- Email: [email protected]
Resolvin D1 regulates bone metabolism
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
(lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) is the most potent RANKL substitute identified to date and in this study we assessed the role of LIGHT in bone destruction in RA. Methods: Synovial fluid (SF) macrophages from RA patients were incubated with 25 ng/ml M-CSF (macrophage-colony stimulating factor) and RANKL or LIGHT (50ng/ml) in the presence and absence of 100ng/ml OPG o(steoprotegerin), a decoy receptor for RANKL. Osteoclast formation was assessed histochemically by assessing the number of multinucleated cells expressing the osteoclast-specific enzyme TRAP (tartrate-resistant acid phosphatase) and by measuring the extent of lacunar resorption. LIGHT concentration in RA and OA synovial fluid was measured by ELISA and the expression of LIGHT in RA and OA (osteoarthritis) synovium determined by immunohistochemistry. Results: The addition of LIGHT to cultures of RA SF macrophages induced significant osteoclast formation (comparable to that of RANKL) as measured by the formation of TRAPþ multinucleated cells and the extent of lacunar resorption. Osteoclast formation was not inhibited by OPG indicating that osteoclastogenesis occurred by a RANKL - independent mechanism. LIGHT also increased (almost four-fold) RANKLinduced osteoclastogenesis and bone resorption. The concentration of LIGHT was increased in RA compared with OA synovial fluid and LIGHT was more strongly expressed in RA synovium. Conclusions: Our findings indicate that LIGHT is present in increased amounts in RA SF and that it is is highly expressed in RA compared with OA synovium. LIGHT independently induces significant osteoclast formation and bone resorption from SF macrophages and dramatically increases RANKL-induced osteoclastogenesis and bone resorption. Osteoclast formation from SF macrophages is likely to play a role in pathological bone resorption in RA and both RANKL - dependent and RANKL - independent mechanisms (largely LIGHT- induced) may be involved in this process.
S143
Conclusions: Our in vitro results clearly show that RvD1 may play an important role in the regulation of bone metabolism. Additionally to our previous data, our findings suggest that RvD1 presents a novel and original perspective to musculoskeletal and bone diseases therapy.
Figure 1. RvD1 inhibits osteoclasts recruitment.
204 RESOLVIN D1 REGULATES BONE METABOLISM H.A. Benabdoune y, z, P.E. Rondon y, z, S. Qin z, J.C. Fernandes y, z, e de Montr eal, Montreal, QC, H. Fahmi y, x, M. Benderdour y, z. y Universit ^pital du Sacr Canada; z Orthopedic research laboratory-Ho e-Cœur de x e Montr eal, Montreal, QC, Canada; Res. Ctr.-Ctr. Hosp.ier de l’universit de Montr eal, Montreal, QC, Canada Purpose: Resolvin-D1 (RvD1) is an omega-3 fatty acids derivative and a potent anti-inflammatory agent synthesized during the resolution phase of inflammation. In human cartilage, we recently reported that RvD1 strongly inhibits a number of factors involved in inflammation, catabolism, oxidative stress, and apoptosis. Thus, the overall objective of this study is to further investigate its effects on bone metabolism. Methods: First, murine macrophages RAW264.7 were used to assess osteoclasts (OC) recruitment and bone resorption. RAW264.7 cells were incubated with 50 ng/ml LPS with or without RvD1 (0 - 10 mM) for 48 hours. Cell viability was verified with the MTS test. OC phenotype markers, namely TRAP and cathepsin-K, were assessed by western blot, enzymatic staining and immunocytochemistry. TNF-a, IL-1b, IL-6, IL-10 levels were measured by ELISA, and PGE2 levels by EIA. NO release was measured by Greiss reaction. To investigate bone resorption, RAW264.7 cells were seeded in hydroxyapatite plates, then treated with 50 ng/ml LPS with or without RvD1 (0.5 and 1 mM) for 48 hours. Plot formation was assessed by Von Kossa staining. Second, human osteoblasts (Ob) were obtained from post-surgery discarded trabecular bone of osteoarthritic (OA) patients who underwent total knee arthroplasty. First passage human OA Ob were treated either with RvD1 (0.1 - 1 mM) alone, or with 20 nM VitD3 with or without RvD1 (0.1 - 1 mM), for 48 hours. Cell viability was evaluated with the MTS test. Alkaline phosphatase (PAL) activity and osteocalcin (OCN) release was determined by colorimetric reaction and ELISA, respectively. Results: Our results clearly show that, in RAW264.7 cell, RvD1 strongly reduces OC recruitment and activation (Figure 1) as indicated by the inhibition of TRAP and cathepsin K expression as well as TNF-a, IL-1b, IL-6, PGE2 and NO release, and a concurrent enhancement of IL-10 levels. Besides, RvD1 decreases bone resorption through the inhibition of plots formation in hydroxyapatite matrix (Figure 2). In human OA Ob, RvD1 partially decreases VitD3-induced PAL activity, while it maintains OCN expression at control levels.
Figure 2. RvD1 inhibits plot formation in hydroxy apatite matrix. 205 EGFR/ MIG-6 SIGNALLING IN OSTEOARTHRITIS M.R. Bellini, M.A. Pest, F. Beier. Univ. of Western Ontario, London, ON, Canada Purpose: Osteoarthritis (OA) is the most common form of arthritis and progresses by degeneration of articular cartilage, amongst others. Despite an increasing awareness of OA as a medical problem, there is a surprising absence of effective medical treatments beyond pain control and surgery. Our laboratory has identified several promising novel targets for therapeutic strategies in OA, such as transforming growth factor alpha (TGFa) and epidermal growth factor receptor (EGFR) in cartilage degeneration. Mitogen-inducible gene 6 (Mig-6) has been
(lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) is the most potent RANKL substitute identified to date and in this study we assessed the role of LIGHT in bone destruction in RA. Methods: Synovial fluid (SF) macrophages from RA patients were incubated with 25 ng/ml M-CSF (macrophage-colony stimulating factor) and RANKL or LIGHT (50ng/ml) in the presence and absence of 100ng/ml OPG o(steoprotegerin), a decoy receptor for RANKL. Osteoclast formation was assessed histochemically by assessing the number of multinucleated cells expressing the osteoclast-specific enzyme TRAP (tartrate-resistant acid phosphatase) and by measuring the extent of lacunar resorption. LIGHT concentration in RA and OA synovial fluid was measured by ELISA and the expression of LIGHT in RA and OA (osteoarthritis) synovium determined by immunohistochemistry. Results: The addition of LIGHT to cultures of RA SF macrophages induced significant osteoclast formation (comparable to that of RANKL) as measured by the formation of TRAPþ multinucleated cells and the extent of lacunar resorption. Osteoclast formation was not inhibited by OPG indicating that osteoclastogenesis occurred by a RANKL - independent mechanism. LIGHT also increased (almost four-fold) RANKLinduced osteoclastogenesis and bone resorption. The concentration of LIGHT was increased in RA compared with OA synovial fluid and LIGHT was more strongly expressed in RA synovium. Conclusions: Our findings indicate that LIGHT is present in increased amounts in RA SF and that it is is highly expressed in RA compared with OA synovium. LIGHT independently induces significant osteoclast formation and bone resorption from SF macrophages and dramatically increases RANKL-induced osteoclastogenesis and bone resorption. Osteoclast formation from SF macrophages is likely to play a role in pathological bone resorption in RA and both RANKL - dependent and RANKL - independent mechanisms (largely LIGHT- induced) may be involved in this process.
S143
Conclusions: Our in vitro results clearly show that RvD1 may play an important role in the regulation of bone metabolism. Additionally to our previous data, our findings suggest that RvD1 presents a novel and original perspective to musculoskeletal and bone diseases therapy.
Figure 1. RvD1 inhibits osteoclasts recruitment.
204 RESOLVIN D1 REGULATES BONE METABOLISM H.A. Benabdoune y, z, P.E. Rondon y, z, S. Qin z, J.C. Fernandes y, z, e de Montr eal, Montreal, QC, H. Fahmi y, x, M. Benderdour y, z. y Universit ^pital du Sacr Canada; z Orthopedic research laboratory-Ho e-Cœur de x e Montr eal, Montreal, QC, Canada; Res. Ctr.-Ctr. Hosp.ier de l’universit de Montr eal, Montreal, QC, Canada Purpose: Resolvin-D1 (RvD1) is an omega-3 fatty acids derivative and a potent anti-inflammatory agent synthesized during the resolution phase of inflammation. In human cartilage, we recently reported that RvD1 strongly inhibits a number of factors involved in inflammation, catabolism, oxidative stress, and apoptosis. Thus, the overall objective of this study is to further investigate its effects on bone metabolism. Methods: First, murine macrophages RAW264.7 were used to assess osteoclasts (OC) recruitment and bone resorption. RAW264.7 cells were incubated with 50 ng/ml LPS with or without RvD1 (0 - 10 mM) for 48 hours. Cell viability was verified with the MTS test. OC phenotype markers, namely TRAP and cathepsin-K, were assessed by western blot, enzymatic staining and immunocytochemistry. TNF-a, IL-1b, IL-6, IL-10 levels were measured by ELISA, and PGE2 levels by EIA. NO release was measured by Greiss reaction. To investigate bone resorption, RAW264.7 cells were seeded in hydroxyapatite plates, then treated with 50 ng/ml LPS with or without RvD1 (0.5 and 1 mM) for 48 hours. Plot formation was assessed by Von Kossa staining. Second, human osteoblasts (Ob) were obtained from post-surgery discarded trabecular bone of osteoarthritic (OA) patients who underwent total knee arthroplasty. First passage human OA Ob were treated either with RvD1 (0.1 - 1 mM) alone, or with 20 nM VitD3 with or without RvD1 (0.1 - 1 mM), for 48 hours. Cell viability was evaluated with the MTS test. Alkaline phosphatase (PAL) activity and osteocalcin (OCN) release was determined by colorimetric reaction and ELISA, respectively. Results: Our results clearly show that, in RAW264.7 cell, RvD1 strongly reduces OC recruitment and activation (Figure 1) as indicated by the inhibition of TRAP and cathepsin K expression as well as TNF-a, IL-1b, IL-6, PGE2 and NO release, and a concurrent enhancement of IL-10 levels. Besides, RvD1 decreases bone resorption through the inhibition of plots formation in hydroxyapatite matrix (Figure 2). In human OA Ob, RvD1 partially decreases VitD3-induced PAL activity, while it maintains OCN expression at control levels.
Figure 2. RvD1 inhibits plot formation in hydroxy apatite matrix. 205 EGFR/ MIG-6 SIGNALLING IN OSTEOARTHRITIS M.R. Bellini, M.A. Pest, F. Beier. Univ. of Western Ontario, London, ON, Canada Purpose: Osteoarthritis (OA) is the most common form of arthritis and progresses by degeneration of articular cartilage, amongst others. Despite an increasing awareness of OA as a medical problem, there is a surprising absence of effective medical treatments beyond pain control and surgery. Our laboratory has identified several promising novel targets for therapeutic strategies in OA, such as transforming growth factor alpha (TGFa) and epidermal growth factor receptor (EGFR) in cartilage degeneration. Mitogen-inducible gene 6 (Mig-6) has been