Background Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and osteoclast (OC) mediated bone erosions. AlphaVbeta3 (αvβ3) integrin is highly expressed in osteoclasts and its inhibition disturbs their function. Avβ3 blocking antibodies can reduce bone resorption and mice lacking β3 are osteopetrotic. However, the role of αvβ3 in the development of collagen induced arthritis (CIA), a well established model for human RA, has not been examined extensively.
Objectives To study the role of the αvβ3 inhibitor cilengitide, a synthetic Arginine-Glycine-Asparagine amino acid peptide (RGD peptide), on osteoclastogenesis and its efficacy in preventing and treating CIA.
Methods For in vitro analysis mouse bone marrow-derived cells (BMCs) were differentiated into tartrate resistant acid phosphatase positive (TRAP+) mononuclear OC precursor cells (pre-OCs) and TRAP+ multinucleated mature OCs in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Cilengitide, was added in increasing concentrations (2nM to 20μM) to the culture. Moreover, we performed these osteoclastogenesis assays on plates coated with RGD containing matrix molecules such as osteopontin, fibronectin and fibrinogen but also on Poly-D-lysine coatings to asses for αvβ3 independent adhesion.
For in vivo analysis CIA was induced in 6-8 week old male DBA/1 mice by immunisation with bovine type II collagen at day 1, followed by boosting at day 21. For the CIA prevention study mice were injected 1,5mg/kg cilengitide (n=15) or placebo (n=15) subcutaneously (s.c.), 5 days per week, starting 1 day prior to CIA induction until day 53. In the CIA treatment study mice with established arthritis were randomized and treated s.c. with 1,5 mg/kg (low dose, n=19) or 75 mg/kg (high dose, n=7) cilengitide or placebo (n=21) 5 days per week until day 59. The preventive and treatment effects were evaluated by investigating the clinical course of arthritis assessed by paw thickness and grip strength.
Results In vitro increasing concentrations of cilengitide (IC50: 250nM) dose-dependently reduced pre-OC numbers on all plate coatings, indicating an inhibiting effect at the early stage of pre-OC proliferation. OCs were significantly reduced between 20nM and 200nM, followed by complete blockade of OC formation above 2μM. At 200nM an intriguing morphological difference was observed with reduction in OC size, suggesting that cilengitide may disrupt spreading and the fusion capacity at the early pre-OC stage. In the in vivo preventive experiment, cilengitide significantly reduced incidence (92,8% vs. 40%) and severity of CIA as evidenced by the reduction of the clinical disease activity scores of paw swelling and grip strength. In the in vivo treatment experiment, both low dose and high dose cilengitide effectively inhibited the progression of established arthritis.
Conclusions Osteoclastogenesis requires intact αvβ3 integrin function. Systemic αvβ3 integrin inhibition with cilengitide potently prevents and treats experimental CIA arthritis. Therefore, cilengitide may be a novel therapeutic target in RA.
Disclosure of Interest D. Sykoutri: None Declared, G. Nisha: None Declared, S. Hayer: None Declared, P. Mandl: None Declared, J. Smolen: None Declared, G. Prager Grant/research support from: Merck KGaA, K. Redlich: None Declared