1. Role of HNE in osteoartritic cartilage degradation.
Barbara J. Morquette, presentator
Dr Mohamed Benderdour, director
Dr Julio Fernandes, director.
06/06/05

2. Plan Osteoarthritis (OA) 4-Hydroxy-2-Nonenal (HNE) Aims
Methods and results
Modulation of MMP-13 and type II collagen
Signalling pathway investigation
Conclusion and perspectives
06/06/05

3. Osteoarthritis (OA)
65% of the population over 60 suffers from OA and this ratio increases with age.
Degenerative disease
gradual loss of articular cartilage
inflammation.
Oxidative stress
ROS
Normal OA
06/06/05

4. Biochemical stages of OA
HNE
MMP-13
ExtraCellular Matrix degradation:
Change in chondrocytes metabolism,
Type II collagen degradation (via MMP-13),
Proteoglycan degradation.
06/06/05

5. HNE: 4 Hydroxy-2-Nonenal
HNE: end product of lipid peroxidation.
Results from free radicals attack on -3 and -6 polyunsaturated fatty acids.
Highly reactive compound.
06/06/05

6. Phospholipidic membrane
HNE Actions
ROS
Nitric Oxide (NO)
Phospholipidic membrane
HNE Proteins Target
HNE
HNE signaling
Enzymes
G-6-P DmNADP-ICDH
Pyruvate deshydrogenase
C T E
Proteins kinase
JNK
Function
Contraction proteins
type II Collagen
Proteins kinase
p38
JNK
Genes
COX-2
iNOS
Transcription Factors
AP-1
NF-kB
BIOLOGICAL
EFFECTS
26/11/04

7. HNE Cartilage degradation AP-1 NF-kB NSAIDs NSAIDs NF-kB MAPK NF-kB
COX-1
COX-2
AP NF-kB
Prostaglandines
Thromboxanes
MMP-13
MMP-1
PGE2 , PGI2 , PGE2-R
Coll type II
Coll type I
Cartilage
degradation
bone erosion
Inflammation
06/06/05

8. Aims . .
We investigate the role of HNE in OA cartilage degradation through the modification of MMP-13 and type II collagen. . .
06/06/05

9. Previous results  HNE/protein adducts formation in
OA chondrocytes treated with ROS
in OA synovial fluid.
HNE seems to initiate OA cartilage degradation via:
 MMP-13 expression through p-38 MAPK phosphorylation.
 type II collagen expression in OA
06/06/05

10. Specimens  from OA patients undergoing total knee replacements
Methods and Results
Specimens  from OA patients undergoing total knee replacements
06/06/05

11. Type II collagen degradation
Human OA cartilage explants
Incubated with  concentration of HNE
Measurement of HYP release
Measurement of type II collagen neoepitopes generation.
Commercial Kit
Action of HNE on coll II turnover.
06/06/05

12. Results:
 HYP release & type II collagen fragments generation in a dose-dependent manner.
Measurement of HYP release by OA human cartilage explants incubated with increasing concentration of HNE.
Measurement of type II neoepitopes formation in OA human cartilage explants incubated with increasing concentration of HNE.
06/06/05

13. Post-translational modifications
Investigation:
Post-translational modifications
Susceptibility of HNE/ type II collagen adducts to the degradation by MMP-13
Incubation of type II collagen with:
 concentrations of HNE
activated MMP-13
HNE/ type II collagen adducts formation
OA cartilage explants
Incubated with ROS, TNF- or HNE
Immunoprecipitation / western blot
Pour tester la conséquence de la modification du COLL II par le HNE en se qui attrait sa degradation par la MMP-13
06/06/05

14. HNE  type II collagen degradation by MMP-13 via adducts formation.
Results:
type II collagen degradation & adducts formation
Control
MMP-13 (2 U/ml)
HNE (mM)
HNE  type II collagen degradation by MMP-13 via adducts formation.
110 kDa
75 kDa
Control
TNFa
H2O2
SIN
HNE
HNE/type II collagen
adducts
06/06/05

15. Post-translational modifications
Investigation:
Post-translational modifications
MMP-13 recombinant
Incubate with  concentrations of HNE
at 37°C for 1h
Measurement of MMP-13 activation
Elisa Kit ( commercial).
Measurement of HNE proteolytic effect & adducts formation
Western blot.
06/06/05

16.  MMP-13 activity with HNE
Results:
*** **
 MMP-13 activity with HNE *
Molar Ratio (MMP-13:HNE)
No proteolytic effects of HNE on MMP-13
CTL
APMA
1:1
1:10
1:102
1:103
55 kDa
46 kDa
HNE/MMP-13 adduct
06/06/05

17. Conclusion et perspectives
HNE seems to be implicated in OA cartilage degradation via:
 MMP-13 activity &  type II collagen degradation via adducts formation.
First study to demonstrate the implication of HNE in OA pathophysiology.
Perspectives:
clarify the role of HNE in inflammatory process and chondrocytes apoptosis in OA.
06/06/05

18. Laboratoire de recherche en
Acknowledgments
Laboratoire de recherche en
orthopédie de l'hôpital du Sacré-Cœur
Dr Julio Fernandes, laboratory director,
Mohamed Benderbour, director,
Shi Qin, Patrick Lavigne
l’équipe de travail….
Fonds de la recherche
en santé
Québec
Thanks
06/06/05