Despoina Sykoutri

Nicotinic acetylcholine receptors modulate osteoclastogenesis

BackgroundOur aim was to investigate the role of nicotinic acetylcholine receptors (nAChRs) in in-vitro osteoclastogenesis and in in-vivo bone homeostasis.MethodsThe presence of nAChR subunits as well as the in-vitro effects of nAChR agonists were investigated by ex vivo osteoclastogenesis assays, real-time polymerase chain reaction, Western blot and flow cytometry in murine bone marrow-derived macrophages differentiated in the presence of recombinant receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). The bone phenotype of mice lacking various nAChR subunits was investigated by peripheral quantitative computed tomography and histomorphometric analysis. Oscillations in the intracellular calcium concentration were detected by measuring the Fura-2 fluorescence intensity.ResultsWe could demonstrate the presence of several nAChR subunits in bone marrow-derived macrophages stimulated with RANKL and M-CSF, and showed that they are capable of producing acetylcholine. nAChR ligands reduced the number of osteoclasts as well as the number of tartrate-resistant acidic phosphatase-positive mononuclear cells in a dose-dependent manner. In vitro RANKL-mediated osteoclastogenesis was reduced in mice lacking α7 homomeric nAChR or β2-containing heteromeric nAChRs, while bone histomorphometry revealed increased bone volume as well as impaired osteoclastogenesis in male mice lacking the α7 nAChR. nAChR ligands inhibited RANKL-induced calcium oscillation, a well-established phenomenon of osteoclastogenesis. This inhibitory effect on Ca2+ oscillation subsequently led to the inhibition of RANKL-induced NFATc1 and c-fos expression after long-term treatment with nicotine.ConclusionsWe have shown that the activity of nAChRs conveys a marked effect on osteoclastogenesis in mice. Agonists of these receptors inhibited calcium oscillations in osteoclasts and blocked the RANKL-induced activation of c-fos and NFATc1. RANKL-mediated in-vitro osteoclastogenesis was reduced in α7 knockout mice, which was paralleled by increased tibial bone volume in male mice in vivo.

Effects of cilengitide in osteoclast maturation and behavior.

Bone metastasis is a common burden in many types of cancer and has a severe impact on the quality of life in 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. For this purpose, we used cilengitide, a cyclic RGD peptide, which blocks integrin αVβ3 and αVβ5-ligand binding. Our results revealed that cilengitide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with cilengitide exhibited reduced cell spreading, cell migration and cell adhesion on RGD-containing matrix proteins, which are ligands of integrin αV. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, FAK and c-Src, were consistently blocked by cilengitide. First evidence suggests that cilengitide might interfere with metastatic bone disease in vivo and this study describes a potential underlying mechanism of the inhibitory effect of cilengitide on αV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that cilengitide, which interferes with αV-integrins on osteoclasts, may represent a novel therapeutic strategy in the treatment of malignant bone disease.

A9.1 αvβ3 Integrin Inhibition with Cilengitide Both Prevents and Treats Collagen Induced Arthritis

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 Methods In vitro 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 with macrophage-colony 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.

Abstract 406: Effects of an RGD peptide in osteoclast maturation and behavior as a therapeutic option for metastatic bone disease

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 aVâ3 and aVâ5-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 aVâ3. 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 aV-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 aV-integrins on osteoclasts may represent a novel therapeutic strategy in the treatment of malignant bone disease. Citation Format: Gerald Prager, Daniela Bianconi, Anastasia Chilla, 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

A1.24 Cilengitide reduces pannus formation and bone erosions in collagen induced arthritis

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.

A9.9 Treatment with BGP-15, a Novel Insulin Sensitiser Attenuates Collagen-Induced Arthritis in DBA/1 Mice

Background and Objectives BGP-15, a small synthetic hydroxylamine derivative is a member of a new class of insulin-sensitising medications also known as chaperone-inducers. Beside its beneficial effects on glycemic control and insulin sensitivity in patients with Type 2 diabetes, BGP-15 is known to induce heat shock protein Hsp72 and heat shock transcription factor HSF1, which in turn are involved in joint inflammation. Moreover, BGP-15 also inhibits poly-ADP-ribose polymerase (PARP) and the phosphorylation of c-JUN N-terminal kinase via Hsp72 overexpression. Therefore it might also play a role in the regulation of inflammatory joint disease. Our objective was to evaluate the in vivo effects of BGP-15 on collagen-induced arthritis (CIA) in DBA/1 mice. Materials and Methods Arthritis was induced by intradermal injection of bovine type II collagen (bCII) and incomplete Freund’s adjuvant (CFA) in male DBA/1 mice. BGP-15 was administered either one week prior to the first immunisation (prophylactic experiment, n:14 in both groups) or upon the appearance of symptoms (therapeutic experiment, n:12 in both groups) in drinking water. Arthritis incidence and severity was assessed for 28 days following the second immunisation (boost) with bCII and CFA on day 21. Histological evaluation was carried out on hind paws using Osteomeasure® software. Anticollagen antibodies were measured by enzyme-linked immunosorbent assay. The cellular composition of the draining lymph nodes was measured by flow cytometry. Results BGP-15 significantly reduced the incidence of CIA by 28% and also reduced both paw swelling (p ≤ 0.01) and grip strength (p ≤ 0.05) in the prophylactic experiment. In the therapeutic experiment BGP-15 significantly attenuated both paw swelling (p ≤ 0.01) and grip strength (p ≤ 0.05). Histological evaluation of the hind paws demonstrated reduced area of inflammation (p ≤ 0.05), area of erosion (p ≤ 0.01) and number of osteoclasts (p ≤ 0.05) in the BGP-15 treated group when compared to the control group. No significant differences were revealed between anti-collagen antibody levels or in the distribution of T-cells, B-cells, dendritic cells and monocyte/macrophages harvested from draining lymph nodes, suggesting an effect predominantly involving the innate immune system. Conclusions Our results demonstrate that the novel chaperone-inducer BGP-15 has a profound prophylactic and therapeutic effect on autoimmune arthritis, likely due to an effect on the effector phase.

THU0031 Blocking αVβ3 integrin with cilengitide affects osteoclastogenesis in vitro and prevents collagen induced arthritis in vivo

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

Multi-targeted kinase inhibitor PKC412 diminishes osteoclastogenesis and counteracts osteoclast function in vitro

Background Rheumatoid arthritis is a chronic inflammatory disease characterised by osteoclast mediated bone erosions. Small molecule multi-kinase inhibitors are being explored as potential therapeutic targets. PKC412 is a small molecule multi-kinase inhibitor targeting class III tyrosine-protein-kinases such as FMS-like tyrosine kinase 3 (FLT-3) and multiple isoforms of serine/threonine protein kinase C. PKC412 has been shown to inhibit macrophage function in vitro. However, the role of PKC412 in modulating the commitment of the monocyte/macrophage lineage to osteoclast precursors and their differentiation into mature osteoclasts has not been fully elucidated. Objectives The authors aimed to investigate the effect of PKC412 on osteoclast differentiation and function. Materials and methods The authors differentiated mouse bone marrow derived cells in the presence of macrophage colony-stimulating factor and RANKL into tartrate-resistant acid phosphatase positive (TRAP+) mononucleated preosteoclasts and TRAP+ multinucleated mature osteoclasts, and added PKC412 in increasing concentrations (10 nM to 10 μM) to the culture. The authors assessed the role of PKC412 in the bone resorbing capacity of osteoclasts by culturing osteoclasts on dentine slices. To further characterise the effect of PKC412 on cell proliferation the authors performed MTT assays. The authors used quantitative PCR to evaluate expression levels of mRNA encoding for osteoclast specific markers such as nuclear factor of activated T cells cytoplasmic 1 (NFATc1), matrix metalloproteinase 9 (MMP9), Cathepsin K and TRAP. Flow cytometry analysis for annexin V and 7 Aminoactinomycin D (7-AAD) was performed to determine potential apoptotic effects of PKC412. Results Increasing concentrations of PKC412 (IC50: 250 nM) dose-dependently reduced osteoclast numbers. Preosteoclasts were also significantly decreased after addition of PKC412, indicating an effect of PKC412 on early stages of osteoclastogenesis. In line with this finding, the authors showed a dose-dependent reduction of preosteoclast proliferation using MTT proliferation assays. Moreover, the authors detected a significant time- and dose-dependent increase in the ratio of apoptotic cells in the PKC412-treated cells by annexin V and 7-AAD staining. In the presence of PKC412 the authors obtained a significant reduction in osteoclast size, nuclei number and resorption pit formation. Consistently, the authors were able to demonstrate a dose dependent downregulation of mRNA coding for osteoclast markers, such as NFATc1, MMP9, Cathepsin K and TRAP in the presence of PKC412. Conclusion These results suggest a regulatory role of PKC412 in preosteoclast differentiation und osteoclastogenesis through apoptosis induction.