-13 (p=0.38) protein levels remained at basal level (Table 2).

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361

Cannabinoid receptor expression in OA chondrocytes

363

To identify the expression of cannabinoid receptors CB1, CB2, PPARα and PPARγ

364

in chondrocytes cultured in monolayer immunocytochemistry was used. Here we

365

observed the expression of CB1, CB2, PPARα and γ (Figure 5) PPARα and γ

366

expression was localised to the cytoplasm and nucleus (Figure 5).

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DISCUSSION

369

Cartilage degradation is a pathological feature of both OA and RA1. The

370

inflammatory cytokine IL-1 plays a key role in cartilage destruction and stimulates

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increased production of MMPs by chondrocytes, resulting in the breakdown of

372

collagen and proteoglycan33. Here, we have shown that treatment of articular

373

chondrocytes from human OA cartilage with cannabinoid WIN-55 reduces the gene

374

and protein expression of MMP-3 and MMP-13 in the presence of IL-1β, suggesting

375

that cannabinoids may have potential in terms of arthritis therapy. In addition we

376

have shown that WIN-55 significantly reduces the gene expression of TIMP-1 and

377

TIMP-2 to below basal levels.

378

In this study we have used both monolayer and a 3D culture system for culture of

379

chondrocytes. Chondrocytes that have been isolated from articular cartilage

380

dedifferentiate in monolayer culture changing their matrix synthesis, with a decrease

381

in type II collagen and aggrecan, developing a fibroblast like phenotype and an

382

increase in collagen type I28-30. Dedifferentiation can be reversed with the key

383

phenotypic features of chondrocytes being preserved when cultured in a 3D system

384

such as alginate beads34. In this study, chondrocytes were treated with IL-1β to

385

mimic inflammatory processes in an in vitro model of OA 35.

386

Synthetic cannabinoids WIN-55 and HU-210 reduce IL-1α induced proteoglycan and

387

collagen degradation in bovine nasal cartilage tissue suggesting a chondroprotective

388

effect of these compounds 9. Here, we have shown a possible mechanism by which

389

WIN-55 may prevent IL-1β induced ECM breakdown in OA cartilage tissue via

390

preventing the expression of MMPs. We have demonstrated that chondrocytes from

391

different grades of OA cartilage modulate MMP-3 and MMP-13 expression in

392

response to WIN-55 with and without IL-1β stimulation. These findings together with

393

others, suggest that cannabinoids may be of importance in the treatment of arthritis 3-

394

5, 7, 8

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. Selvi et al 2008 demonstrated that WIN-55 and CP55,940 inhibit IL-1β induced

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395

secretion of IL-6 and IL-8 in RA fibroblast like synovial cells, suggesting an anti-

396

inflammatory

397

cannabinoid ajulemic acid (AJA) reduced MMP-1, MMP-3 and MMP-9 release from

398

fibroblast like synovial cells stimulated with IL-1α and TNFα 8. In vivo, AJA has also

399

been shown to reduce the severity of adjuvant-induced arthritis

400

psychoactive cannabinoids, cannabidiol (CBD) and HU-320 reduced inflammation

401

and joint damage in murine collagen-induced arthritis 3, 5.

402

The effects of WIN-55 on articular chondrocytes did not appear to be influenced by

403

the grade of the cartilage they were isolated from when cultured in monolayer.

404

Chondrocytes cultured in monolayer express MMP-3 and MMP-13 at very low levels

405

following WIN-55 treatment. However when comparing the effects of WIN-55 in

406

monolayer to alginate bead culture, a greater inhibitory effect on MMP-3 and MMP-

407

13 gene expression was seen in alginate bead cultured chondrocytes. Interestingly a

408

biphasic expression pattern of MMP-3 and MMP-13 in response to WIN-55 was

409

observed. MMP-3 was expressed in grade 0 and grade 3 cartilage derived

410

chondrocytes and abolished in grade 2 cartilage chondrocytes and MMP-13 was

411

abolished in chondrocytes from all grades of cartilage when cultured in alginate

412

beads. These varying responses to WIN-55 treatment in alginate bead culture may

413

indicate that the expression of MMPs may be differentially regulated depending on

414

the grade and extent of cartilage degradation and the culture method utilised.

415

Studies have shown that cartilage tissue derived from different OA grades or normal

416

aged cartilage may influence the response of the chondrocytes to different

417

treatments40-42. Interestingly biphasic effects have also been seen with other

418

cannabinoids namely AJA 43.

cannabinoids7.

In

another

study

non-psychoactive

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and other non-

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During OA there is thought to be an imbalance between MMP and TIMP expression

420

which in part contributes to cartilage breakdown21. We have shown that WIN-55

421

inhibits expression of both destructive MMPs and protective TIMPs involved in the

422

pathogenesis of OA, indicating that inhibition may occur via a signalling pathway

423

which regulates both at the transcription level. Human MMPs and TIMPs share a

424

common activating protein 1 (AP-1) site in their promoters which regulates their

425

transcription44. WIN-55 may have a differential effect on AP-1 activation via

426

peroxisome proliferator activated receptors (PPARs). WIN-55 has been shown to

427

activate AP-1 via PPARα. In addition AP-1 may be involved in the activation of

428

interferon β (IFNβ) 45. Production of IFNβ may result in reduced levels of MMPs and

429

TIMPs. IFNβ reduced MMP-1, -3 and TIMP-1 in fibroblast-like synovial cells both

430

with and without IL-1β stimulation and synovial tissue from patients with RA, treated

431

with IFNβ, showed reduced levels of MMP-1 and TIMP-146. Furthermore IFNβ has

432

been shown to have anti-inflammatory properties in the treatment of arthritis47-49.

433

Conversely PPARγ agonists have been shown to reduce IL-1β induced MMP-1

434

expression in human synovial fibroblasts via inhibiting DNA binding of AP-150. WIN-

435

55 also binds to PPARγ so could also act in this way51. The biological activities of

436

cannabinoids and the signal transduction pathways they induce or inhibit need to be

437

further investigated in OA. Since TIMP-1 and -2 are decreased by WIN-55 in human

438

OA chondrocytes it is unclear whether there is a change in MMP and TIMP balance

439

following cannabinoid treatment. However the inhibitory effect of WIN-55 on MMP-3

440

and -13 expression would indicate a possible role of cannabinoids in supressing IL-

441

1β induced ECM degradation by MMPs.

442

We have observed the expression of both classical cannabinoid receptors CB1 and

443

CB2 in human chondrocytes from OA cartilage at passage 2. CB1 and CB2 have

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previously been shown to be expressed at similar levels both at the protein and RNA

445

level in synovia of patients with OA and RA and their expression is thought to play a

446

role in the pathology of joint disease52. PPARα and γ were also expressed in OA

447

chondrocytes at passage 2 and their expression appears to be both cytoplasmic and

448

nucleus. PPARs are nuclear receptors however studies have also shown that the

449

localisation of PPARα receptors in chondrocytes is also cytoplasmic

450

activates CB1and CB2 with Kis of 1.89-123 nM and 0.28-16.2 nM respectively 24. In

451

addition, WIN-55 has also been shown to activate PPARα and γ25,

452

PPARs using both specific and cannabinoid agonists for the treatment of OA and RA

453

has been reported previously8, 26, 36, 50, 54-56. The observed effects of WIN-55 on MMP

454

and TIMP expression may be mediated by one or more of these receptors or by

455

receptors as yet unknown.

456

In conclusion, in human OA chondrocytes, the synthetic cannabinoid WIN-55 inhibits

457

the expression of matrix degrading enzymes MMP-3 and -13 and their inhibitors

458

TIMP-1 and -2 in the presence or absence of IL-1β. This suggests a possible

459

mechanism by which cannabinoids may act to prevent ECM breakdown in arthritis.

460

Cannabinoids could provide a dual role in the treatment of arthritis as disease

461

modifying agents in addition to having anti-inflammatory properties10. Although there

462

is increasing evidence to suggest that cannabinoids may be of therapeutic value in

463

the treatment of arthritis, further studies into the receptors and signalling pathways

464

involved in the actions of cannabinoids need to be investigated in order to elucidate

465

their effects on catabolic and anabolic mediators in both normal and arthritic cartilage.

466 467

53

. WIN-55

26

. Targeting

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Acknowledgements:

469

This work was funded by a PhD Studentship from the Biomedical Research Centre,

470

Faculty of Health and Well Being, Sheffield Hallam University, Sheffield, UK. Tissue

471

samples were supplied by the Sheffield Biorepository.

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472 Authors contributions:

474

SD participated in its design, performed all the laboratory work and analysis and co-

475

wrote the manuscript. JMW helped to conceive the study, secure funding,

476

contributed to its design and co-ordination, participated in interpretation of data and

477

co-wrote the manuscript. AC helped to conceive the study, secure funding,

478

contributed to its design and co-ordination and co-wrote the manuscript. CLM helped

479

to conceive the study, secure funding, contributed to its design and co-ordination,

480

participated in interpretation of data and co-wrote the manuscript. RAB helped to

481

conceive the study, secure funding, contributed to its design and co-ordination,

482

participated in interpretation of data and co-wrote the manuscript. All authors read

483

and approved the final manuscript.

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484

Competing Interest

486

Competing Interest: None Declared.

487

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488

References

489

1. Goldring MB and Marcu KB. Cartilage homeostasis in health and rheumatic

490

diseases. Arthritis Res.Ther. 2009;11:224. doi: 10.1186/ar2592.

ACCEPTED MANUSCRIPT

2. Blake DR, Robson P, Ho M, Jubb RW, McCabe CS. Preliminary assessment of

492

the efficacy, tolerability and safety of a cannabis-based medicine (Sativex) in the

493

treatment of pain caused by rheumatoid arthritis. Rheumatology (Oxford)

494

2006;45:50-2. doi: 10.1093/rheumatology/kei183.

495

3. Sumariwalla PF, Gallily R, Tchilibon S, Fride E, Mechoulam R, Feldmann M. A

496

novel synthetic, nonpsychoactive cannabinoid acid (HU-320) with antiinflammatory

497

properties in murine collagen-induced arthritis. Arthritis Rheum. 2004;50:985-98. doi:

498

10.1002/art.20050.

499

4. Zurier RB, Rossetti RG, Lane JH, Goldberg JM, Hunter SA, Burstein SH.

500

Dimethylheptyl-THC-11 oic acid: a nonpsychoactive antiinflammatory agent with a

501

cannabinoid template structure. Arthritis Rheum. 1998;41:163-70. doi: 2-9.

502

5. Malfait AM, Gallily R, Sumariwalla PF, Malik AS, Andreakos E, Mechoulam R, et

503

al. The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic

504

therapeutic in murine collagen-induced arthritis. Proc.Natl.Acad.Sci.U.S.A.

505

2000;97:9561-6. doi: 10.1073/pnas.160105897.

506

6. Zurier RB, Rossetti RG, Burstein SH, Bidinger B. Suppression of human monocyte

507

interleukin-1beta production by ajulemic acid, a nonpsychoactive cannabinoid.

508

Biochem.Pharmacol. 2003;65:649-55.

509

7. Selvi E, Lorenzini S, Garcia-Gonzalez E, Maggio R, Lazzerini PE, Capecchi PL, et

510

al. Inhibitory effect of synthetic cannabinoids on cytokine production in rheumatoid

511

fibroblast-like synoviocytes. Clin.Exp.Rheumatol. 2008;26:574-81.

AC C

EP

TE D

M AN U

SC

RI PT

491

ACCEPTED MANUSCRIPT

8. Johnson DR, Stebulis JA, Rossetti RG, Burstein SH, Zurier RB. Suppression of

513

fibroblast metalloproteinases by ajulemic acid, a nonpsychoactive cannabinoid acid.

514

J.Cell.Biochem. 2007;100:184-90. doi: 10.1002/jcb.21046.

515

9. Mbvundula EC, Bunning RA, Rainsford KD. Arthritis and cannabinoids: HU-210

516

and Win-55,212-2 prevent IL-1alpha-induced matrix degradation in bovine articular

517

chondrocytes in-vitro. J.Pharm.Pharmacol. 2006;58:351-8. doi:

518

10.1211/jpp.58.3.0009.

519

10. Dunn SL, Wilkinson JM, Crawford A, Le Maitre CL, Bunning RA. Cannabinoids:

520

novel therapies for arthritis? Future Med.Chem. 2012;4:713-25. doi:

521

10.4155/fmc.12.20.

522

11. Goldring MB and Otero M. Inflammation in osteoarthritis. Curr.Opin.Rheumatol.

523

2011;23:471-8. doi: 10.1097/BOR.0b013e328349c2b1.

524

12. Davidson RK, Waters JG, Kevorkian L, Darrah C, Cooper A, Donell ST, et al.

525

Expression profiling of metalloproteinases and their inhibitors in synovium and

526

cartilage. Arthritis Res.Ther. 2006;8:R124. doi: 10.1186/ar2013.

527

13. Bau B, Gebhard PM, Haag J, Knorr T, Bartnik E, Aigner T. Relative messenger

528

RNA expression profiling of collagenases and aggrecanases in human articular

529

chondrocytes in vivo and in vitro. Arthritis Rheum. 2002;46:2648-57. doi:

530

10.1002/art.10531.

531

14. Hembry RM, Bagga MR, Reynolds JJ, Hamblen DL. Immunolocalisation studies

532

on six matrix metalloproteinases and their inhibitors, TIMP-1 and TIMP-2, in synovia

533

from patients with osteo- and rheumatoid arthritis. Ann.Rheum.Dis. 1995;54:25-32.

AC C

EP

TE D

M AN U

SC

RI PT

512

ACCEPTED MANUSCRIPT

15. Okada Y, Shinmei M, Tanaka O, Naka K, Kimura A, Nakanishi I, et al.

535

Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and

536

synovium. Lab.Invest. 1992;66:680-90.

537

16. Wolfe GC, MacNaul KL, Buechel FF, McDonnell J, Hoerrner LA, Lark MW, et al.

538

Differential in vivo expression of collagenase messenger RNA in synovium and

539

cartilage. Quantitative comparison with stromelysin messenger RNA levels in human

540

rheumatoid arthritis and osteoarthritis patients and in two animal models of acute

541

inflammatory arthritis. Arthritis Rheum. 1993;36:1540-7.

542

17. Chubinskaya S, Kuettner KE, Cole AA. Expression of matrix metalloproteinases

543

in normal and damaged articular cartilage from human knee and ankle joints.

544

Lab.Invest. 1999;79:1669-77.

545

18. Yoshihara Y, Nakamura H, Obata K, Yamada H, Hayakawa T, Fujikawa K, et al.

546

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial

547

fluids from patients with rheumatoid arthritis or osteoarthritis. Ann.Rheum.Dis.

548

2000;59:455-61.

549

19. Koshy PJ, Lundy CJ, Rowan AD, Porter S, Edwards DR, Hogan A, et al. The

550

modulation of matrix metalloproteinase and ADAM gene expression in human

551

chondrocytes by interleukin-1 and oncostatin M: a time-course study using real-time

552

quantitative reverse transcription-polymerase chain reaction. Arthritis Rheum.

553

2002;46:961-7.

554

20. Tetlow LC, Adlam DJ, Woolley DE. Matrix metalloproteinase and

555

proinflammatory cytokine production by chondrocytes of human osteoarthritic

AC C

EP

TE D

M AN U

SC

RI PT

534

ACCEPTED MANUSCRIPT

cartilage: associations with degenerative changes. Arthritis Rheum. 2001;44:585-94.

557

doi: 2-C.

558

21. Dean DD, Martel-Pelletier J, Pelletier JP, Howell DS, Woessner JF,Jr. Evidence

559

for metalloproteinase and metalloproteinase inhibitor imbalance in human

560

osteoarthritic cartilage. J.Clin.Invest. 1989;84:678-85. doi: 10.1172/JCI114215.

561

22. Martel-Pelletier J, McCollum R, Fujimoto N, Obata K, Cloutier JM, Pelletier JP.

562

Excess of metalloproteases over tissue inhibitor of metalloprotease may contribute to

563

cartilage degradation in osteoarthritis and rheumatoid arthritis. Lab.Invest.

564

1994;70:807-15.

565

23. Murphy G and Nagase H. Reappraising metalloproteinases in rheumatoid

566

arthritis and osteoarthritis: destruction or repair? Nat.Clin.Pract.Rheumatol.

567

2008;4:128-35. doi: 10.1038/ncprheum0727.

568

24. Pertwee RG, Howlett AC, Abood ME, Alexander SP, Di Marzo V, Elphick MR, et

569

al. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid

570

receptors and their ligands: beyond CB and CB. Pharmacol.Rev. 2010;62:588-631.

571

doi: 10.1124/pr.110.003004.

572

25. Sun Y, Alexander SP, Kendall DA, Bennett AJ. Cannabinoids and PPARalpha

573

signalling. Biochem.Soc.Trans. 2006;34:1095-7. doi: 10.1042/BST0341095.

574

26. O'Sullivan SE and Kendall DA. Cannabinoid activation of peroxisome proliferator-

575

activated receptors: potential for modulation of inflammatory disease. Immunobiology

576

2010;215:611-6. doi: 10.1016/j.imbio.2009.09.007.

AC C

EP

TE D

M AN U

SC

RI PT

556

ACCEPTED MANUSCRIPT

27. Cameron ML, Briggs KK, Steadman JR. Reproducibility and reliability of the

578

outerbridge classification for grading chondral lesions of the knee arthroscopically.

579

Am.J.Sports Med. 2003;31:83-6.

580

28. Mayne R, Vail MS, Mayne PM, Miller EJ. Changes in type of collagen

581

synthesized as clones of chick chondrocytes grow and eventually lose division

582

capacity. Proc.Natl.Acad.Sci.U.S.A. 1976;73:1674-8.

583

29. von der Mark K, Gauss V, von der Mark H, Muller P. Relationship between cell

584

shape and type of collagen synthesised as chondrocytes lose their cartilage

585

phenotype in culture. Nature 1977;267:531-2.

586

30. Benya PD, Padilla SR, Nimni ME. Independent regulation of collagen types by

587

chondrocytes during the loss of differentiated function in culture. Cell 1978;15:1313-

588

21.

589

31. Le Maitre CL, Freemont AJ, Hoyland JA. The role of interleukin-1 in the

590

pathogenesis of human intervertebral disc degeneration. Arthritis Res.Ther.

591

2005;7:R732-45. doi: 10.1186/ar1732.

592

32. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using

593

real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods

594

2001;25:402-8. doi: 10.1006/meth.2001.1262.

595

33. Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: role in arthritis.

596

Front.Biosci. 2006;11:529-43.

597

34. Caron MM, Emans PJ, Coolsen MM, Voss L, Surtel DA, Cremers A, et al.

598

Redifferentiation of dedifferentiated human articular chondrocytes: comparison of 2D

AC C

EP

TE D

M AN U

SC

RI PT

577

ACCEPTED MANUSCRIPT

and 3D cultures. Osteoarthritis Cartilage 2012;20:1170-8. doi:

600

10.1016/j.joca.2012.06.016.

601

35. Goldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum.

602

2000;43:1916-26. doi: 2-I.

603

36. Clockaerts S, Bastiaansen-Jenniskens YM, Feijt C, Verhaar JA, Somville J, De

604

Clerck LS, et al. Peroxisome proliferator activated receptor alpha activation

605

decreases inflammatory and destructive responses in osteoarthritic cartilage.

606

Osteoarthritis Cartilage 2011;19:895-902. doi: 10.1016/j.joca.2011.03.010.

607

37. Freemont AJ, Hampson V, Tilman R, Goupille P, Taiwo Y, Hoyland JA. Gene

608

expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic

609

human knee articular cartilage is zone and grade specific. Ann.Rheum.Dis.

610

1997;56:542-9.

611

38. Vincenti MP. The matrix metalloproteinase (MMP) and tissue inhibitor of

612

metalloproteinase (TIMP) genes. Transcriptional and posttranscriptional regulation,

613

signal transduction and cell-type-specific expression. Methods Mol.Biol.

614

2001;151:121-48.

615

39. Page CE, Smale S, Carty SM, Amos N, Lauder SN, Goodfellow RM, et al.

616

Interferon-gamma inhibits interleukin-1beta-induced matrix metalloproteinase

617

production by synovial fibroblasts and protects articular cartilage in early arthritis.

618

Arthritis Res.Ther. 2010;12:R49. doi: 10.1186/ar2960; 10.1186/ar2960.

AC C

EP

TE D

M AN U

SC

RI PT

599

ACCEPTED MANUSCRIPT

40. Dozin B, Malpeli M, Camardella L, Cancedda R, Pietrangelo A. Response of

620

young, aged and osteoarthritic human articular chondrocytes to inflammatory

621

cytokines: molecular and cellular aspects. Matrix Biol. 2002;21:449-59.

622

41. Hickery MS, Bayliss MT, Dudhia J, Lewthwaite JC, Edwards JC, Pitsillides AA.

623

Age-related changes in the response of human articular cartilage to IL-1alpha and

624

transforming growth factor-beta (TGF-beta): chondrocytes exhibit a diminished

625

sensitivity to TGF-beta. J.Biol.Chem. 2003;278:53063-71. doi:

626

10.1074/jbc.M209632200.

627

42. Fan Z, Bau B, Yang H, Soeder S, Aigner T. Freshly isolated osteoarthritic

628

chondrocytes are catabolically more active than normal chondrocytes, but less

629

responsive to catabolic stimulation with interleukin-1beta. Arthritis Rheum.

630

2005;52:136-43. doi: 10.1002/art.20725.

631

43. Burstein S. PPAR-gamma: a nuclear receptor with affinity for cannabinoids. Life

632

Sci. 2005;77:1674-84. doi: 10.1016/j.lfs.2005.05.039.

633

44. Borden P and Heller RA. Transcriptional control of matrix metalloproteinases and

634

the tissue inhibitors of matrix metalloproteinases. Crit.Rev.Eukaryot.Gene Expr.

635

1997;7:159-78.

636

45. Downer EJ, Clifford E, Amu S, Fallon PG, Moynagh PN. The Synthetic

637

Cannabinoid R(+)WIN55,212-2 Augments Interferon-beta Expression via

638

Peroxisome Proliferator-activated Receptor-alpha. J.Biol.Chem. 2012;287:25440-53.

639

doi: 10.1074/jbc.M112.371757.

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EP

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46. Smeets TJ, Dayer JM, Kraan MC, Versendaal J, Chicheportiche R, Breedveld

641

FC, et al. The effects of interferon-beta treatment of synovial inflammation and

642

expression of metalloproteinases in patients with rheumatoid arthritis. Arthritis

643

Rheum. 2000;43:270-4. doi: 2-H.

644

47. Tak PP, Hart BA, Kraan MC, Jonker M, Smeets TJ, Breedveld FC. The effects of

645

interferon beta treatment on arthritis. Rheumatology (Oxford) 1999;38:362-9.

646

48. van Holten J, Plater-Zyberk C, Tak PP. Interferon-beta for treatment of

647

rheumatoid arthritis? Arthritis Res. 2002;4:346-52.

648

49. van Holten J, Reedquist K, Sattonet-Roche P, Smeets TJ, Plater-Zyberk C,

649

Vervoordeldonk MJ, et al. Treatment with recombinant interferon-beta reduces

650

inflammation and slows cartilage destruction in the collagen-induced arthritis model

651

of rheumatoid arthritis. Arthritis Res.Ther. 2004;6:R239-49. doi: 10.1186/ar1165.

652

50. Fahmi H, Pelletier JP, Di Battista JA, Cheung HS, Fernandes JC, Martel-Pelletier

653

J. Peroxisome proliferator-activated receptor gamma activators inhibit MMP-1

654

production in human synovial fibroblasts likely by reducing the binding of the

655

activator protein 1. Osteoarthritis Cartilage 2002;10:100-8. doi:

656

10.1053/joca.2001.0485.

657

51. O'Sullivan SE. Cannabinoids go nuclear: evidence for activation of peroxisome

658

proliferator-activated receptors. Br.J.Pharmacol. 2007;152:576-82. doi:

659

10.1038/sj.bjp.0707423.

660

52. Richardson D, Pearson RG, Kurian N, Latif ML, Garle MJ, Barrett DA, et al.

661

Characterisation of the cannabinoid receptor system in synovial tissue and fluid in

AC C

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640

ACCEPTED MANUSCRIPT

patients with osteoarthritis and rheumatoid arthritis. Arthritis Res.Ther. 2008;10:R43.

663

doi: 10.1186/ar2401.

664

53. Bordji K, Grillasca JP, Gouze JN, Magdalou J, Schohn H, Keller JM, et al.

665

Evidence for the presence of peroxisome proliferator-activated receptor (PPAR)

666

alpha and gamma and retinoid Z receptor in cartilage. PPARgamma activation

667

modulates the effects of interleukin-1beta on rat chondrocytes. J.Biol.Chem.

668

2000;275:12243-50.

669

54. Fahmi H, Martel-Pelletier J, Pelletier JP, Kapoor M. Peroxisome proliferator-

670

activated receptor gamma in osteoarthritis. Mod.Rheumatol. 2011;21:1-9. doi:

671

10.1007/s10165-010-0347-x.

672

55. Giaginis C, Giagini A, Theocharis S. Peroxisome proliferator-activated receptor-

673

gamma (PPAR-gamma) ligands as potential therapeutic agents to treat arthritis.

674

Pharmacol.Res. 2009;60:160-9. doi: 10.1016/j.phrs.2009.02.005;

675

10.1016/j.phrs.2009.02.005.

676

56. Fahmi H, Di Battista JA, Pelletier JP, Mineau F, Ranger P, Martel-Pelletier J.

677

Peroxisome proliferator--activated receptor gamma activators inhibit interleukin-

678

1beta-induced nitric oxide and matrix metalloproteinase 13 production in human

679

chondrocytes. Arthritis Rheum. 2001;44:595-607. doi: 2-8.

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681

Figure Legends

682

Figure 1: A-C. The effect of WIN-55 on IL-1β induced MMP-3 gene expression in

683

human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in

684

monolayer. D-F The effect of WIN-55 on IL-1β induced MMP-13 gene expression in

ACCEPTED MANUSCRIPT

human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in

686

monolayer. Each point represents the average of the repeats per patient. IL-1β (10

687

ng/ml) induced the gene expression of MMP-3 and -13. Following WIN-55 (10 µM)

688

treatment in combination with IL-1β (10 ng/ml) MMP-3 and -13 gene expression was

689

decreased. WIN-55 (10 µM) alone decreased MMP-3 and -13 gene expression.

690

***p<0.001, **p<0.01 compared to DMSO (0.1 %) vehicle control and

691

compared to IL-1β (10 ng/ml) treatment. N=9 samples for each treatment group,

692

obtained from 3 separate patient samples for each grade of tissue (0, 2 and 3) tested

693

in triplicate.

694

Figure 2: A-C. The effect of WIN-55 on IL-1β induced MMP-3 gene expression in

695

human chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate

696

beads. D-F. The effect of WIN-55 on IL-1β induced MMP-13 gene expression in

697

human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in

698

alginate beads. Each point represents the average of the repeats per patient. IL-1β

699

(10 ng/ml) induced the gene expression of MMP-3 and -13, following WIN-55 (10 µM)

700

treatment in combination with IL-1β (10 ng/ml) MMP-3 and -13 gene expression was

701

decreased or abolished. WIN-55 (10 µM) alone decreased or abolished MMP-3 and -

702

13 gene expression. *p<0.05, *p<0.01, ***p<0.001 compared to DMSO vehicle

703

control and

704

treatment group, obtained from 3 separate patient samples for each grade of tissue

705

(0, 2 and 3) tested in triplicate.

706

Figure 3: A-C The effect of WIN-55 on TIMP-1 gene expression in human OA

707

chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. D-F.

708

The effect of WIN-55 on TIMP-2 gene expression in human OA chondrocytes

709

isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. Each point

RI PT

685

+++

+++

EP

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M AN U

SC

p<0.001

AC C

p<0.001 compared to IL-1β treatment. N=9 samples for each

ACCEPTED MANUSCRIPT

710

represents the average of the repeats per patient. IL-1β (10 ng/ml) had no effect on

711

TIMP-1 and -2 gene expression. WIN-55 treatment (10 µM) both alone and in

712

combination with IL-1β (10 ng/ml) reduced TIMP-1 and -2 gene expression.

713

***p<0.001 compared to DMSO vehicle control and

714

treatment. N=9 samples for each treatment group, obtained from 3 separate patient

715

samples for each grade of tissue (0, 2 and 3) tested in triplicate.

716

Figure 4: A-C. The effect of WIN-55 on TIMP-1 gene expression in human OA

717

chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads.

718

D-F. The effect of WIN-55 on TIMP-2 gene expression in OA human chondrocytes

719

isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads. Each point

720

represents the average of the repeats per patient.

721

increased TIMP-1 gene expression and decreased TIMP-2 gene expression below

722

basal levels. WIN-55 treatment (10 µM) both alone and in combination with IL-1β (10

723

ng/ml) reduced TIMP-1 and -2 gene expression. *p<0.05, **p<0.01, ***p<0.001

724

compared to DMSO vehicle control and

725

N=9 samples for each treatment group, obtained from 3 separate patient samples for

726

each grade of tissue (0, 2 and 3) tested in triplicate.

727

Figure 5: Cannabinoid receptor expression in OA chondrocytes cultured in

728

monolayer. Representative photomicrographs of immunocytochemistry of (A) CB1

729

(B) CB2 (C) PPARα (D) PPARγ (E) IgG control (10 µg) (n=4 cultures).

730

Supplementary Figures

731

Figure 1: A-B The time dependent effect of 10 µM WIN-55 on MMP-3 and -13 gene

732

expression in chondrocytes isolated from grade 0 cartilage and cultured in

733

monolayer. C-D. The time dependant effect of 10 µM WIN-55 on TIMP-1 and -2 gene

RI PT

p<0.001 compared to IL-1β

+++

IL-1β (10 ng/ml) stimulation

p<0.001 compared to IL-1β treatment.

AC C

EP

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+++

ACCEPTED MANUSCRIPT

expression in grade 0 human chondrocytes cultured in monolayer. Each point

735

represents each treatment replicates in culture. WIN-55 (10 µM) decreases the gene

736

expression of MMP-3, -13, TIMP-1 and -2 in a time dependant manner, with maximal

737

decrease obtained following 48 hours treatment. N=3 samples for each treatment

738

group obtained from 1 patient sample 1 and tested in triplicate.

SC

739

RI PT

734

Figure 2: A-B. The concentration dependent effect of WIN-55 on IL-1β induced

741

MMP-3 and -13 gene expression in chondrocytes isolated from grade 0 cartilage and

742

cultured in monolayer. C-D. The concentration dependent effect of WIN-55 on IL-1β

743

induced TIMP-1 and -2 gene expression in grade 0 human chondrocytes cultured in

744

monolayer. Each point represents each treatment replicates in culture. IL-1β (10

745

ng/ml) induces the gene expression of MMP-3 and -13 and has no effect on TIMP-1

746

and -2. MMP-3, -13, TIMP-1 and -2 gene expression is decreased in a WIN-55

747

concentration dependent manner both alone and in combination with IL-1β (10

748

ng/ml). N=3 samples for each treatment group obtained from 1 patient sample and

749

tested in triplicate.

751

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EP AC C

750

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ACCEPTED MANUSCRIPT

Table 1:Taqman Gene Expression Assay IDs Assay ID

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Hs9999905_m1

18S

Hs99999901_s1

MMP-3

Hs00968305_m1

MMP-13

Hs00233992_m1

TIMP-1

Hs00171558_m1

TIMP-2

Hs00234278_m1

AC C

EP

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Taqman Gene Expression Assay

ACCEPTED MANUSCRIPT

Table 1: The effect of WIN-55 on pro and active MMP-3 and pro-MMP-13 protein release into culture media from grade 3 OA chondrocytes cultured in alginate beads. MMP-3 (ng/alginate bead) 1.4 (0.0659)

MMP-13 (pg/alginate bead)

IL-1β (10 ng/ml)+DMSO (0.1%)

38.4 *** (0.5092)

1092.0* (16.8345)

WIN-55 (10 µM)+IL-1β (10 ng/ml)

1.4+++ (0.0178)

219.4+++ (1.2038)

WIN-55 (10 µM)

0.2* (0.0051)

RI PT

327.5 (1.6407)

M AN U

DMSO (0.1%)

SC

Treatment (48 hours)

270.7 (3.8866)

AC C

EP

TE D

Results are expressed as mean and 95% confidence interval; *p<0.05, ***p<0.001,compared to DMSO vehicle control and +++p<0.001 compared to IL-1β treatment alone.

Treatment (48 hours)

5

M

)+

M

)

l)

0.1

(1 0

ng /m

100

5



-5 5 (1 0 M )+

W IN

.1 %

)

)

l) M

ng /m

(0 .1 % )

(0

(1 0



-5 5



M SO



D

IL -1

l)+

SC

Treatment (48 hours)

IN

W

-5

5

(1 0



)+

W







M SO

ng /m IN

-5 5

(1 0

M

)

l)

(0 .1 % )

)

ol .1 %

on tr (0

C M SO l)+ D IL -1

ng /m M



D

RI PT

Treatment (48 hours)

-5



(0 .1 % )

)

ng /m



0.001

Relative Gene Expression (Log Scale) 0.1

IN



.1 %



IL -1

SO ng (0 /m .1 l)+ % (1 D ) 0 M M SO )+ (0 .1 IL % -1 )    ng /m W l) IN -5 5 (1 0 M )



1000

W

IL -1

(0

F

M SO

IN

M SO

1000

l)+ D

W

D

E

(1 0

0.001

-5

***

M SO

0.1

Patient 7 Patient 8 Patient 9

ng /m

***



0.01

Patient 4 Patient 5 Patient 6



***

IN

+++ *** ol

Grade 2 MMP-3

Relative Gene Expression (Log Scale)

5

D

W

0.0001

ol

10

on tr

***

C

+++ ***

M AN U

)

l)

M

ng /m

-5

0.0001

D

)

W IN

***



.1 %

(0 .1 % )

(0

(1 0



5



-5



M SO

IN

W

IL -1

D

ol

0.001

IL -1

)+

l)+

on tr

0.01

on tr

M

ng /m

M SO

on tr ol

+++ ***

0.0001

Relative Gene Expression (Log Scale)

Treatment (48 hours)

TE D

(1 0

D

C

10

Patient 1 Patient 2 Patient 3

C

M )

Grade 3 MMP 3

EP

5



D M

100



0

(1

1

l)

)

-5



1.010 4 1.010 3 1.010 2 1.010 1 1.010 0 1.010 - 1 1.010 - 2 1.010 - 3 1.010 - 4 1.010 - 5 1.010 - 6

5

ng /m

IN





1

-5



W



0.1

W IN



100



1000

-1

.1 %

(0

)

.1 %

(0

on tr ol

AC C

SO

SO

M

l)+ D

D M

C

IL

ng /m

M )+



IL -1

1000

***



0



C

B

(1



IL -1

Grade 0 MMP-3

IL -1

5

-1

C

Relative Gene Expression (Log Scale)

A

W IN -5

IL

Relative Gene Expression (Log Scale)

Relative Gene Expression (Log Scale)

ACCEPTED MANUSCRIPT Grade 0 MMP-13

100

***

10 1

Patient 1 Patient 2 Patient 3

+++ ***

0.01

***

Grade 2 MMP-13 *

100

10

+++

1

**

Treatment (48 hours)

Patient 4 Patient 5 Patient 6

0.01

0.1

0.001

Treatment (48 hours)

Grade 3 MMP-13

10

***

1

+++ *** Patient 7 Patient 8 Patient 9

0.001

0.01

***

1

Treatment (48 hours)

ng

 

IN -5 5

W

IL -1 ng

)

M )

/m l)

(0 .1 %

(1 0



M SO

(0 .1 % )

Grade 2 MMP-13

SC

Treatment (48 hours)

IN -5 5

 (1 0



ng

W

IL -1







M SO

IN -5 5

)

)

ng

(0 .1 %

(1 0

Treatment (48 hours)

M )

/m l)

)

)

on tr ol

M )

/m l)

(0 .1 %

C M SO /m l)+ D M )+



D

ng

(1 0



.1 %

(0 .1 %

(0

RI PT

W

1



IN -5 5



M SO

on tr ol

Relative Gene Expression (Log Scale) 0.1

M )

/m l)

)

1

(1 0



100

IN -5 5



M )+

/m l)+ D

IL -

W

IL -1

/m l)+ D

M SO

C

10

W



F

(0 .1 %

(1 0

ng

100

IL -1

IN -5 5



Treatment (48 hours)

M SO

.1 % )

(0

W

ng

M )+



D

D

M )+

0.001

/m l)+ D

0.01

ng

0.1

M SO

E

(1 0

+++

IN -5 5

1

Patient 7 Patient 8 Patient 9

M SO

***



0.01

on tr ol

0.1



+++

D

1

C

10

Patient 4 Patient 5 Patient 6



D

100



Grade 2 MMP-3

0.01

Relative Gene Expression (Log Scale)



(1 0



0.01

1

***

M AN U

IN -5 5

1

0.1

IL -

/m l)

)

M )

ng

(1 0



W

IL -

1

on tr ol



(0 .1 %

.1 % )

(0

on tr ol

Patient 1 Patient 2 Patient 3

0.001

Relative Gene Expression (Log Scale)

Treatment (48 hours)

TE D



M SO

IN -5 5

W

IL -1

/m l)+ D

M )+

ng

M SO

+++ *

C

Grade 3 MMP-3

EP

100

/m l)

1000

M )

ng

)

(1 0



C

10

(1 0



(0 .1 %

)

.1 %

(0

 D

100





M SO



10

AC C 

1000

W IN -5 5

IL -1

/m l)+ D

1

W IN -5 5

IL -

1000

***

1

ng

M SO

C

M )+



D

on tr ol

Relative Gene Expression (Log Scale)

Grade 0 MMP-3

W



C

B

(1 0



Relative Gene Expression (Log Scale)

A

IL -

IN -5 5

W

IL -

Relative Gene Expression (Log Scale)

ACCEPTED MANUSCRIPT Grade 0 MMP-13 ***

1

+++ ** Patient 1 Patient 2 Patient 3

0.01

0.001

10

***

1

Patient 4 Patient 5 Patient 6

0.1

Treatment (48 hours)

Grade 3 MMP-13

10

***

+++ Patient 7 Patient 8 Patient 9

0.1

0.01

Grade 3 TIMP-1

1

Treatment (48 hours) Patient 7 Patient 8 Patient 9 W IN -5 5

SO ng (0 /m .1 l ) % +D (1 ) 0 M M SO )+ (0 .1 IL % -1 )    ng /m W l) IN -5 5 (1 0 M )



0.001

D M SO ng (0 /m .1 5 l ) % +D (1 ) 0 M  SO M )+ (0 .1 IL % -1 )    ng /m W l) IN -5 5 (1 0 M )

0.01



10

Patient 4 Patient 5 Patient 6



Treatment (48 hours) ***

M

0.1

D

Grade 2 TIMP-1



+++ ***

on tr ol

1

C

100



E

10

Grade 2 TIMP-2

SC

5





M

D

C

on tr ol

SO ng (0 /m .1 l ) % + (1 D ) 0 M M SO )+ (0 .1 IL % -1 )    ng /m W l) IN -5 5 (1 0 M )



Relative Gene Expression (Log Scale)

D

Treatment (48 hours)

Treatment (48 hours)

0.1

F

0.1

RI PT

-5

W IN

IL -1

on tr ol

SO ng (0 /m .1 l ) % +D (1 ) 0 M M SO )+ (0 . IL 1% -1 )    ng /m W l) IN -5 5 (1 0 M )



C

0.001

Relative Gene Expression (Log Scale)

5

M

D

0.01

IL -1

10

M AN U

-5

W IN



0.1

Patient 1 Patient 2 Patient 3

-5





***

Relative Gene Expression (Log Scale)



B

TE D

W IN

IL -1

+++ ***

on tr ol

C

EP



on tr ol

C

D M SO ng (0 /m -5 .1 5 l ) % +D (1 ) 0 M M SO )+ (0 .1 IL % -1 )    ng /m W l) IN -5 5 (1 0 M )

IL -1

1

C

ng (0 /m .1 l) + % D ) 0 M M SO )+ (0 . IL 1% -1 )    ng /m W l) IN -5 5 (1 0 M )

SO

Grade 0 TIMP-1

W IN

on tr ol

+++ **

AC C

C

Relative Gene Expression (Log Scale) 10



(1



M

D

Relative Gene Expression (Log Scale)

A

IL -1

5



0.01

-5



0.1

W IN

IL -1

Relative Gene Expression (Log Scale)

ACCEPTED MANUSCRIPT

10

Grade 0 TIMP-2

1

0.1

+++ ***

Treatment (48 hours)

***

1

+++ ***

1

+++ *** ***

0.01

Patient 1 Patient 2 Patient 3

0.01

0.001

Patient 4 Patient 5 Patient 6

***

0.01

0.001

Treatment (48 hours)

10

Grade 3 TIMP-2

Patient 7 Patient 8 Patient 9

Treatment (48 hours)

(1 0

ng

IN -5 5

W



***

0.1 /m l) M )

ng

(1 0



)

0.1

Treatment (48 hours)

M )

/m l)



(0 .1 %

)

Grade 2 TIMP-2

SC



Treatment (48 hours)

ng

W

IL -1





M SO

M )

/m l)

)

)

ng

/m l)

)

) (0 .1 %

.1 %

on tr ol (0

C M SO



/m l)+ D M )+



D

ng

(1 0



.1 %

(0 .1 %

(0

IN -5 5

(1 0

M )

RI PT

(1 0





IN -5 5



M SO

on tr ol

Relative Gene Expression (Log Scale) 0.1

(1 0

ng

IL -1

M SO

(0 .1 %

1

IN -5 5



)

/m l)+ D

M )+

ng

1 W IN -5 5

IL -

W

IL -1

/m l)+ D

M SO

C

1

W



F



(1 0

10

(0 .1 %

IN -5 5

M SO

E

M SO

.1 % )

(0



Treatment (48 hours)

IL -1

/m l)+ D

W

on tr ol

ng

M )+



D

D

M )+



Patient 7 Patient 8 Patient 9

IN -5 5

W

***

M SO

+++ *** C

0.01



D

0.1



*** Patient 4 Patient 5 Patient 6



+++ ***

1

*** Relative Gene Expression (Log Scale)

Grade 2 TIMP-1

IL -

10

M AN U



(1 0



0.01

on tr ol

/m l)

M )

ng

(1 0



)

1

W IN -5 5

IL -

***

C



(0 .1 %

)

(0 .1 %

on tr ol

+++ *** Patient 1 Patient 2 Patient 3

Relative Gene Expression (Log Scale)

Treatment (48 hours)

TE D



M SO

W IN -5 5

IL -1

/m l)+ D

M SO

C

1

D

Grade 3 TIMP-1

EP

ng

M )+



D

1

M )

/m l)



(1 0



10



ng

W IN -5 5

1

0.1

(1 0



)

*

IN -5 5



(0 .1 %

10

W



M SO

.1 % )

(0

AC C

C

IL -1

/m l)+ D

M SO

IL -

Relative Gene Expression (Log Scale)

Grade 0 TIMP-1

1

ng

on tr ol

B

M )+



C

Relative Gene Expression (Log Scale)

A

IL -



D

0.01

(1 0



0.1

IN -5 5

1

1

W

IL -

Relative Gene Expression (Log Scale)

ACCEPTED MANUSCRIPT

10

Grade 0 TIMP-2

** ***

** +++ ***

1

***

+++

***

0.01

Patient 1 Patient 2 Patient 3

+++ ***

0.01

Patient 4 Patient 5 Patient 6

***

0.01

Treatment (48 hours)

10

Grade 3 TIMP-3

Patient 7 Patient 8 Patient 9

B

C

D

SC

A

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ACCEPTED MANUSCRIPT

AC C

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E

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Figure Legends Figure 1: A-C. The effect of WIN-55 on IL-1β induced MMP-3 gene expression in

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human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. D-F The effect of WIN-55 on IL-1β induced MMP-13 gene expression in human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in

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monolayer. Each point represents the average of the repeats per patient. IL-1β (10 ng/ml) induced the gene expression of MMP-3 and -13. Following WIN-55 (10 µM)

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treatment in combination with IL-1β (10 ng/ml) MMP-3 and -13 gene expression was decreased. WIN-55 (10 µM) alone decreased MMP-3 and -13 gene expression. ***p<0.001, **p<0.01 compared to DMSO (0.1 %) vehicle control and

+++

p<0.001

compared to IL-1β (10 ng/ml) treatment. N=9 samples for each treatment group, obtained from 3 separate patient samples for each grade of tissue (0, 2 and 3) tested

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in triplicate.

Figure 2: A-C. The effect of WIN-55 on IL-1β induced MMP-3 gene expression in human chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate

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beads. D-F. The effect of WIN-55 on IL-1β induced MMP-13 gene expression in

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human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads. Each point represents the average of the repeats per patient. IL-1β (10 ng/ml) induced the gene expression of MMP-3 and -13, following WIN-55 (10 µM) treatment in combination with IL-1β (10 ng/ml) MMP-3 and -13 gene expression was decreased or abolished. WIN-55 (10 µM) alone decreased or abolished MMP-3 and 13 gene expression. *p<0.05, *p<0.01, ***p<0.001 compared to DMSO vehicle control and

+++

p<0.001 compared to IL-1β treatment. N=9 samples for each

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treatment group, obtained from 3 separate patient samples for each grade of tissue (0, 2 and 3) tested in triplicate. Figure 3: A-C The effect of WIN-55 on TIMP-1 gene expression in human OA

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chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. D-F. The effect of WIN-55 on TIMP-2 gene expression in human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. Each point

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represents the average of the repeats per patient. IL-1β (10 ng/ml) had no effect on TIMP-1 and -2 gene expression. WIN-55 treatment (10 µM) both alone and in

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combination with IL-1β (10 ng/ml) reduced TIMP-1 and -2 gene expression. ***p<0.001 compared to DMSO vehicle control and

+++

p<0.001 compared to IL-1β

treatment. N=9 samples for each treatment group, obtained from 3 separate patient samples for each grade of tissue (0, 2 and 3) tested in triplicate.

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Figure 4: A-C. The effect of WIN-55 on TIMP-1 gene expression in human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads. D-F. The effect of WIN-55 on TIMP-2 gene expression in OA human chondrocytes

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isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads. Each point represents the average of the repeats per patient.

IL-1β (10 ng/ml) stimulation

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increased TIMP-1 gene expression and decreased TIMP-2 gene expression below basal levels. WIN-55 treatment (10 µM) both alone and in combination with IL-1β (10 ng/ml) reduced TIMP-1 and -2 gene expression. *p<0.05, **p<0.01, ***p<0.001 compared to DMSO vehicle control and

+++

p<0.001 compared to IL-1β treatment.

N=9 samples for each treatment group, obtained from 3 separate patient samples for each grade of tissue (0, 2 and 3) tested in triplicate.

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Figure 5: Cannabinoid receptor expression in OA chondrocytes cultured in monolayer. Representative photomicrographs of immunocytochemistry of (A) CB1 (B) CB2 (C) PPARα (D) PPARγ (E) IgG control (10 µg) (n=4 cultures).

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Supplementary Figures

Figure 1: A-B The time dependent effect of 10 µM WIN-55 on MMP-3 and -13 gene expression in chondrocytes isolated from grade 0 cartilage and cultured in

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monolayer. C-D. The time dependant effect of 10 µM WIN-55 on TIMP-1 and -2 gene expression in grade 0 human chondrocytes cultured in monolayer. Each point

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represents each treatment replicates in culture. WIN-55 (10 µM) decreases the gene expression of MMP-3, -13, TIMP-1 and -2 in a time dependant manner, with maximal decrease obtained following 48 hours treatment. N=3 samples for each treatment

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group obtained from 1 patient sample 1 and tested in triplicate.

Figure 2: A-B. The concentration dependent effect of WIN-55 on IL-1β induced

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MMP-3 and -13 gene expression in chondrocytes isolated from grade 0 cartilage and cultured in monolayer. C-D. The concentration dependent effect of WIN-55 on IL-1β

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induced TIMP-1 and -2 gene expression in grade 0 human chondrocytes cultured in monolayer. Each point represents each treatment replicates in culture. IL-1β (10 ng/ml) induces the gene expression of MMP-3 and -13 and has no effect on TIMP-1 and -2. MMP-3, -13, TIMP-1 and -2 gene expression is decreased in a WIN-55 concentration dependent manner both alone and in combination with IL-1β (10 ng/ml). N=3 samples for each treatment group obtained from 1 patient sample and tested in triplicate.

SO

Treatment

Treatment

W IN )4 -5 8 5 hr (1 0 W  IN M -5 )3 5 hr (1 W 0 IN M -5 )6 5 (1 hr 0 W M IN -5 )2 5 4 (1 hr 0 M )4 8 hr

Treatment

SC

hr

Relative Gene Expression (Log Scale) 0.01

0.001

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on tr (0 ol .1 W % IN )4 -5 8 5 hr (1 0 W M IN -5 )3 5 (1 hr W 0 IN M -5 )6 5 (1 hr 0 W  IN M -5 ) 24 5 (1 hr 0 M )4 8 hr

C

0.01

ol

M SO

-5

D

M )6

hr

0.1

Relative Gene Expression (Log Scale)

TIMP-1

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0

(1

8

M )3

)4

hr M ) 5 24 (1 hr 0 M )4 8 hr

5

0

(1

0

(1

1

C on tr

1

W IN

5

-5

-5

W IN

W IN

5

.1 %

on tr ol

10

(0 .1 %

0.01

-5

(0

C

MMP-3

SO

0.1

W IN

SO

M

D

Relative Gene Expression (Log Scale)

A

D M

10

TE D

C

EP

C on tr (0 ol .1 W % IN )4 -5 8 5 hr (1 0 W M IN -5 )3 5 hr (1 W 0 IN M -5 )6 5 (1 hr 0 W M IN -5 )2 5 4 (1 hr 0 M )4 8 hr

AC C

D M

Relative Gene Expression (Log Scale)

ACCEPTED MANUSCRIPT B MMP-13

10 1

0.1

D TIMP-2

10

1

0.1

0.01

Treatment

Relative Gene Expression (Log Scale) 1

0.1

0.01

TIMP-1

10

Treatment (48 hours)

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1

0.1

0.01

0.001

Treatment (48 hours)

SC

10

Relative Gene Expression (Log Scale)

100

MMP-3

10000

0.01

0.001

D

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IL W 1 W IN-  IN 5 5  n -5 ( g W 5 ( 1  /m DM C W IN- 2.5 M) l)+ S on IN 55  +I DM O tr W -55 (5 M) L-1 SO (0.1 ol IN (7  +IL  % -5 .5 M) -1  (0. ) 5  +I   1% (1 M L  n ) 0 )+ -1  g/  M IL   n m  l )+ -1  g/m ) IL    n l -1  g/ )   m W  ng/ l) W IN- n ml) IN 55 g/ m -5 W 5 ( (1  l) I 2 W N- .5 M) IN 5 5  W -55 (5 M) IN (7  -5 .5 M) 5  (1 M 0 ) M )

Relative Gene Expression (Log Scale) 1000

Relative Gene Expression (Log Scale)

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C

EP

IL W 1 W IN-  I N 55  n -5 g W 5 ( (1  /m DM C W IN- 2.5 M) l)+ S on IN 55  +I DM O tr W -55 (5 M) L-1 SO (0.1 ol IN (7  +IL  % -5 .5 M) -1  (0. ) 5  + I    1% (1 M L  ng ) 0 )+ -1  /m  M IL   n  l )+ -1  g/m ) IL   n l -1  g/ )   m W  ng/ l) W IN-  n ml) I N 55 g / m -5 W 5 ( (1  l) I 2 W N- .5 M) I N 55  W -55 (5 M) IN (7  -5 .5 M) 5  (1 M 0 ) M )

A

IL W 1 W IN-  IN 55  n -5 ( g W 5 ( 1  /m DM C W IN- 2.5 M) l)+ S on IN 55  +I DM O tr W -55 (5 M) L-1 SO (0.1 ol IN (7  +IL  ( % -5 .5 M) -1  0. ) 5  +I   1% (1 M L   n ) 0 )+ -1  g/m M IL  n l )+ -1  g/m ) IL  n l -1  g/ )   m W  ng/ l) I W N- n ml) IN 55 g/m -5 ( W 5 ( 1  l) W IN- 2.5 M) IN 55  W -55 (5 M) IN (7  -5 .5 M) 5  (1 M 0 ) M )

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IL W 1 W IN-  IN 55  n -5 g W 5 ( (1  /m DM C W IN- 2.5 M) l)+ S on IN 55  +I DM O tr W -55 (5 M) L-1 SO (0.1 ol IN (7  +IL  ( % -5 .5 M) -1  0. ) 5  +I   1% (1 M L-  ng ) 0 ) + 1   /m  M IL   n  l )+ -1  g/m ) IL    n l  -1  g/ )   m W  ng/ l) W IN- n ml) IN 55 g/ m W 55 ( (1  l) I 2 W N- .5 M) IN 55  W -55 (5 M) IN (7  -5 .5 M) 5  (1 M 0 ) M )

ACCEPTED MANUSCRIPT B 1000

MMP-13

100 10 1

0.1

Treatment (48 hours)

10

TIMP-2

1

0.1

0.01

Treatment (48 hours)

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Anatomic compartment Posterior condyle

Age

Sex

Method

HC5

Macroscopic Grade 0

57

Female

HC6

0

Lateral tibial plateau

72

Female

Cell culture treatment Cell culture treatment

HC7

0

Posterior femoral condyle

72

Male

HC1

2

Femoral condyle

64

Male

HC5

2

57

Female

HC17

2

Lateral femoral condyle Trochlea

60

Female

HC13

2

83

HC23

2

Medial femoral condyle Posterior condyle femoral medial

HC11

3

Posterior condyle

72

Male

HC15

3

74

Female

HC16

3

81

Male

HC3

3

Medial femoral condyle Posterior femoral condyle Tibia

73

Female

HC5

2-3

57

Female

HC21

2

64

Female

Medial femoral condyle Lateral femoral condyle

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Supplementary table 1: Patient sample information

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Cell culture treatment

Cell culture treatment Cell culture treatment Cell culture treatment Cell culture treatment Cell culture treatment and receptor investigation Cell culture treatment Cell culture treatment Cell culture treatment Receptor investigation Receptor investigation Receptor investigation

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73

Female Female

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Sample ID