Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and...

Accepted Manuscript

Cannabinoid WIN-55,212-2 Mesylate Inhibits Interleukin-1β Induced MatrixMetalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase Expression inHuman 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, CannabinoidWIN-55,212-2 Mesylate Inhibits Interleukin-1β Induced Matrix Metalloproteinase and Tissue Inhibitor ofMatrix Metalloproteinase Expression in Human Chondrocytes, Osteoarthritis and Cartilage (2013), doi:10.1016/j.joca.2013.10.016.

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http://dx.doi.org/10.1016/j.joca.2013.10.016

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Cannabinoid WIN-55,212-2 Mesylate Inhibits Interleukin-1 β 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 *†

†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

SL Dunn, e-mail: [email protected]

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

3066 E-mail: [email protected]

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

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

(TIMPs)

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

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

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

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

mesylate (WIN-55) may inhibit cartilage degradation.

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

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.

Immunocytochemistry was used to investigate the expression of cannabinoid

receptors in chondrocytes cultures.

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

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

which cannabinoids may act to prevent cartilage breakdown in arthritis.

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

Metalloproteinase and Tissue Inhibitor of Matrix Metalloproteinase Expression 2

in Human Chondrocytes. 3

4 S L Dunn †, J M Wilkinson ‡, A Crawford $, C L Le Maitre † & RAD Bunning *† 5 6 †Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam 7

University, Sheffield, S1 1WB, UK 8 ‡Academic Unit of Bone Metabolism, Department of Human Metabolism, University 9

of Sheffield, UK 10 $Centre for Biomaterials & Tissue Engineering, University of Sheffield, School of 11

Clinical Dentistry, UK 12

SL Dunn, e-mail: [email protected] 13

JM Wilkinson, e-mail: [email protected] 14

A Crawford, e-mail: [email protected] 15

CL Le Maitre, e-mail: [email protected] 16

*Author for correspondence: RAD Bunning,Tel: +44 114 225 3012 Fax: +44 114 225 17

3066 E-mail: [email protected] 18

19

KEY WORDS: Cannabinoid, Cartilage degradation, Chondrocytes, Interleukin 1 (IL-20

1), Matrix metalloproteinases (MMPs), Tissue Inhibitors of Matrix Metalloproteinases 21

(TIMPs) 22

Running title : Inhibition of MMP and TIMP production by WIN-55 23

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24

ABSTRACT 25

Objective Interleukin-1β (IL-1β) is involved in the up-regulation of matrix 26

metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-27

inflammatory and reduce joint damage in animal models of arthritis. This study aimed 28

to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 29

mesylate (WIN-55) may inhibit cartilage degradation. 30

Methods Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 31

and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes 32

were obtained from articular cartilage of patients undergoing total knee replacement. 33

Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time 34

PCR was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and 35

ELISA to measure the amount of MMP-3 and MMP-13 protein released into media. 36

Immunocytochemistry was used to investigate the expression of cannabinoid 37

receptors in chondrocytes cultures. 38

Results Treatment with WIN-55 alone or in combination with IL-1β, decreased or 39

abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte 40

monolayer and alginate bead cultures in both a concentration and time dependent 41

manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 42

and -13 protein production by chondrocytes cultured in alginate beads. 43

Immunocytochemisty demonstrated the expression of cannabinoid receptors in 44

chondrocyte cultures. 45

Conclusion Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene 46

and protein expression of MMP-3 and -13. However WIN-55 also decreased basal 47

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levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by 48

which cannabinoids may act to prevent cartilage breakdown in arthritis. 49

50

INTRODUCTION 51

Osteoarthritis (OA) and rheumatoid arthritis (RA) are debilitating joint diseases, and 52

although they have a different aetiology a key pathological feature of both is the loss 53

of articular cartilage1. Cannabis-based medicine Sativex has been shown to have 54

analgesic effects and to suppress disease activity in patients with RA 2. 55

Cannabinoids also have anti-inflammatory effects and reduce joint damage in animal 56

models of arthritis 3-5. In vitro studies have shown that cannabinoids reduce cytokine 57

production from RA fibroblasts and the release of matrix metalloproteinases (MMPs) 58

from fibroblast-like synovial cells6-8. Cannabinoids also have direct effects on 59

cartilage extracellular matrix (ECM) breakdown; reducing interleukin 1 (IL-1) induced 60

proteoglycan and collagen degradation in bovine cartilage9. There is thus increasing 61

evidence to suggest that cannabinoids have chondroprotective effects and may be of 62

value in the treatment of arthritis10. 63

64

During OA and RA there is a shift in the equilibrium between catabolic and anabolic 65

activities1. As a result the breakdown of collagen and proteoglycans may exceed the 66

rate of synthesis of new matrix molecules resulting in cartilage degradation. Another 67

contributing factor in cartilage breakdown in OA and RA is an increase in 68

inflammatory cytokines particularly IL-1 and tumour necrosis factor (TNF) produced 69

by the articular chondrocytes or cells of the synovium11. This results in an increase in 70

MMPs particularly MMP-3 and MMP-13, which are expressed in RA and OA 71

cartilage and synovial tissue,12-20 without an increase in their tissue inhibitors of 72

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matrix metalloproteinases (TIMPs)21, 22. MMP inhibition has been proposed as a 73

possible mechanism to prevent breakdown of cartilage tissue in arthritis, provided 74

the necessary functional specificity can be achieved 23. 75

76

To investigate further the potential of cannabinoids to regulate cartilage breakdown 77

we have studied the effects of synthetic cannabinoid WIN-55,212-2 mesylate (WIN-78

55) on basal and IL-1β stimulated MMP-3, -13, TIMP-1 and -2 expression in human 79

articular chondrocytes from human OA cartilage tissue in monolayer and 3D alginate 80

bead culture. 81

82

To investigate further the potential of cannabinoids to regulate cartilage breakdown 83

we have studied the effects of synthetic cannabinoid WIN-55,212-2 mesylate (WIN-84

55) on basal and IL-1β stimulated MMP-3, -13, TIMP-1 and -2 expression in human 85

articular chondrocytes from human OA cartilage tissue in monolayer and 3D alginate 86

bead culture. WIN-55 is an agonist at the classical cannabinoid receptors, 87

cannabinoid receptor 1 and 2 (CB1 and 2), but also has been shown to activate 88

other receptors including peroxisome proliferator activated receptors alpha and 89

gamma (PPARα and γ)24-26. Thus the expression of these receptors in cultured OA 90

chondrocytes was determined to identify potential targets of WIN-55. 91

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MATERIALS AND METHODS 98

Human cartilage tissue 99

Human chondrocytes were obtained from articular cartilage (n=9) removed from 100

patients with symptomatic OA at the time of total knee replacement (Supplementary 101

table 1). Cartilage was obtained under the National Research Ethics Service 102

approval held by the Sheffield Musculoskeletal Biobank. All patients provided written, 103

informed consent prior to participation. Cartilage blocks were taken from each 104

anatomic compartment within the knee (n=6-7) (medial and lateral tibio-femoral and 105

patello-femoral compartments). Cartilage tissue was graded macroscopically 0-4 106

using the Outerbridge classification27. Chondrocytes were isolated from grade 0 107

(n=3), grade 2 (n=6) and grade 3 (n=5) cartilage tissue as representative of non-108

degenerate, low degenerate and intermediate degenerate cartilage tissue. Cartilage 109

from grade 4 severe degenerate tissue was not used in the study as the cell yield 110

obtained was not sufficient. 111

112

Isolation of human chondrocytes 113

Cartilage tissue was digested in 0.25% trypsin (Sigma-Aldrich, Poole, UK) at 37°C 114

for 30 minutes followed by digestion in 3 mg/ml collagenase type I (Sigma-Aldrich, 115

Poole UK) in Dulbecco's modified Eagle's medium (DMEM)/F-12 (1:1) (Gibco, 116

Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS) 117

(Gibco, Paisley UK), 2mM glutamine (Gibco, Paisley, UK), 100 U/ml penicillin, 100 118

µg/ml streptomycin (Gibco, Paisley UK), 2.5 µg/ml amphotericin B (Sigma-Aldrich, 119

Poole, UK), and 50 µg/ml ascorbic acid (Sigma-Aldrich, Poole, UK) (complete media), 120

at 37°C for 16 hours. Cells were passed through a 7 0 µM cell strainer and washed 121

twice in DMEM/F12 supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 122

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µg/ml, streptomycin, 2.5 µg/ml amphotericin B and 50 µg/ml ascorbic acid (serum 123

free media). Cells were counted and the viability checked using trypan blue staining. 124

Chondrocytes were cultured in monolayer in complete media in a humidified 125

atmosphere of 5% CO2 at 37˚C and harvested at passage 2. 126

127

Monolayer Culture 128

Chondrocytes harvested at passage 2 were seeded at a cell density of 1x106 129

cells/well in a 6 well plate and maintained in complete media in a humidified 130

atmosphere of 5% CO2 at 37˚C for 24 hours prior to treatment to allow cells to 131

adhere to the cell culture plate. 132

133

Alginate Bead Culture 134

When cultured in monolayer, chondrocytes dedifferentiate into fibroblast like cells, 28-135

30 therefore alginate beads were used to redifferentiate the chondrocytes to their 136

native phenotype. Chondrocytes harvested at passage 2 were encapsulated in 137

alginate beads at a cell density of 2x106 cells/ml, as previously described 31. Alginate 138

beads were cultured in complete media in a humidified atmosphere of 5% CO2 at 139

37°C and the media changed every other day. Chondro cytes were redifferentiated in 140

alginate beads for 4 weeks prior to treatment. 141

142

Cytotoxicity studies 143

The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, 144

Southampton, UK) was used to determine the effects of 10µM WIN-55 on the cell 145

viability of human chondrocytes according to the manufacturer’s instructions. 146

147

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Cannabinoid WIN-55 and IL-1 β treatments 148

Prior to stimulation complete media was removed cells washed with serum free 149

media and serum free media containing 500 µg/ml bovine serum albumin (BSA) 150

(Sigma-Aldrich, Poole, UK) added. Chondrocytes cultured in monolayer were 151

unstimulated or stimulated with 10 ng/ml IL-1β (Peprotech, London, UK) with and 152

without 1 µM, 2.5 µM, 5 µM, 7.5 µM and 10 µM WIN-55 (Sigma-Aldrich, Poole, UK) 153

for 48 hours at 37°C. Chondrocytes in monolayer we re treated with 10 µM WIN-55 154

for 3, 6, 24 and 48 hours at 37°C. Chondrocytes cul tured in alginate beads were 155

unstimulated or stimulated with 10 ng/ml IL-1β with and without 10 µM WIN-55. 156

Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Poole, UK) was used as a vehicle 157

control at 0.1% (equivalent to that present in 10 µM WIN-55). 158

159

MMP-3 and -13 Enzyme Linked Immunosorbent Assay (ELISA) 160

ELISA (R&D Systems, Abingdon, UK) were used according to the manufacturer's 161

instructions to measure pro and active MMP-3 protein expression (ng/ml) and pro 162

MMP-13 protein expression (pg/ml) in alginate bead conditioned culture media 163

following treatment with 10 µM WIN-55 with and without 10ng/ml IL-1β stimulation for 164

48 hours. 165

166

RNA extraction from monolayers 167

For each of the 3 patient samples obtained from Outerbridge grade 0, 2 and 3 (n=9), 168

patient samples were tested in triplicate. Following treatments, media was removed, 169

cells washed in 1xPBS and RNA extracted in 1 ml of TRIzol reagent (Invitrogen, 170

Paisley, UK) following the manufacturer's instructions. RNA was resuspended in 14 171

µl sterile deionised water. 172

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RNA extraction from alginate beads 173

For each of the 3 patient samples obtained from Outerbridge grade 0, 2 and 3 (n=9), 174

patient samples were tested in triplicate RNA was extracted from alginate beads 175

using 2 beads per extraction as described previously 31. The resulting RNA was then 176

resuspended in 100µl of sterile deionised water and the RNA was purified using 177

RNeasy clean up columns (Qiagen, Crawley, UK) according to the manufacturer's 178

instructions and RNA eluted in 14 µl of sterile deionised water. 179

180

Reverse transcription-real-time polymerase chain reaction (qRT-PCR) 181

cDNA was formed as described previously31. Real-time PCR was used to investigate 182

MMP-3, -13, TIMP-1, and -2 gene expression using Applied Biosystems Taqman 183

Gene Expression Assays (Table 1). The reactions were performed for 40 cycles 184

using Taqman Fast Universal PCR Master Mix (Applied Biosystems, Paisley, UK) on 185

the ABI StepOnePlus real-time PCR machine (Applied Biosystems, Paisley, UK). 186

The data were collected and the fold changes in gene expression analysed using the 187

2-∆∆Ct method 32. Gene expression was normalised to housekeeping genes GAPDH 188

and 18S together with untreated controls. 189

190

Cannabinoid receptor expression in OA chondrocytes 191

Chondrocytes isolated from 4 patient samples of grade 2-3 cartilage at passage 2 192

were plated at a density of 1x105 cells in 8 well chamber slides and allowed to 193

adhere overnight. Chondrocytes were fixed in 4% formalin and endogenous 194

peroxidases were quenched. Following 0.01% chymotrypsin antigen retrieval at 37°C 195

for 20 minutes cells were blocked in 25% v/v goat serum/BSA. Cells were incubated 196

with rabbit polyclonal antibodies (Abcam) overnight at 4°C against CB1 (1/100), CB2 197

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(1/50), PPARα (1/150) and PPARγ (1/50). A rabbit isotype IgG antibody (Abcam) 198

was used as negative control. Following washing cells were incubated with 199

biotinylated goat anti-rabbit secondary antibody (1/300: Abcam) and binding 200

detected using streptavidin-biotinylated horse radish peroxide complex (Vector 201

Laboratories, Peterbrough, UK) with 3,3’-diaminobenzidine tetrahydrochloride 202

substrate (Sigma-Aldrich). Cells were counterstained using Mayers Haematoxylin 203

(Leica Microsystems, Milton Keynes, UK) dehydrated, cleared and mounted in 204

Pertex (Leica Microsystems). Immunopositivity was visualised and images captured 205

using an Olympus BX60 microscope and QCapture Pro v8.0 software 206

(MediaCybernertic, Marlow, UK). 207

Statistical Analysis 208

Data was shown to be non-parametric via a Shapiro–Wilk test hence statistical 209

significance between DMSO vehicle control and treatment groups was determined 210

using the non-parametric Kruskall-Wallis multiple comparisons test and Conover-211

Inman post hoc test; p<0.05 was considered statistically significant for real-time PCR 212

and ELISA analysis. All statistical analysis was performed using StatsDirect. All data 213

analysis was performed using individual replicates for each treatment group. 214

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RESULTS 222

Cytotoxicity studies 223

Cytotoxicity studies showed that WIN-55 was not toxic to chondrocytes at the 224

concentrations used (Data not shown). 225

226

Time-course and concentration dependent effects of WIN-55 on MMP-3, MMP-227

13, TIMP-1 and TIMP-2 gene expression in monolayer 228

Chondrocytes were treated with WIN-55 (10 µM) for 3, 6, 24 and 48 hours to 229

determine the time point at which a maximum decrease in gene expression was 230

observed. MMP-3, -13, TIMP-1 and -2 gene expression was decreased in a time-231

dependent manner, the largest decrease was observed at 48 hours (Supplementary 232

Figure 1A-D); therefore this incubation time was used for all subsequent treatments. 233

Gene expression of MMP-3, -13, TIMP-1 and -2 was decreased in a WIN-55 234

concentration dependent manner both with and without IL-1β (10 ng/ml) stimulation 235

(Supplementary Figure 2A-D). A significant decrease in gene expression was 236

observed at concentrations ≥ 2.5 µM WIN-55 compared to DMSO (0.1%) vehicle 237

controls (Supplementary Figure 2A-D). WIN-55 was used at 10 µM in all following 238

treatments as this was the maximal inhibitory non-toxic concentration. 239

240

Effects of DMSO in monolayer and alginate bead culture 241

No differences in gene or protein expression were observed between untreated cells 242

compared to cells treated with 0.1% DMSO and 10 ng/ml IL-1β treatment in 243

combination with 0.1% DMSO compared to IL-1β treatment alone (Data not shown). 244

Therefore 10 ng/ml IL-1β in combination with 0.1% DMSO and 0.1% DMSO controls 245

were used for all real-time PCR and ELISA analysis. 246

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247

Effect of WIN-55 on IL-1 β induced MMP-3 and MMP-13 gene expression in 248

monolayer 249

In order to determine the effects of WIN-55 on MMPs in the presence of the 250

inflammatory cytokine IL-1β, real-time PCR was used to measure the gene 251

expression of MMP-3 and -13 in chondrocytes obtained from macroscopically graded 252

OA cartilage. For this purpose chondrocytes were stimulated with IL-1β to induce 253

MMP expression and co-treated with WIN-55. The effects of WIN-55 alone were also 254

investigated. IL-1β stimulation significantly induced MMP-3 gene expression in 255

chondrocytes isolated from grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) 256

cartilage and MMP-13 in chondrocytes isolated from grade 0 (p<0.0001), grade 2 257

(p=0.02) and grade 3 cartilage (p<0.0001) compared to DMSO vehicle control 258

(Figure 1A-F). Treatment with WIN-55 in combination with IL-1β significantly reduced 259

MMP-3 and -13 gene expression in chondrocytes derived from grade 0 (p<0.0001), 2 260

(p<0.0001) and 3 (p<0.0001) cartilage compared to IL-1β stimulation alone (Figure 261

1A-F). WIN-55 treatment in combination with IL-1β also significantly reduced MMP-3 262

gene expression in chondrocytes isolated from grade 0 (p<0.0001), 2 (p<0.0001) 263

and 3 (p<0.0001) cartilage and MMP-13 gene expression in chondrocytes isolated 264

from grade 0 (p<0.0001), grade 2 (p=0.01) and grade 3 (p<0.0001) compared to 265

DMSO vehicle control (Figure 1A-F). WIN-55 treatment alone significantly reduced 266

MMP-3 gene expression in grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) 267

cartilage derived chondrocytes below basal levels compared to DMSO vehicle 268

control (Figure 1 A-C). WIN-55 treatment alone also significantly decreased MMP-269

13 gene expression in grade 0 (p=0.002) and 3 (p<0.0001) cartilage derived 270

chondrocytes compared to DMSO control (Figure 1D, 2F). MMP-13 was only 271

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expressed in two samples in chondrocytes isolated from grade 2 cartilage following 272

WIN-55 treatment therefore statistical analysis could not be performed (Figure 1E). 273

274

Effects of WIN-55 on IL-1 β induced MMP-3 and MMP-13 gene expression on 275

chondrocytes cultured in alginate beads 276

The effects of WIN-55 on IL-1β induced MMP-3 and -13 gene expression were 277

determined in chondrocytes cultured in alginate beads in order to determine gene 278

expression in chondrocytes following redifferentiation back to their native phenotype. 279

IL-1β stimulation significantly induced MMP-3 gene expression in chondrocytes from 280

grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage and MMP-13 gene 281

expression in grade 0 (p=0.018), 2 (p<0.0001) and 3 (p<0.0001) cartilage derived 282

chondrocytes compared to DMSO vehicle control (Figure 2A-F). Similarly WIN-55 283

treatment in combination with IL-1β significantly reduced MMP-3 gene expression in 284

grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage derived chondrocytes 285

and MMP-13 gene expression in grade 0 (p<0.0001) and 3 (p<0.0001) cartilage 286

derived chondrocytes compared to IL-1β stimulation alone (Figure 2A, 2B, 2C, 2D, 287

2F). MMP-13 was not expressed in chondrocytes from grade 2 cartilage treated with 288

IL-1β in combination with WIN-55 (Figure 2E). WIN-55 treatment in combination with 289

IL-1β significantly reduced MMP-3 (p=0.03) and -13 (p=0.003) gene expression in 290

chondrocytes from grade 0 chondrocytes compared to DMSO vehicle control (Figure 291

2A-D). WIN-55 treatment in combination with IL-1β reduced MMP-3 gene expression 292

in chondrocytes from grade 2 (p=0.11) cartilage and both MMP-3 (p=0.58) and -13 293

(p=0.11) gene expression in chondrocytes from grade 3 cartilage compared to 294

DMSO vehicle control; however this was not significant (Figure 2A, 2C, 2F). There 295

was no significant difference from basal levels of MMP-3 gene expression in grade 0 296

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(p=0.052) and 3 (p=0.16) cartilage derived chondrocytes when treated with WIN-55 297

alone (Figure 2A, 2C). MMP-3 was not expressed in grade 2 cartilage chondrocytes 298

treated with WIN-55 alone (Figure 2B). MMP-13 was abolished in chondrocytes from 299

grade 0, 2 and 3 cartilage treated with WIN-55 alone (Figure 2D-F). 300

301

Effects of WIN-55 on IL-1 β induced TIMP-1 and TIMP-2 gene expression in 302

monolayer 303

In order to determine the effects of WIN-55 on the inhibitors of MMP-3 and -13 the 304

gene expression of TIMP-1 and -2 was investigated following WIN-55 treatment both 305

alone and in combination with IL-1β. IL-1β stimulation had no significant effect on 306

TIMP-1 gene expression in chondrocytes derived from grade 0 (p=0.75), 2 (p=0.60) 307

and 3 (p=0.12) cartilage or TIMP-2 gene expression in chondrocytes isolated from 308

grade 0 (p=0.77), 2 (p=0.49) and 3 (p=0.98) cartilage (Figure 3A-F). However, WIN-309

55 treatment in combination with IL-1β resulted in a significant decrease in TIMP-1 310

gene expression compared to DMSO vehicle control and IL-1β stimulated 311

chondrocytes derived from grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p=0.001) 312

cartilage (Figure 3A-C). WIN-55 alone also significantly reduced TIMP-1 gene 313

expression in chondrocytes derived from grade 0 (p<0.0001) and 2 (p<0.0001) 314

cartilage below basal levels (Figure 3A, 3B). TIMP-1 gene expression was 315

decreased in chondrocytes derived from grade 3 cartilage following WIN-55 316

treatment however this was not significant (p=0.11) (Figure 3C). TIMP-2 gene 317

expression was significantly reduced following WIN-55 treatment in combination with 318

IL-1β compared to DMSO vehicle control and IL-1β stimulation in chondrocytes 319

derived from grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage (Figure 320

3D-F). WIN-55 alone significantly reduced the gene expression of TIMP-2 below 321

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basal levels in chondrocytes isolated from grade 0 (p<0.0001), 2 (p<0.0001) and 3 322

(p<0.0001) cartilage (Figure 3D-F). 323

324

Effects of WIN-55 on IL-1 β induced TIMP-1 and TIMP-2 gene expression on 325

chondrocytes cultured in alginate beads 326

TIMP-1 gene expression was significantly increased in grade 2 (p<0.0009) and 3 327

cartilage derived chondrocytes (p=0.02) following IL-1β stimulation (Figure 4B-C) but 328

not in grade 0 (p=0.24) cartilage chondrocytes compared to DMSO vehicle control 329

(Figure 4A). In contrast TIMP-2 gene expression was significantly decreased 330

following IL-1β treatment in chondrocytes extracted from grade 0 (p=0.009), 2 331

(p=0.001) and 3 (p<0.0001) cartilage compared to DMSO vehicle control (Figure 4D-332

F). WIN-55 treatment in combination with IL-1β resulted in a significant decrease in 333

TIMP-1 gene expression in chondrocytes derived from grades 0 (p<0.0001), 2 334

(p<0.0001), and 3 (p<0.0001), cartilage and TIMP-2 gene expression in 335

chondrocytes isolated from grades 0 (p<0.0001), 2 (p<0.0001), and 3 (p=0.0007), 336

cartilage compared to IL-1β stimulation (Figure 4A-F). WIN-55 treatment in 337

combination with IL-1β also resulted in a significant decrease in TIMP-1 gene 338

expression in chondrocytes derived from grades 0 (p<0.0001), 2 (p<0.0001), and 3 339

(p<0.0001), cartilage and TIMP-2 gene expression in chondrocytes isolated from 340

grades 0 (p<0.0001), 2 (p<0.0001), and 3 (p<0.0001) cartilage compared to DMSO 341

control (Figure 4A-F). WIN-55 treatment alone significantly reduced both TIMP-1 342

gene expression in chondrocytes derived from grade 0 (p=0.0005), 2 (p<0.0001) and 343

3 (p=0.0003) cartilage below basal levels (Figure 4A-C). WIN-55 treatment alone 344

significantly reduced both TIMP-2 gene expression in chondrocytes derived from 345

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grade 0 (p<0.0001), 2 (p<0.0001) and 3 (p<0.0001) cartilage below basal levels 346

(Figure 4D-F). 347

348

The effects of WIN-55 on MMP-3 and -13 protein expression 349

In order to determine whether WIN-55 also inhibited MMP-3 and -13 expression at 350

the protein level, pro and active MMP-3 and pro MMP-13 were measured in culture 351

media using ELISA. Culture media obtained from chondrocytes isolated from grade 3 352

cartilage which had been cultured in alginate beads were used, as these 353

demonstrated clear gene expression effects. Following stimulation of chondrocytes 354

with IL-1β there was a significant increase in MMP-3 (p<0.0001) and -13 (p=0.046) 355

protein release into the media (Table 2). Treatment of chondrocytes with WIN-55 in 356

combination with IL-1β significantly reduced both MMP-3 (p=0.0007) and -13 357

(p=0.0005) protein compared to IL-1β treatment alone (Table 2). WIN-55 treatment 358

alone significantly reduced MMP-3 protein release to below basal levels (p=0.04) 359

and MMP-13 (p=0.38) protein levels remained at basal level (Table 2). 360

361

Cannabinoid receptor expression in OA chondrocytes 362

To identify the expression of cannabinoid receptors CB1, CB2, PPARα and PPARγ 363

in chondrocytes cultured in monolayer immunocytochemistry was used. Here we 364

observed the expression of CB1, CB2, PPARα and γ (Figure 5) PPARα and γ 365

expression was localised to the cytoplasm and nucleus (Figure 5). 366

367

DISCUSSION 368

Cartilage degradation is a pathological feature of both OA and RA1. The 369

inflammatory cytokine IL-1 plays a key role in cartilage destruction and stimulates 370

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increased production of MMPs by chondrocytes, resulting in the breakdown of 371

collagen and proteoglycan33. Here, we have shown that treatment of articular 372

chondrocytes from human OA cartilage with cannabinoid WIN-55 reduces the gene 373

and protein expression of MMP-3 and MMP-13 in the presence of IL-1β, suggesting 374

that cannabinoids may have potential in terms of arthritis therapy. In addition we 375

have shown that WIN-55 significantly reduces the gene expression of TIMP-1 and 376

TIMP-2 to below basal levels. 377

In this study we have used both monolayer and a 3D culture system for culture of 378

chondrocytes. Chondrocytes that have been isolated from articular cartilage 379

dedifferentiate in monolayer culture changing their matrix synthesis, with a decrease 380

in type II collagen and aggrecan, developing a fibroblast like phenotype and an 381

increase in collagen type I28-30. Dedifferentiation can be reversed with the key 382

phenotypic features of chondrocytes being preserved when cultured in a 3D system 383

such as alginate beads34. In this study, chondrocytes were treated with IL-1β to 384

mimic inflammatory processes in an in vitro model of OA 35. 385

Synthetic cannabinoids WIN-55 and HU-210 reduce IL-1α induced proteoglycan and 386

collagen degradation in bovine nasal cartilage tissue suggesting a chondroprotective 387

effect of these compounds 9. Here, we have shown a possible mechanism by which 388

WIN-55 may prevent IL-1β induced ECM breakdown in OA cartilage tissue via 389

preventing the expression of MMPs. We have demonstrated that chondrocytes from 390

different grades of OA cartilage modulate MMP-3 and MMP-13 expression in 391

response to WIN-55 with and without IL-1β stimulation. These findings together with 392

others, suggest that cannabinoids may be of importance in the treatment of arthritis 3-393

5, 7, 8. Selvi et al 2008 demonstrated that WIN-55 and CP55,940 inhibit IL-1β induced 394

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secretion of IL-6 and IL-8 in RA fibroblast like synovial cells, suggesting an anti-395

inflammatory activity of cannabinoids7. In another study non-psychoactive 396

cannabinoid ajulemic acid (AJA) reduced MMP-1, MMP-3 and MMP-9 release from 397

fibroblast like synovial cells stimulated with IL-1α and TNFα 8. In vivo, AJA has also 398

been shown to reduce the severity of adjuvant-induced arthritis 4 and other non-399

psychoactive cannabinoids, cannabidiol (CBD) and HU-320 reduced inflammation 400

and joint damage in murine collagen-induced arthritis 3, 5. 401

The effects of WIN-55 on articular chondrocytes did not appear to be influenced by 402

the grade of the cartilage they were isolated from when cultured in monolayer. 403

Chondrocytes cultured in monolayer express MMP-3 and MMP-13 at very low levels 404

following WIN-55 treatment. However when comparing the effects of WIN-55 in 405

monolayer to alginate bead culture, a greater inhibitory effect on MMP-3 and MMP-406

13 gene expression was seen in alginate bead cultured chondrocytes. Interestingly a 407

biphasic expression pattern of MMP-3 and MMP-13 in response to WIN-55 was 408

observed. MMP-3 was expressed in grade 0 and grade 3 cartilage derived 409

chondrocytes and abolished in grade 2 cartilage chondrocytes and MMP-13 was 410

abolished in chondrocytes from all grades of cartilage when cultured in alginate 411

beads. These varying responses to WIN-55 treatment in alginate bead culture may 412

indicate that the expression of MMPs may be differentially regulated depending on 413

the grade and extent of cartilage degradation and the culture method utilised. 414

Studies have shown that cartilage tissue derived from different OA grades or normal 415

aged cartilage may influence the response of the chondrocytes to different 416

treatments40-42. Interestingly biphasic effects have also been seen with other 417

cannabinoids namely AJA 43. 418

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During OA there is thought to be an imbalance between MMP and TIMP expression 419

which in part contributes to cartilage breakdown21. We have shown that WIN-55 420

inhibits expression of both destructive MMPs and protective TIMPs involved in the 421

pathogenesis of OA, indicating that inhibition may occur via a signalling pathway 422

which regulates both at the transcription level. Human MMPs and TIMPs share a 423

common activating protein 1 (AP-1) site in their promoters which regulates their 424

transcription44. WIN-55 may have a differential effect on AP-1 activation via 425

peroxisome proliferator activated receptors (PPARs). WIN-55 has been shown to 426

activate AP-1 via PPARα. In addition AP-1 may be involved in the activation of 427

interferon β (IFNβ) 45. Production of IFNβ may result in reduced levels of MMPs and 428

TIMPs. IFNβ reduced MMP-1, -3 and TIMP-1 in fibroblast-like synovial cells both 429

with and without IL-1β stimulation and synovial tissue from patients with RA, treated 430

with IFNβ, showed reduced levels of MMP-1 and TIMP-146. Furthermore IFNβ has 431

been shown to have anti-inflammatory properties in the treatment of arthritis47-49. 432

Conversely PPARγ agonists have been shown to reduce IL-1β induced MMP-1 433

expression in human synovial fibroblasts via inhibiting DNA binding of AP-150. WIN-434

55 also binds to PPARγ so could also act in this way51. The biological activities of 435

cannabinoids and the signal transduction pathways they induce or inhibit need to be 436

further investigated in OA. Since TIMP-1 and -2 are decreased by WIN-55 in human 437

OA chondrocytes it is unclear whether there is a change in MMP and TIMP balance 438

following cannabinoid treatment. However the inhibitory effect of WIN-55 on MMP-3 439

and -13 expression would indicate a possible role of cannabinoids in supressing IL-440

1β induced ECM degradation by MMPs. 441

We have observed the expression of both classical cannabinoid receptors CB1 and 442

CB2 in human chondrocytes from OA cartilage at passage 2. CB1 and CB2 have 443

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previously been shown to be expressed at similar levels both at the protein and RNA 444

level in synovia of patients with OA and RA and their expression is thought to play a 445

role in the pathology of joint disease52. PPARα and γ were also expressed in OA 446

chondrocytes at passage 2 and their expression appears to be both cytoplasmic and 447

nucleus. PPARs are nuclear receptors however studies have also shown that the 448

localisation of PPARα receptors in chondrocytes is also cytoplasmic 53. WIN-55 449

activates CB1and CB2 with Kis of 1.89-123 nM and 0.28-16.2 nM respectively 24. In 450

addition, WIN-55 has also been shown to activate PPARα and γ25, 26. Targeting 451

PPARs using both specific and cannabinoid agonists for the treatment of OA and RA 452

has been reported previously8, 26, 36, 50, 54-56. The observed effects of WIN-55 on MMP 453

and TIMP expression may be mediated by one or more of these receptors or by 454

receptors as yet unknown. 455

In conclusion, in human OA chondrocytes, the synthetic cannabinoid WIN-55 inhibits 456

the expression of matrix degrading enzymes MMP-3 and -13 and their inhibitors 457

TIMP-1 and -2 in the presence or absence of IL-1β. This suggests a possible 458

mechanism by which cannabinoids may act to prevent ECM breakdown in arthritis. 459

Cannabinoids could provide a dual role in the treatment of arthritis as disease 460

modifying agents in addition to having anti-inflammatory properties10. Although there 461

is increasing evidence to suggest that cannabinoids may be of therapeutic value in 462

the treatment of arthritis, further studies into the receptors and signalling pathways 463

involved in the actions of cannabinoids need to be investigated in order to elucidate 464

their effects on catabolic and anabolic mediators in both normal and arthritic cartilage. 465

466

467

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Acknowledgements: 468

This work was funded by a PhD Studentship from the Biomedical Research Centre, 469

Faculty of Health and Well Being, Sheffield Hallam University, Sheffield, UK. Tissue 470

samples were supplied by the Sheffield Biorepository. 471

472

Authors contributions: 473

SD participated in its design, performed all the laboratory work and analysis and co-474

wrote the manuscript. JMW helped to conceive the study, secure funding, 475

contributed to its design and co-ordination, participated in interpretation of data and 476

co-wrote the manuscript. AC helped to conceive the study, secure funding, 477

contributed to its design and co-ordination and co-wrote the manuscript. CLM helped 478

to conceive the study, secure funding, contributed to its design and co-ordination, 479

participated in interpretation of data and co-wrote the manuscript. RAB helped to 480

conceive the study, secure funding, contributed to its design and co-ordination, 481

participated in interpretation of data and co-wrote the manuscript. All authors read 482

and approved the final manuscript. 483

484

Competing Interest 485

Competing Interest: None Declared. 486

487

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680

Figure Legends 681

Figure 1: A-C. The effect of WIN-55 on IL-1β induced MMP-3 gene expression in 682

human OA chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in 683

monolayer. D-F The effect of WIN-55 on IL-1β induced MMP-13 gene expression in 684

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monolayer. Each point represents the average of the repeats per patient. IL-1β (10 686

ng/ml) induced the gene expression of MMP-3 and -13. Following WIN-55 (10 µM) 687

treatment in combination with IL-1β (10 ng/ml) MMP-3 and -13 gene expression was 688

decreased. WIN-55 (10 µM) alone decreased MMP-3 and -13 gene expression. 689

***p<0.001, **p<0.01 compared to DMSO (0.1 %) vehicle control and +++p<0.001 690

compared to IL-1β (10 ng/ml) treatment. N=9 samples for each treatment group, 691

obtained from 3 separate patient samples for each grade of tissue (0, 2 and 3) tested 692

in triplicate. 693

Figure 2: A-C. The effect of WIN-55 on IL-1β induced MMP-3 gene expression in 694

human chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate 695

beads. D-F. The effect of WIN-55 on IL-1β induced MMP-13 gene expression in 696


alginate beads. Each point represents the average of the repeats per patient. IL-1β 698

(10 ng/ml) induced the gene expression of MMP-3 and -13, following WIN-55 (10 µM) 699

treatment in combination with IL-1β (10 ng/ml) MMP-3 and -13 gene expression was 700

decreased or abolished. WIN-55 (10 µM) alone decreased or abolished MMP-3 and -701

13 gene expression. *p<0.05, *p<0.01, ***p<0.001 compared to DMSO vehicle 702

control and +++p<0.001 compared to IL-1β treatment. N=9 samples for each 703

treatment group, obtained from 3 separate patient samples for each grade of tissue 704

(0, 2 and 3) tested in triplicate. 705

Figure 3: A-C The effect of WIN-55 on TIMP-1 gene expression in human OA 706

chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. D-F. 707

The effect of WIN-55 on TIMP-2 gene expression in human OA chondrocytes 708

isolated from grade 0, 2 and 3 cartilage and cultured in monolayer. Each point 709

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represents the average of the repeats per patient. IL-1β (10 ng/ml) had no effect on 710

TIMP-1 and -2 gene expression. WIN-55 treatment (10 µM) both alone and in 711

combination with IL-1β (10 ng/ml) reduced TIMP-1 and -2 gene expression. 712

***p<0.001 compared to DMSO vehicle control and +++p<0.001 compared to IL-1β 713

treatment. N=9 samples for each treatment group, obtained from 3 separate patient 714

samples for each grade of tissue (0, 2 and 3) tested in triplicate. 715

Figure 4: A-C. The effect of WIN-55 on TIMP-1 gene expression in human OA 716

chondrocytes isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads. 717

D-F. The effect of WIN-55 on TIMP-2 gene expression in OA human chondrocytes 718

isolated from grade 0, 2 and 3 cartilage and cultured in alginate beads. Each point 719

represents the average of the repeats per patient. IL-1β (10 ng/ml) stimulation 720

increased TIMP-1 gene expression and decreased TIMP-2 gene expression below 721

basal levels. WIN-55 treatment (10 µM) both alone and in combination with IL-1β (10 722

ng/ml) reduced TIMP-1 and -2 gene expression. *p<0.05, **p<0.01, ***p<0.001 723

compared to DMSO vehicle control and +++p<0.001 compared to IL-1β treatment. 724

N=9 samples for each treatment group, obtained from 3 separate patient samples for 725

each grade of tissue (0, 2 and 3) tested in triplicate. 726

Figure 5: Cannabinoid receptor expression in OA chondrocytes cultured in 727

monolayer. Representative photomicrographs of immunocytochemistry of (A) CB1 728

(B) CB2 (C) PPARα (D) PPARγ (E) IgG control (10 µg) (n=4 cultures). 729

Supplementary Figures 730

Figure 1: A-B The time dependent effect of 10 µM WIN-55 on MMP-3 and -13 gene 731

expression in chondrocytes isolated from grade 0 cartilage and cultured in 732

monolayer. C-D. The time dependant effect of 10 µM WIN-55 on TIMP-1 and -2 gene 733

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expression in grade 0 human chondrocytes cultured in monolayer. Each point 734

represents each treatment replicates in culture. WIN-55 (10 µM) decreases the gene 735

expression of MMP-3, -13, TIMP-1 and -2 in a time dependant manner, with maximal 736

decrease obtained following 48 hours treatment. N=3 samples for each treatment 737

group obtained from 1 patient sample 1 and tested in triplicate. 738

739

Figure 2: A-B. The concentration dependent effect of WIN-55 on IL-1β induced 740

MMP-3 and -13 gene expression in chondrocytes isolated from grade 0 cartilage and 741

cultured in monolayer. C-D. The concentration dependent effect of WIN-55 on IL-1β 742

induced TIMP-1 and -2 gene expression in grade 0 human chondrocytes cultured in 743

monolayer. Each point represents each treatment replicates in culture. IL-1β (10 744

ng/ml) induces the gene expression of MMP-3 and -13 and has no effect on TIMP-1 745

and -2. MMP-3, -13, TIMP-1 and -2 gene expression is decreased in a WIN-55 746

concentration dependent manner both alone and in combination with IL-1β (10 747

ng/ml). N=3 samples for each treatment group obtained from 1 patient sample and 748

tested in triplicate. 749

750

751

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Table 1:Taqman Gene Expression Assay IDs

Taqman Gene Expression Assay Assay ID

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Hs9999905_m1

18S Hs99999901_s1

MMP-3 Hs00968305_m1

MMP-13

TIMP-1

TIMP-2

Hs00233992_m1

Hs00171558_m1

Hs00234278_m1

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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.

Treatment (48 hours) MMP-3 (ng/alginate bead)

MMP-13 (pg/alginate bead)

DMSO (0.1%) 1.4 (0.0659)

327.5 (1.6407)

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)

270.7 (3.8866)

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.

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Grade 0 MMP-3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.0001

0.001

0.01

0.1

1

10

100

1000Patient 1

Patient 2

Patient 3

***

***

***

+++

Treatment (48 hours)

Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 MMP-3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

1.010 - 6

1.010 - 5

1.010 - 4

1.010 - 3

1.010 - 2

1.010 - 1

1.0100

1.0101

1.0102

1.0103

1.0104

Patient 4

Patient 5

Patient 6

+++

***

*** ***


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 MMP 3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.0001

0.001

0.01

0.1

1

10

100

1000Patient 7

Patient 8

Patient 9

***

***

***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 0 MMP-13

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

1000

Patient 1

Patient 2

Patient 3

+++

***

******


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 MMP-13

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

1000

Patient 4

Patient 5

Patient 6

+++

*

**


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 MMP-13

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.0001

0.001

0.01

0.1

1

10

100

Patient 7

Patient 8

Patient 9

***

***

***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

A

B

C

D

E

F

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Grade 0 MMP-3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

100

1000

Patient 1

Patient 2

Patient 3+++

***

*


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 MMP-3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

100

1000

Patient 4

Patient 5

Patient 6

***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 MMP-3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

1000

Patient 7

Patient 8

Patient 9

+++

***


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)Grade 0 MMP-13

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

Patient 1

Patient 2

Patient 3

***

+++**


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 MMP-13

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

100

Patient 4

Patient 5

Patient 6

***


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 MMP-13

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

Patient 7

Patient 8

Patient 9

***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

A

B

C

D

E

F

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Grade 0 TIMP-1

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

Patient 1

Patient 2

Patient 3

***

***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 TIMP-1

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

Patient 4

Patient 5

Patient 6+++*** ***


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 TIMP-1

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 7

Patient 8

Patient 9+++**


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)Grade 0 TIMP-2

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

Patient 1

Patient 2

Patient 3*** ***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 TIMP-2

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.001

0.01

0.1

1

10

Patient 4

Patient 5

Patient 6

+++

******


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 TIMP-2

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 7

Patient 8

Patient 9

***

***+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

A

B

C

D

E

F

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Grade 0 TIMP-1

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 1

Patient 2

Patient 3+++

***

***


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 TIMP-1

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 4

Patient 5

Patient 6

***

*** ***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 TIMP-1

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 7

Patient 8

Patient 9

*

***

***+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)Grade 0 TIMP-2

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 1

Patient 2

Patient 3

**

***

***+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 2 TIMP-2

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 4

Patient 5

Patient 6

+++

**

***

***


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Grade 3 TIMP-3

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M) +

IL-1

WIN

-55

(10

M)

WIN

-55

(10

0.01

0.1

1

10

Patient 7

Patient 8

Patient 9***

***

***

+++


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

A

B

C

D

E

F

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A

C D

E

B

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Figure Legends

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

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


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)

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

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

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

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

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

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.

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

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

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).

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

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

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

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

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

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.

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Contr

ol

DM

SO (0

.1%

) 48

hr

M) 3

hr

WIN

-55

(10

M) 6

hr

WIN

-55

(10

M) 2

4 hr

WIN

-55

(10

M) 4

8 hr

WIN

-55

(10

0.01

0.1

1

10

MMP-3

Treatment

Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Contr

ol

DM

SO (0

.1%

) 48

hr

M) 3

hr

WIN

-55

(10

M) 6

hr

WIN

-55

(10

M) 2

4 hr

WIN

-55

(10

M) 4

8 hr

WIN

-55

(10

0.01

0.1

1

10

TIMP-1

Treatment

Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Contr

ol

DM

SO (0

.1%

) 48

hr

M) 3

hr

WIN

-55

(10

M) 6

hr

WIN

-55

(10

M) 2

4 hr

WIN

-55

(10

M) 4

8 hr

WIN

-55

(10

0.001

0.01

0.1

1

10

MMP-13

Treatment

Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Contr

ol

DM

SO (0

.1%

) 48

hr

M) 3

hr

WIN

-55

(10

M) 6

hr

WIN

-55

(10

M) 2

4 hr

WIN

-55

(10

M) 4

8 hr

WIN

-55

(10

0.01

0.1

1

10

TIMP-2

Treatment

Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

A B

C D

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO

(0.1

%)

IL-1

ng/ml)

M)+

IL-1

WIN

-55

(1

ng/ml)

M)+

IL-1

WIN

-55

(2.5

ng/ml)

M)+

IL-1

WIN

-55

(5

ng/ml)

M)+

IL-1

WIN

-55

(7.5

ng/ml)

M)+

IL-1

WIN

-55

(10

M)

WIN

-55

(1

M)

WIN

-55

(2.5

M

)

WIN

-55

(5

M)

WIN

-55

(7.5

M

)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

1000

10000

MMP-3


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M)+

IL-1

WIN

-55

(1

ng/ml)

M)+

IL-1

WIN

-55

(2.5

ng/ml)

M)+

IL-1

WIN

-55

(5

ng/ml)

M)+

IL-1

WIN

-55

(7.5

ng/ml)

M)+

IL-1

WIN

-55

(10

M)

WIN

-55

(1

M)

WIN

-55

(2.5

M

)

WIN

-55

(5

M)

WIN

-55

(7.5

M

)

WIN

-55

(10

0.01

0.1

1

10

TIMP-1


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M)+

IL-1

WIN

-55

(1

ng/ml)

M)+

IL-1

WIN

-55

(2.5

ng/ml)

M)+

IL-1

WIN

-55

(5

ng/ml)

M)+

IL-1

WIN

-55

(7.5

ng/ml)

M)+

IL-1

WIN

-55

(10

M)

WIN

-55

(1

M)

WIN

-55

(2.5

M

)

WIN

-55

(5

M)

WIN

-55

(7.5

M

)

WIN

-55

(10

0.001

0.01

0.1

1

10

100

1000

MMP-13


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

Contr

ol

DM

SO (0

.1%

)

ng/ml)+

DM

SO (0

.1%

)

IL-1

ng/ml)

M)+

IL-1

WIN

-55

(1

ng/ml)

M)+

IL-1

WIN

-55

(2.5

ng/ml)

M)+

IL-1

WIN

-55

(5

ng/ml)

M)+

IL-1

WIN

-55

(7.5

ng/ml)

M)+

IL-1

WIN

-55

(10

M)

WIN

-55

(1

M)

WIN

-55

(2.5

M

)

WIN

-55

(5

M)

WIN

-55

(7.5

M

)

WIN

-55

(10

0.01

0.1

1

10

TIMP-2


Rela

tive G

en

e E

xp

ressio

n

(Lo

g S

cale

)

A B

C D

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Sample ID Macroscopic Grade

Anatomic compartment

Age Sex Method

HC5 0 Posterior condyle

57 Female Cell culture treatment

HC6

0 Lateral tibial plateau


HC7 0 Posterior femoral condyle

72 Male Cell culture treatment

HC1

2 Femoral condyle 64 Male Cell culture treatment

HC5 2 Lateral femoral condyle


HC17 2 Trochlea 60 Female Cell culture treatment

HC13 2 Medial femoral condyle


HC23 2 Posterior condyle femoral medial

73 Female Cell culture treatment and receptor investigation

HC11 3 Posterior condyle 72 Male Cell culture treatment

HC15 3 Medial femoral condyle


HC16 3 Posterior femoral condyle

81 Male Cell culture treatment

HC3 3 Tibia 73 Female Receptor investigation

HC5 2-3 Medial femoral condyle

57 Female Receptor investigation

HC21 2 Lateral femoral condyle

64 Female Receptor investigation

Supplementary table 1: Patient sample information

Cannabinoid WIN-55,212-2 mesylate inhibits interleukin-1β induced matrix metalloproteinase and...

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