Background Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and osteoclast mediated bone erosions. AlphaVbeta3 (αvβ3) Integrin is highly expressed in osteoclasts and its inhibition disturbs osteoclast function in vivo. However, the role of αvβ3 Integrin 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, an Arginine-Glycine-Asparagine amino acid peptide (RGD peptide), on osteoclastogenesis in vitro and to assess its efficacy in preventing CIA in vivo.
Methods For in vitro validation of osteoclastogenesis mouse bone marrow derived cells were differentiated into tartrate-resistant acid phosphatase positive (TRAP+) mononuclear osteoclasts (pre-osteoclasts) and TRAP+ multinucleated mature osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Cilengitide, kindly provided by Merck KGaA, was added in increasing concentrations (2nM to 20μM) to the culture. Moreover, we performed osteoclastogenesis assays on osteopontin, fibronectin and fibrinogen matrix coated plates. In order to asses for αvβ3 integrin independent adhesion, osteolastogenesis assays were performed on Poly-D-lysine coated plates. CIA was induced in male DBA/1 mice by immunisation with bovine type II collagen (CII) at day 1, followed by boosting at day 21. For the CIA prevention study mice were injected 15mg/kg cilengitide subcutaneously, 5 days per week, starting 1 day prior to CIA induction until day 50. The preventive effect was 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-osteoclast numbers on all plate coatings, indicating an inhibiting effect on early stages of pre-osteoclast proliferation. Interestingly, with respect to osteoclast numbers a severe reduction was observed between 20nM and 200nM cilengitide, followed by a complete blockade of osteoclast formation at 2μM and 20μM. In the presence of 200nM cilengitide compared to the 20nM an intriguing morphological difference was observed with significant reduction in osteoclast size, suggesting that cilengitide may disrupt spreading and the fusion capacity at early pre-osteoclast stage. In vivo administration of cilengitide significantly reduced incidence (92,8% vs. 46,7%) and severity of CIA as evidenced by the reduction of clinical disease activity scores such as paw swelling and reduction of grip strength.
Conclusions These findings demonstrate that the αvβ3 integrin is instrumental for osteoclastogenesis. Moreover, in vivo systemic αvβ3 integrin inhibition with cilengitide potently arrests experimental arthritis onset and progression. Interfering with αvβ3 integrin may thus be an efficient target in diseases with disrupted bone metabolism such as rheumatoid arthritis.
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
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