Transcript What destroys cartilage in osteoarthritis and how can we stop it?"

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.
Osteoarthritis
NORMAL
OSTEOARTHRITIC
• > 6 million people in UK with
moderate to severe OA
• predominantly age > 45 with
major morbidity in age > 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
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
Collagen
Aggrecan
IGD
G2
G1
G3
Y
Y
Link
Protein
Keratan Sulphate
Rich Region
Hyaluronan
Chondroitin Sulphate
Rich Region
Cartilage
Synovial fluid
Cartilage
Cross section of
cartilage
Bone
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
The balancing act of cartilage turnover…
(M)MP
breakdown
TIMP
build up
TIMP
(M)MP
build up
breakdown
Cartilage turnover
Cartilage degradation
Which metalloproteinases are
expressed by cartilage? Does
this change in disease?
Kevorkian, Davidson, Swingler
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
Cartilage samples
Osteoarthritis (OA)
femoral head from THR for osteoarthritis
(n=18, age 38-81)
‘Normal’
femoral head from THR following fracture to
the neck of femur ‘NOF’ (n=15, age 52-93)
(Mr Simon Donell, Consultant Orthopaedic Surgeon, NNUH)
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 >1 exon)
(products verified by sequencing)
Real time PCR
Expression profile of MMP, ADAMTS and TIMP family
in normal vs. OA cartilage (mean Ct values)
MMP
1
2 3
7
8 9 10 11 12 13 14 15 16 17 19 20 21 23 24 25 26 27 28
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 CT=40
TIMP
1
OA
N
2
Low expression CT 36-39.
3
4
Moderate expression CT 31-35.
High expression CT= 26-30
Very high expression CT< 25
Genes that are up-regulated in OA
P< 0.001
MMP-28/18S
(arbitrary units)
MMP-13/18S
(arbitrary units)
300
250
200
150
100
50
0
OA
MMP-13
MMP
MMP-2
MMP-9
MMP-16
Normal
1.00
45
40
35
30
25
20
15
10
5
0
ADAMTS-16/18S
(arbitrary units)
350
0.75
0.50
0.25
0.00
OA
Normal
MMP-28
ADAMTS
ADAMTS-2
ADAMTS-12
ADAMTS-14
OA
Normal
ADAMTS-16
TIMP
TIMP-3
Genes that are down-regulated in OA
P< 0.001
2
2500
5.0
2.5
0.0
2000
ADAMTS-1/18S
(arbitrary units)
MMP-3/18S
(arbitrary units)
MMP-1/18S
(arbitrary units)
7.5
1500
1000
500
0
0
OA
MMP-1
MMP
MMP-10
Normal
1
OA
Normal
MMP-3
ADAMTS
ADAMTS-5
ADAMTS-9
ADAMTS-15
OA
Normal
ADAMTS-1
TIMP
TIMP-1
TIMP-4
MMP28 expression in upregulated in RA cartilage
Momohara et al. Arth Rheum (2004) 50:4074
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
The human degradome
570 proteases
Threonine (28)
Aspartate (21)
Serine (176)
Cysteine (154)
Metallo- (191)
How can we stop cartilage degradation?
Dietary factors
Association between osteoarthritis and
obesity suggests a possibility of an
association with diet
Observational Study
• St Thomas’ Twins UK registry
• Matched co-twin case control design
compares discordant exposuredisease status
• Radiographic OA determined at the
hand hip and knee
• Lumbar and cervical degeneration
assessed by MRI
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
Peripheral Joints
Knee
Hand
A B C D E
A B C D E
Odds ratio
Hip
A B C D E
Dietary pattern
Odds ratio
Fruit and vegetable pattern at the hip
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
Allium
vegetables
Cruciferous
vegetables
Laboratory studies
Organosulphur compounds derived from garlic oil
Sulforaphane
1-isothiocyanato-(4R)-(methylsulfinyl) butane
myrosinase
glucoraphanin
Plant injury
Pathogen attack
Chewing
Food prep
SFN
Histone acetylation
AcH3
AcH3
Total H3
Total H3
AcLys
AcLys
SFN (M)
IL-1/OSM
SW1353 cell line
DADS (M)
IL-1/OSM
Chondroprotection – sulforaphane
46kDa
MMP-1
43kDa
MMP-13
SFN (µM)
IL-1/OSM
0
+
5
+
10
+
15
+
30
+
IL-1/OSM
DADS (M)
IL-1
4
8
16
32
0
4
8
16
32
1
4
8
16
32
2
Hydroxyproline Loss (%)
4
on
tr
o
IL I l
1/ L1
O
SM
Relative MMP-13 Expression
(arbitrary units)
5
C
IL-1
4
8
16
32
4
8
16
32
4
8
16
32
on
tr
o
IL I l
1/ L1
O
SM
C
Chondroprotection – diallyl disulphide
6
70
40
10
1.6
1.2
0.8
0.4
0.0
IL-1/OSM
DADS (M)
Mechanism?
SPN
Ub
Keap1
Nrf2
Nrf2
Nrf2
ARE
Cytoprotective genes
HO-1
GSH
NQO1
- Anti-inflammatory effects
Haem oxygenase-1 expression
primary human articular chondrocytes
***
***
*
***
Nrf2 dependent
SFN does not regulate MMP1 expression
via Nrf2
Nrf2 independent
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
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
Acknowledgements
Clark lab
Kirsty Culley
Rose Davidson
Orla Jupp
Janine Morris
Ursula Rodgers
Sarah Snelling
Tracey Swingler
Institute of
Orthopaedics, NNUH
Simon Donell
Clare Darrah
Adele Cooper
School of Medicine,
UEA
Yongping Bao
Aedin Cassidy
Alex MacGregor
Kings College London
Frances Williams
Tim Spector
MMP-28
Kevorkian, Rodgers
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 119:14-21
Epilysin (MMP-28) induces EMT in A549 cells
Control
MMP-28
EA mutant
Illman, S. A. et al. J Cell Sci 2006;119:3856-3865
Expression of MMP-28 in HeLa cells
(pcDNA4-FLAG, transient transfection)
Cell lysate
Vector MMP-28-FLAG
Conditioned medium
Vector MMP-28-FLAG
75
50
75
50
37
Pro
37
25
Pro
Extracellular matrix
Vector MMP-28-FLAG
CTD
75
50
37
Pre Pro Fu Catalytic Zn
Western blot, Anti-FLAG
Pro
Active
Haemopexin
Expression of MMP-28 in SW1353 cells
(pcDNA4-FLAG, stable transfection)
Conditioned medium
V MMP28
Pro
Cell lysate
V MMP28
Extracellular matrix
V MMP28
Pro
Pro
Active
CTD
Pre Pro Fu Catalytic Zn
Western blot, Anti-FLAG
Haemopexin
Activation of proMMP-28 require furin activity
Extracellular matrix
Vector only
+
MMP-28-FLAG
+ furin inhibitor
75
50
Pro
Active
37
Western blot, anti-FLAG antibody
Furin inhibitor = Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone
Expression of MMP-28 in SW1353 cells
Impact on other metalloproteinases
3.5
MMP28/18S
Arbitrary Units
3
2.5
2
1.5
1
0.5
0
1
2
1
2
1
2
1
2
Vector only Wild type EA mutant Pro-cat
3.5
Arbitrary Units
MMP2/18S
3
**
2.5
2
***
1.5
***
1
****
0.5
0
1
2
1
2
1
2
1
2
Vector only Wild type EA mutant Pro-cat
Expression of MMP-28 in SW1353 cells
Impact on other metalloproteinases
3.5
Arbitrary Units
MMP2/18S
3
**
2.5
2
***
1.5
***
1
****
0.5
0
1
2
1
2
1
2
1
2
Vector only Wild type EA mutant Pro-cat
Gelatin
zymography
Vector
only Wild type
EA mutant
Pro-cat
Expression of MMP-28 in SW1353 cells
Immunocytochemistry
Wild-type
EA mutant
Pro-cat
Vector only
Permeabilized
Non-permeabilized
Wild-type
EA mutant
Pro-cat
Vector only
Expression of MMP-28 in SW1353 cells
Actin cytoskeleton (phalloidin)
Vector
only
EA mutant
Wildtype
Pro-cat
Expression of MMP-28 in SW1353 cells
Adhesion and migration
4
3.5
3
****
3
****
2.5
****
****
2
*
**
1.5
Fold change
Fold change
3.5
0.5
1
2
EA mutant
1
2
Pro-cat
μm/hour
1
2
Wild type
1
2
EA mutant
Type II collagen
Fibronectin
Migration
40
35
****
30
**
**
****
25
****
****
15
10
5
0
1
2
Vector only
1
2
Vector only
Adhesion
45
20
0
Adhesion
50
1
2
1
2
Wild type EA mutan
1
2
Pro-cat
****
***
1.5
0.5
1
2
Wild type
****
*
2
1
1
2
Vector only
****
2.5
1
0
****
1
2
Pro-cat
Summary
• recombinant proMMP-28 is a secreted protein, but fulllength 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