1. Role of Leptin in the Pathophysiology of Osteoarthritis Mohamed Aoulad Aissa, Aline Delalandre Daniel Lajeunesse Centre Hospitalier de l’Université de Montréal, Hôpital Notre-Dame,

2. Introduction Osteoarthritis (OA ) is characterized by: - Progressive articular cartilage loss - Bone sclerosis of the subchondral trabeculae and growth plate - formation of osteophytes OA risk factors: - Age (1/3 over 65 ) - Sex ( > women over 50) - Heredity - Obesity Adipocytes share a common mesenchymal stem cell precursor with osteoblasts, chondrocytes, tenocytes, and myoblasts, all affected by OA.

3. OA bone:Bone sclerosis: increased osteoid tissue Increased trabecular thickness (Tb.Th) Decreased trabecular space (Tb. Sp.) Hence:Altered mecanical properties Increased tissue rigidity Decreased shock absorbing capacities Caracteristics of osteoarthritic subchondral bone Normal OA

4. Role of Leptin in Joint Tissues Metabolism Leptin has dual effects, both central and local, depending on bone tissue, skeletal maturity and/or signaling. Leptin plays a role in endochondral ossification possibly via its influence on angiogenesis due to enhanced MMP-2 activity. Leptin acts as a growth factor for chondrocytes in young individuals yet its role on mature chondrocytes is unclear. Leptin enhances the metabolic markers of osteoblasts i.e. ALPase, osteocalcin, Coll1, IGF-1 and TGF- , all of which are elevated in OA osteoblasts.

5. HYPOTHESIS The local increase in leptin production and/or leptin signaling in OA Ob leads to the abnormal osteoblast cell function observed in OA and possibly abnormal bone remodeling in this pathology. This also facilitates cartilage deterioration and loss.

6. Phenotypic characteristics of normal and OA Subchcondral osteoblasts NormalOA 0 200 400 600 800 1000 1200 1400 NormalOA 0 100 200 300 400 500 Alkaline phosphatase (nmol/mg protein/30 min Osteocalcin (ng/mg protein/48 h p<0.01 p<0.02

7. Leptin expression by OA osteoblasts, chondrocytes and Synoviocytes by Real-time PCR OsteoblastsChondrocytesSynoviocytes 0.000e+0 1.000e-4 2.000e-4 3.000e-4 4.000e-4 Lep1 Lep2 Leptin/GAPDH ratio

8. NormalOA 0.00 0.05 0.10 0.15 0.20 0.25 Leptin/GAPDH (relative value) Leptin release by normal and OA Osteoblasts by Real-time PCR

9. Leptin release by normal and OA Osteoblasts Normal OA 0 20 40 60 80 100 Leptin (pg/mg protein/48 h) p<0.025

10. Regulation of Leptin Expression in OA Osteoblasts Measured by Real-time PCR BasalHGFTGF  D3D3 D 3 + HGF 0 2 4 6 8 10 12 Leptin expression (relative value) p<0.01

11. Basal+ anti OB-RL 0 100 200 300 400 Osteocalcin (ng/mg protein/48 h p<0.05 Effect of anti leptin receptor (OB-RL) antibodies on osteocalcin secretion by OA osteoblasts

12. Effect of anti leptin receptor (OB-RL) antibodies on alkaline phosphatase activity in OA osteoblasts Basal+ anti OB-RL Alkaline phosphatase (nmol/mg protein/30 min) 0 200 400 600 800 1000 1200 p<0.05

13. BasalD3D3 + leptinD3 + anti leptin 0 200 400 600 800 1000 1200 1400 Alkaline phosphatase (nmol/mg protein/30 min) Effect of leptin on alkaline phosphatase activity in OA osteoblasts

14. 85 60 50 Ctr 1,25(OH) 2 D 3 Lep Exo TGF  HGF M Induction of Leptin Receptor (OB-RB) in human osteoblasts treated with leptin, D 3, TGF , or HGF. Leptin Receptors were detected with a monoclonal anti-OB-RB antibody. Regulation of Leptin Receptor Production in OA Osteoblasts Measured by Western blot

15. DISCUSSION Leptin expression was restricted to subchondral Ob whereas we could not detect significant expression in chondrocytes nor synoviocytes. This would suggest that leptin found in articular cartilage must be due to this production by Ob. OA Ob produced more leptin than normal Ob. Enhanced leptin production by OA subchondral bone tissue may contribute to both abnormal expression of cellular markers by Ob and inflammation, leading to loss of cartilage. The interaction between TGF- , HGF, 1,25(OH) 2 D 3 and leptin that exist in OA bone tissue needs to be studied further.

16. Osteoblasts Osteoclast IL-6 IGF-1 TGF-  PGE 2 Tidemark Micro-fracture uPA, IGF-1 IL-1  IL-6 IGF-1 TNF  TGF-   bFGF Leptin HGF Collagenases Gelatinases Stromelysin NO, free radicals HGF/NK2 Trabecular bone Subchondral bone Cartilage Osteoblasts Osteoclast IL-6 IGF-1 TGF-  PGE 2 Tidemark Micro-fracture uPA, IGF-1 IL-1  IL-6 IGF-1TNF  TGF-  bFGF Leptin Collagenases Gelatinases Stromelysin NO, free radicals Trabecular bone Subchondral bone Cartilage ? ? Inflammation IL-1/IFN 

17. Acknowledgments Daniel Lajeunesse Aline Delalandre Denis Couchourel