Background Rheumatoid arthritis (RA) is characterised by synovial inflammation and osteoclast (OC) mediated bone erosions. AlphaVbeta3 (αvβ3) integrin is highly expressed in OCs. Avβ3 blocking antibodies reduce bone resorption and mice lacking β3 are osteopetrotic. Objectives Efficacy testing of the αvβ3 inhibitor cilengitide, a synthetic Arginine-Glycine-Asparagine amino acid peptide (RGD peptide), on osteoclastogenesis and the collagen induced arthritis (CIA) model for human RA. Materials and MethodsIn vitro mouse bone marrow-derived cells (BMCs) were differentiated into tartrate resistant acid phosphatase positive (TRAP+) mononuclear OC precursor cells (preOCs) and TRAP+ multinucleated mature OCs with macrophagecolony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Cilengitide, was added in increasing concentrations (2 nM to 20 µM) to the culture. These osteoclastogenesis assays were performed on plates coated with RGD containing matrixes osteopontin, fibronectin and fibrinogen but also on Poly-D-lysine to assess αvβ3 independent adhesion. In vivo 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 CIA prevention mice received subcutaneously (s.c.) 1.5 mg/kg cilengitide (n = 15) or placebo (n = 15), 5 days per week, 1 day prior to CIA induction until day 53. For CIA treatment, mice with arthritis were randomised and received 1.5 mg/kg (low dose, n = 19) or 75 mg/kg (high dose, n = 7) cilengitide or placebo (n = 21), until day 59. Preventive and treatment effects were evaluated by assessing paw thickness and grip strength. Results In vitro increasing concentrations of cilengitide (IC50: 250 nM) dose-dependently reduced pre-OCs on all coatings, indicating early inhibition at the pre-OC proliferation stage. OCs were reduced above 200 nM, followed by complete disappearance above 2 µM. At 200 nM an intriguing morphological difference with reduction in OC size suggested that cilengitide may disrupt spreading and fusion capacity at the early pre-OC stage. In vivo CIA prevention with cilengitide effectively reduced incidence (92.8% versus 40%) and severity of arthritis as evidenced by reduction of clinical disease activity scores. Low and high dose cilengitide effectively inhibited progression of established arthritis. Conclusions Osteoclastogenesis requires intact αvβ3 integrin function. Systemic αvβ3 integrin inhibition with cilengitide potently prevents and treats experimental CIA. Cilengitide may be a novel therapeutic target in RA. THE PHOSPHOINOSITIDE 3-KINASE PATHWAY REGULATES FIBROBLAST-LIKE SYNOVIOCYTES INVASION
Metastatic bone disease is a common feature of many types of cancer and has a severe impact on the quality of life of patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process, including osteoclastogenesis as well as osteoclast/bone matrix interaction. For this purpose, we used a cyclic RGD peptide which blocks integrin áVâ3 and áVâ5-ligand binding. Our results revealed that the RGD peptide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with the RGD peptide exhibited reduced cell spreading, migration and adhesion on RGD-containing matrix proteins, such as osteopontin and fibrinogen, which are ligands of integrin áVâ3. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, such as FAK and c-Src, were consistently blocked by the RGD peptide. First evidence has suggested that the RGD peptide might interfere with metastatic bone disease in vivo and the evidence presented herein describes the underlying mechanisms of the inhibitory effect of the RGD peptide on áV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that using an RGD peptide to interfere with áV-integrins on osteoclasts may represent a novel therapeutic strategy in the treatment of malignant bone disease. Citation Format: Gerald Prager, Daniela Bianconi, Anastasia Chillà, Alexandra Dorda, Nisha Geetha, Matthias Unseld, Despoina Sykoutri, Marina Poettler, Kurt Redlich, Christoph Zielinski. Effects of an RGD peptide in osteoclast maturation and behavior as a therapeutic option for metastatic bone disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2015-406
Background and Objectives Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by synovial inflammation and osteoclast (OC) mediated bone erosions. AlphaVbeta3 integrin (αvβ3) is highly expressed in OCs and αvβ3 blocking antibodies reduce bone resorption. We have shown that Cilengitide, a synthetic Arginine-Glycine-Asparagine peptide (RGD-peptide), effectively inhibits osteoclastogenesis in vitro and reduces clinical signs of arthritis in Collagen Induced Arthritis (CIA). We aimed to further characterise the effect of cilengitide in arthritis. Materials and Methods CIA was induced in 6-8 week old male DBA/1 mice by immunisation with bovine type II collagen (CII) at day 1 and boosting at day 21. For CIA prevention mice received 1.5 mg/kg cilengitide (n = 15) or placebo (n = 15) subcutaneously (s.c.), 5 days/week, starting 1 day prior to CIA induction until day 53. For CIA treatment mice with established arthritis were randomised and received 1.5 mg/kg (low dose) or 75mg/kg (high dose) cilengitide or placebo s.c. 5 days/week until day 59. Incidence and severity of arthritis was assessed by weekly clinical scoring of paw swelling and grip strength. In the end histological staining was performed on hind paws with haematoxylin and eosin (H&E) for quantification of pannus, tartrate-resistant acid phosphatase (TRAP) for detection of bone erosions and OCs, toluidine blue (TB) for determination of cartilage breakdown. Blood vessel formation was assessed with endomucin staining. Cellular composition of joint infiltrates was determined with immunohistochemistry with anti-CD3, CD45RO, F4/80 and Neutrophil 7/4 antibodies. Results In the preventive experiment, cilengitide significantly reduced incidence (92.8% vs. 40%) and severity of CIA. Histological examination revealed significantly reduced numbers of synovial OCs, extent of bone destruction, cartilage damage as well as reduction of inflammatory pannus formation. In addition, blood vessel density in pannus was significantly reduced. Similarly, in the treatment experiment, low and high dose cilengitide effectively inhibited progression of established arthritis as evidenced by amelioration of clinical disease activity scores and histopathology. Conclusions Our findings demonstrate that cilengitide potently prevents and treats experimental CIA, reducing osteoclast-mediated bone erosions and inflammation. Further investigation of its mechanism of action in arthritis, in respect to neovascularisation and osteoclast function, may optimise its effect and provide a novel therapeutic target in RA.
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