What destroys cartilage in osteoarthritis and how can we

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What destroys cartilage in osteoarthritis and how
can we stop it?"
Ian M Clark, PhD Biomedical Research
Centre, University of East Anglia, Norwich, NR4
7TJ. United Kingdom.
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Osteoarthritis
NORMAL
OSTEOARTHRITIC

• gt 6 million people in UK with moderate to severe
  OA
• predominantly age gt 45 with major morbidity in
  age gt 60
• 3 million GP visits for OA in yr 2000
• approx. 3 billion in lost productivity

Arthritis The Big Picture. Arthritis Research
Campaign report May 2002
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Changes in cartilage during OA
NORMAL
OSTEOARTHRITIC

• Biochemical changes
• decrease in aggrecan
• damage to the type II collagen network
• Morphological changes
• fibrillations/pitting
• softening and loss of cartilage thickness
• Enzymatic changes

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Collagen
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Aggrecan
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Cartilage
Cross section of cartilage
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Metalloproteinases and their inhibitors
Matrix metalloproteinases 23 human
enzymes (Collagenases MMP-1, -8, -13, -2, -14?)
ADAMTSs 19 human enzymes (Aggrecanases ADAMTS-1,
-4, -5, -8, -9, -15?)
TIMPs 4 human inhibitors
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The balancing act of cartilage turnover
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Which metalloproteinases are expressed by
cartilage? Does this change in disease?
Kevorkian, Davidson, Swingler
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Hip replacement
In Europe, a joint is replaced due to
osteoarthritis every 1.5 minutes. Wieland et al
(2005)
2006/7 in the UK, one hip or knee replaced every
4 minutes
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Cartilage samples Osteoarthritis (OA) femoral
head from THR for osteoarthritis (n18, age
38-81) Normal femoral head from THR
following fracture to the neck of femur NOF
(n15, age 52-93)
(Mr Simon Donell, Consultant Orthopaedic Surgeon,
NNUH)
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Assay

• Taqman quantitative real-time RT-PCR
• (steady state mRNA levels measured)
• primer/probe sets for
• 23 human MMPs, 19 human ADAMTSs, 4 TIMPs
• (designed across gt1 exon)
• (products verified by sequencing)

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Real time PCR
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Expression profile of MMP, ADAMTS and TIMP family
in normal vs. OA cartilage (mean Ct values)
MMP
1
2
3
8
9
10
12
11
13
14
15
16
17
19
20
21
23
24
25
26
27
28
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OA
N
ADAMTS
1
2
3
4
5
6
7
8
9
10
12
13
14
15
16
17
18
19
20
OA
N
Not detected CT40
TIMP
Low expression CT 36-39.
1
2
3
4
Moderate expression CT 31-35.
OA
High expression CT 26-30
N
Very high expression CTlt 25
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Genes that are up-regulated in OA
MMP ADAMTS
TIMP MMP-2
ADAMTS-2
TIMP-3 MMP-9 ADAMTS-12
MMP-16
ADAMTS-14
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Genes that are down-regulated in OA
Plt 0.001
MMP-1 MMP-3
ADAMTS-1
MMP ADAMTS
TIMP MMP-10
ADAMTS-5
TIMP-1
ADAMTS-9
TIMP-4
ADAMTS-15
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MMP28 expression in upregulated in RA cartilage
Momohara et al. Arth Rheum (2004) 504074
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Summary

• First expression profile to assay all MMPs,
  ADAMTSs and TIMPs in cartilage
• MMP-28 and ADAMTS-16 expression is
  significantly increased in end-stage OA

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The human degradome 570 proteases
Aspartate (21)
Threonine (28)
Cysteine (154)
Serine (176)
Metallo- (191)
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How can we stop cartilage degradation? Dietary
factors
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• Association between osteoarthritis and obesity
  suggests a possibility of an association with diet

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Observational Study

• St Thomas Twins UK registry
• Matched co-twin case control design compares
  discordant exposure- disease status
• Radiographic OA determined at the hand hip and
  knee
• Lumbar and cervical degeneration assessed by MRI

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Dietary intake

• Food frequency questionnaire (EPIC format)
• Prior analysis indicated 5 discrete patterns of
  intake, that were used as the main variables in
  the analysis
• A Fruit and vegetable pattern
• B High alcohol pattern
• C Traditional English
• D Dieting pattern score
• E Low meat pattern score
• Teucher et al 2008

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Peripheral Joints
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Fruit and vegetable pattern at the hip
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Comment

• Matched analysis indicates that these findings
  are less likely to be confounded by other
  lifestyle factors
• However, the effects are small effects and
  (despite the dietary pattern approach) not robust
  to multiple testing

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Allium vegetables
Cruciferous vegetables
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Laboratory studies Organosulphur compounds
derived from garlic oil
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Sulforaphane1-isothiocyanato-(4R)-(methylsulfinyl
) butane
myrosinase
Plant injury Pathogen attack Chewing Food prep
glucoraphanin
myrosinase
glucoraphanin
SFN
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Histone acetylation
SW1353 cell line
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Chondroprotection sulforaphane
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Chondroprotection diallyl disulphide
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Mechanism?
Keap1
Ub
Nrf2
Nrf2
Nrf2
ARE
HO-1 GSH NQO1 - Anti-inflammatory effects
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Haem oxygenase-1 expressionprimary human
articular chondrocytes




Nrf2 dependent
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SFN does not regulate MMP1 expression via Nrf2
Nrf2 independent
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Summary

• Epidemiology shows a protective association
  between allium intake and osteoarthritis
• In chondrocytes, SFN and DADS can attenuate the
  expression of metalloproteinase genes in a dose
  dependent manner
• This is likely not through the inhibition of
  histone deacetylases and not via Nrf2
• Both compounds induces HO-1 which is itself
  chondroprotective
• Both compounds can abrogate cartilage destruction
  in the BNC model in vitro
• At least SFN can abrogate MMP expression and
  cartilage destruction in human cartilage in vitro

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Conclusions

• A complete understanding of protease expression
  and activity in the joint will allow drug
  development in this area
• Dietary bioactives may represent an alternative
  strategy in the prevention or treatment of
  osteoarthritis
• A thorough understanding of disease association,
  mechanism(s), metabolism and pharmacodynamics is
  needed to underpin this premise

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Acknowledgements
Institute of Orthopaedics, NNUH Simon
Donell Clare Darrah Adele Cooper
Clark lab Kirsty Culley Rose Davidson Orla
Jupp Janine Morris Ursula Rodgers Sarah
Snelling Tracey Swingler
Kings College London Frances Williams Tim Spector
School of Medicine, UEA Yongping Bao Aedin
Cassidy Alex MacGregor
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MMP-28
Kevorkian, Rodgers
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Expression of MMP-28 in skin

• expressed strongly in keratinocytes in vitro and
  in vivo

• induced by TNF?
• Saarialho-Kere et al. (2002) J Invest Dermatol
  11914-21

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Epilysin (MMP-28) induces EMT in A549 cells
Control MMP-28 EA mutant
Illman, S. A. et al. J Cell Sci 20061193856-3865
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Expression of MMP-28 in HeLa cells (pcDNA4-FLAG,
transient transfection)
Western blot, Anti-FLAG
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Expression of MMP-28 in SW1353 cells (pcDNA4-FLAG,
stable transfection)
Conditioned medium V MMP28
Cell lysate V MMP28
Extracellular matrix V MMP28
Pro CTD
Pro Active
Pro
Western blot, Anti-FLAG
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Activation of proMMP-28 require furin activity
Western blot, anti-FLAG antibody Furin inhibitor
Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone
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Expression of MMP-28 in SW1353 cells Impact on
other metalloproteinases
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Expression of MMP-28 in SW1353 cells Impact on
other metalloproteinases
Gelatin zymography
Vector only Wild type EA mutant
Pro-cat
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Expression of MMP-28 in SW1353 cells Immunocytoche
mistry
Permeabilized Non-permeabilized
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Expression of MMP-28 in SW1353 cells Actin
cytoskeleton (phalloidin)
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Expression of MMP-28 in SW1353 cells Adhesion and
migration
Adhesion Fibronectin
Adhesion Type II collagen Migration
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Summary

• recombinant proMMP-28 is a secreted protein, but
  full-length MMP-28 is also associated with the
  cell surface
• activation of proMMP-28 is via a pro-protein
  convertase with the active form predominantly
  bound to ECM
• expression of active MMP-28 induces MMP-2
  expression and activity
• expression of MMP-28 alters cytoskeleton
• MMP-28 expression increases adhesion to type II
  collagen and fibronectin, but only decreases
  migration on the former
• MMP-28 expression alters the cell proteome