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Role of HNE in osteoartritic cartilage degradation.
Barbara J. Morquette, presentator Dr Mohamed Benderdour, director Dr Julio Fernandes, director. 06/06/05
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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
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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
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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
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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
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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
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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
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Aims . . We investigate the role of HNE in OA cartilage degradation through the modification of MMP-13 and type II collagen. . . 06/06/05
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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
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Specimens from OA patients undergoing total knee replacements
Methods and Results Specimens from OA patients undergoing total knee replacements 06/06/05
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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
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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
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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
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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
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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
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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
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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
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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
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