0815 Lilla Simonaro VancouverFinal-pc

Chondrocyte Characterization
in MPS Disease:
The Role of GAG - Mediated Inflammation
Lilla Simonaro, PhD
Department of Genetics & Genomic Sciences
The Mount Sinai School of Medicine, New York
10th International Symposium on Mucopolysaccharide & Related Diseases
June 26-29, 2008 Vancouver, British Columbia, Canada
- GOAL OF OUR RESEARCH Investigate The Pathobiology Of MPS
Joints & Bones, & To Develop New
Therapeutic Approaches
Using Animal Models
FOCUS ON JOINT DISEASE
SINCE THIS IS A MAJOR CLINICAL
MANIFESTATION IN ALL
MPS PATIENTS
Sites Of Pathology
Articular Cartilage
Synovial Membranes
- WORKING HYPOTHESIS GAG Storage In MPS Connective
Tissue Cells & Matrices
Activation Of TLR4 Signaling Pathways
Inflammation
Proliferation
Apoptosis
Joint & Bone Disease
SIGNALING VIA TOLL - LIKE RECEPTOR 4
(Gallo et al., JBC 282, 2007)
Joint Injury
Bacteria
Joint Injury /
Matrix Breakdown
LPS-derived oligosaccharides
GAG-derived oligosaccharides
p38
MMP-13
TGF-β2
Inflammatory Cytokines
(TNF-α, IL-1β)
GAGs & INFLAMMATION
Wang, JY & Roehrl, PNAS, 22:14362-14367, 2002
Glycosaminoglycans are potential cause of rheumatoid arthritis.
Trowbridge JM, Gallo, RL. Glycobiology 12(9):117-125, 2002
Dermatan sulfate: new functions from an old glycosaminoglycan.
Kawashima et al. J Biol Chem 277(15):12921-12930, 2002
Oversulfated chondroitin/dermatan sulfates containing GlcAβ1/IdoAα13GalNAc(4,6-O-disulfate) interact with L- and P-selectin and chemokines.
Cripps et al. Int Immunopharmocol 5(11):1622-32, 2005
Modulation of acute inflammation by targeting glycosaminoglycan cytokine interactions.
Taylor KR, Gallo, RL. FASEB 20(1):9-22, 2006
Glycosaminoglycans and their proteoglycans: host-associated
molecular patterns for initiation and modulation of inflammation.
LIPOPOLYSACCARIDE (LPS)
Repeating Polymer of Uronic Acids & Amine Sugars
Potent Apoptotic Agent
Core glycolipid
O-specific polysaccharide chain
lipid A
n
(outer)
(inner)
O-specificn
oligosaccharide Core oligosaccharide
subunit
Uronic Acid
GlcN
GalN
Heptose
Glucose
INITIAL FOCUS ON MPS CHONDROCYTES
Simonaro et al., Lab Invest 81:1389, 2001
Enhanced chondrocyte apoptosis, proteoglycan depletion and inreased
collagen type II in mucopolysaccharidosis types I and VI.
Simonaro et al., Ped Res 57:701, 2005
Joint and bone disease in the mucopolysaccharidoses: Identification
of new therapeutic targets and biomarkers using animal models.
INITIAL CONCLUSIONS
•
GAGs Induce Apoptosis In Chondrocytes At A Level Equivalent Or
Greater Than LPS
•
MPS Chondrocytes Exhibit Excess, Age - Dependent Apoptosis
•
In Response To Apoptosis, A Preponderance Of Immature
Chondrocytes Is Seen In MPS
•
MPS Chondrocytes Release Numerous Inflammatory Cytokines
(TNF-α, IL-1β, etc.) & Degradative Proteases (MMPs) Resulting in
Inflammation & Matrix Destruction
Overall, The Process Of Apoptosis &
Inflammation Induced By GAG Storage
Leads To Massive Disorganization &
Break - Down Of Articular Cartilage
RECENT STUDIES HAVE FOCUSED ON
INVESTIGATING MPS SYNOVIAL TISSUE
A Major Site Of Pathology In Arthritis & Other
Inflammatory Bone Diseases
Simonaro et al., Amer J Path 72(1):112, 2008
Mechanism of glycosaminoglycan-mediated bone and joint disease:
implications for the mucopolysaccharidoses and other connective tissue
diseases.
GENE EXPRESSION ARRAYS OF NORMAL & MPS VI
RAT SYNOVIAL FIBROBLASTS
• 375 Genes Over - Expressed In MPS VI Cells
• Numerous Inflammatory & Signaling - Related
Genes, Including Many Related To The LPS Pathway
• Many Of The Over - Expressed Genes Confirmed
By Protein Expression Studies
A Similar Inflammatory Disease Process Occurs In
Synovial Tissue As Cartilage
MOST OVER - EXPRESSED GENES IN MPS SYNOVIUM
ASSOCIATED WITH INFLAMMATION / LPS ACTIVATION
Fold Change Gene
13.6
12.6
11.8
10.6
10.4
10.2
9.8
9.6
9.6
8.2
7.0
5.8
C1qb
β2 integrin
CXCR4
Aif1
Mpeg1
KFMS
L-CA
LCR1
FcγRrIII
MIP-1α
CD14
LBP
Gene Description
complement component 1, q subcomponent,b
integrin β2 α subunit
CXC chemokine receptor
allograft inflammatory factor 1
macrophage expressed gene 1
macrophage CSF receptor precursor
leukocyte common antigen
chemokine receptor LCR1
Ig-epsilon receptor γ precursor
macrophage inflammatory protein-1α
CD14 antigen
lipopolysaccharide binding protein
TLR4, MyD88, LBP & CD44 EXPRESSION IN
RAT SYNOVIOCYTES & CHONDROCYTES
Fibroblast-Like
Synoviocytes
Normal
TLR4
MyD88
LPS
INDUCED
LBP
GAG INDUCED
CD44
PKC
Loading Control
MPS VI
Chondrocytes
Normal
MPS VI
CO-LOCALIZATION OF TLR-4 IN RAT SYNOVIAL FIBROBLASTS
MPS VI
Normal
Hoechst
TLR-4
Lysotracker
Merged
MMP-13 IN RAT SYNOVIAL FIBROBLASTS
A
Normal
MMP-13(μg/μl)
0.3
MPS VI
*
MMP-13
0.2
GAPDH
0.1
Normal
0.0
Normal
MPS VI
MPS VI
TNF-α MEDIATED OSTEOCLASTOGENESIS
TNF-α
p38
RANKL (synovial cells, T cells, osteoblast/stromal cells)
M-CSF
RANK
Proliferation
HSC
Differentiation
TRAP+ Mononuclear
osteoclast precursor
Multinucleated osteoclast
RANKL IN RAT SYNOVIOCYTES & CHONDROCYTES
RANKL (pmo/l)
4
*
3
2
0
Normal
MPS VI
Chondrocytes
MPS VI
Normal
Synoviocytes
Normal
MPS VI
RANKL
529 bp
GAPDH
496 bp
POTENTIAL BIOMARKERS FOR THE
MPS DISORDERS
TNF-α
TGF-β
IL-1β
MMPs
RANKL
CD44
Others?
Can Altered Expression Be Observed In
Human Serum and/or Synovial Fluid As A Function
Of Disease Progression and / or Treatment?
PRO-INFLAMMATORY CYTOKINES
IN DOG SYNOVIAL FLUID
75
30
*
IL1-β (pg/ml)
TNF-α (pmol/l)
60
45
30
*
20
10
15
0
0
Normal
MPS VI
Normal
MPS VI
Synovial Fluid Might Be An Excellent Source
Of Biomarkers In MPS Patients
IL-1β RELEASED INTO MPS SYNOVIAL FLUID
40
IL1-β (pg/ml)
30
*
*
20
10
*
0
Normal
Cat
Normal
Cat
MPS VI MPS VII
Cat
Dog
MPS I
Dog
MPS & SPHINGOLIPID SIGNALING
LPS SIGNALING & SPHINGOLIPIDS
- Link To Arthritis • Cuscheri et al., Surg Infect, 2007. Acid sphingomyelinase is required for lipid
raft TLR4 complex. F
ormation
• Kim et al., Biochem Biophys Res Commun, 2006. Sphingosine 1- phosphate
stimulates rat primary chondrocyte proliferation.
• Kitano et al., Arthritis & Rheum, 2006. Sphingosine 1-phosphate/sphingosine
1-phosphate receptor 1 signaling in rheumatoid synovium.
• Migita et al., Biochem Biophys Res Commun, 2000. Regulation of rheumatoid
synovial cell growth by ceramide.
SPHINGOMYELIN
Smases
Cdases
CERAMIDE
APOPTOSIS
SPHINGOSINE
S1P
PROLIFERATION
CERAMIDE LEVELS IN NORMAL RAT CHONDROCYTES
TREATED WITH DERMATAN SULFATE
120
% Increase
100
80
60
Consistent With GAGInduced Apoptosis
40
20
0
0
10
30
120
Time (min) Post-Addition of Dermatan Sulfate
CERAMIDE LEVELS IN RAT SERUM
% of Normal
200
*
150
100
50
0
Normal
MPS VI
ACID SPHINGOMYELINASE ACTIVITY IN RAT SERUM
12000
ASM Activity (pmol/ml/h)
*
10000
8000
6000
4000
2000
0
Normal
MPS VI
SPHINGOSINE-1-PHOSPHATE LEVELS IN MPS VI RATS
Synovial Fibroblasts
900
Serum
*
750
0.20
600
S1P (μM)
S1P (pmol/mg)
*
0.25
450
300
0.15
0.10
0.05
150
0.00
0
Normal
MPS VI
Normal
MPS VI
PROLIFERATION IN RAT SYNOVIAL FIBROBLASTS
*
Absorbance (490nm)
0.4
*
0.3
0.2
*
*
0.1
0
6
12
24
48
Time (Hours)
Normal
MPS VI
SUPPLEMENTAL THERAPIES
FOR THE MPS DISORDERS
Enzyme Replacement Therapy
+
Cytokine Inhibitors
Anti TNF-α (Remicade™, Enbrel ™)
Infliximab (Remicade™)
• Mouse-Human Chimeric Anti Human TNF-α Antibody
9 FDA APPROVED
• Targets, Binds & Blocks TNF-α
• Helps To Control The Inflammation Process
• Works Quickly. Many RA Patients Begin To Experience Relief In
As Soon As 2 Weeks
• Administered By A 2-Hour IV Infusion (3 mg/kg) & Is Used In
Patients With Moderate To Severe RA
TNF-α LEVELS IN REMICADE™ TREATED MPS VI RATS
2.0
1.8
1.6
1.6
1.4
1.4
1.2
1.2
1
1.0
0.8
0.8
0.6
0.6
0.4
0.4
Serum TNF-α(pg/ml)
1.8
0.2
0.2
0
0.0
1
6
Animals injected at 6 months of age
Animals injected at 1 month of age
Normal Serum
0
2
4
6
8
10
12
14
Weeks Post-Injection of 3 mg/kg I.V. of Remicade™
16
p38 & COX-2 EXPRESSION IN RAT SYNOVIOCYTES
Fibroblast-Like Synoviocytes
Normal
p38
Cox-2
PKC
MPS VI
MPS VI Treated
RANKL LEVELS IN REMICADE™ TREATED MPS VI RATS
6.0
6
Serum RANKL pmol/l
5.0
1.8
1.6
4.0
1.4
1.2
1
3.0
Animals injected at 6 months of age
2.0
0.8
0.6
1.0
Animals injected at 1 month of age
Normal
0.4
0.2
0.0
1
0
0
2
4
6
8
10
12
14
Weeks Post-Injection of 3 mg/kg I.V. of Remicade™
16
SIX MONTH TREATMENT OF MPS VI RATS WITH REMICADE™
Normal
MPS Treated*
No Gross
Differences
MPS
Joint Pathology?
Age (Months)
Normal (n=4)
MPS VI (n=4)
MPS VI Treated (n=3)
7
7
7
Weight (Grams)
418
260
267
Length (Inches)
9
7
7
OUR HYPOTHESIS: “MPS” SIGNALING PATHWAY
GAGs
TNF-α
CHONDROCYTES
ECM
CD44, MyD88 ,p38
LPS
LBP, MyD88
MMP-13, TGF-β2
APOPTOSIS
TLR-4
TNFR-1
Ceramide
FAS
TNF-α
TNF-α
p38
ASM
S1P
NF-κB
COX-2
INFLAMMATION & PROLIFERATION
SYNOVIOCYTES
ACKNOWLEDGEMENTS
Edward H. Schuchman, PhD
Efrat Eliyahu, PhD
Yi Ge, PhD
Xingxuan He, MD
Nataly Shtraizent, MS
Schuchman Lab
Collaborators:
Mark Haskins, VMD, PhD, University of Pennsylvania
Marina D’Angelo, PhD, Philadelphia College of Osteopathic Medicine
Research Funded by:
The NIH, The National MPS Society, and The Isaac Foundation