Synovial tissue–resident macrophages regulate immune homeostasis within the joint, but can adopt an inflammation-permissive phenotype that promotes immune cell infiltration in rheumatoid arthritis (RA). Understanding the factors that drive this phenotypic switch may help prevent the localisation of inflammation in the joints of individuals at-risk of RA. We identified the mechanosensitive ion channel PIEZO1 as a potential regulator of lining-layer synovial tissue macrophage (STM) function. PIEZO1 was highly expressed in homeostatic, tissue resident TREM2pos lining-layer STMs and in its pathogenic chemokine producing TREM2low phenotype that characterises the hyperplastic lining-layer in active RA. Intra-articular injection of a PIEZO1 agonist in mice induced neutrophil and monocyte infiltration, whereas inhibition of PIEZO1 signalling restored the protective macrophage phenotype. Thus, mechanosensing via PIEZO1 is a defining feature of the joint lining-layer, and its aberrant activation by mechanical stress may lead to the localisation of inflammation within the joint, facilitating a transition from asymptomatic autoimmunity of at-risk RA to clinical disease. ### Competing Interest Statement The authors have declared no competing interest. All raw data are available to readers in ArrayExpress under accession number E-MTAB-15203 (bulk RNAseq of PIEZO1 activated macrophages) and E-MTAB-15200 (scRNASeq data of PIEZO1 activated macrophages.

Objective IĸB protein B cell lymphoma 3‐encoded protein (BCL3) is a regulator of the NF‐κB family of transcription factors. NF‐κB signaling fundamentally influences the fate of bone‐forming osteoblasts and bone‐resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. Methods To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n = 6–14) lacking BCL3 ( Bcl3 −/− ) and wild‐type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3 −/− mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n = 3–7) were assessed. Osteoclast differentiation and function in Bcl3 −/− mice (n = 3–5) was assessed. Adult 20‐week Bcl3 −/− and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3 −/− mice (n = 11–13). Results Evaluation of Bcl3 −/− mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3 −/− cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis‐induced osteophytogenesis, Bcl3 −/− mice exhibited decreased pathological osteophyte formation ( P < 0.05). Conclusion Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology. image

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

Objectives The aim of this study was to determine the presence of protease-activated receptor 2 (PAR2) and matriptase proteins and quantify PAR2 and matriptase mRNA expression in the articular cartilage and synovial membrane of cats with and without osteoarthritis (OA). Methods A total of 28 articular cartilage samples from adult cats (14 OA and 14 normal), 10 synovial membranes from adult cats (five OA and five normal) and three cartilage samples from 9-week-old fetal cats were used. The presence of PAR2 and matriptase in the cartilage and synovial membrane of the adult samples was detected by immunohistochemical (IHC) staining, while real-time PCR was used for mRNA expression analyses in all samples. Results PAR2 was detected in all OA and normal articular cartilage and synovial membrane samples but confined to only a few superficial chondrocytes in the normal samples. Matriptase was only detected in OA articular cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression were, however, detected in all cartilage and synovial membrane samples. PAR2 and matriptase mRNA expression levels in OA articular cartilage were five ( P <0.001) and 3.3 ( P <0.001) times higher than that of the healthy group, respectively. There was no significant difference ( P = 0.05) in the OA synovial membrane PAR2 and matriptase mRNA expression compared with the normal samples. Conclusions and relevance Detection of PAR2 and matriptase proteins and gene expression in feline articular tissues is a novel and important finding, and supports the hypothesis that serine proteases are involved in the pathogenesis of feline OA. The consistent presence of PAR2 and matriptase protein in the cytoplasm of OA chondrocytes suggests a possible involvement of proteases in cartilage degradation. Further investigations into the PAR2 and matriptase pathobiology could enhance our understanding of the proteolytic cascades in feline OA, which might lead to the development of novel therapeutic strategies.

Angiotensin II-type 1 receptor stimulation is recognised to promote inflammation, a state central to the development and maintenance of rheumatoid arthritis. Herein we examined the use of losartan, an angiotensin II-type 1 receptor antagonist, on vascular reactivity, knee joint diameter and behavioural assessment of pain in a Freund's complete adjuvant (FCA) mouse model of joint inflammation. Monoarthritis was induced via FCA in the presence or absence of losartan with naive mice serving as controls. Knee joint swelling, joint pain (assessed by dynamic weight bearing of limb use), knee joint artery reactivity (assessed ex vivo) and blood perfusion of the knee joint (assessed in vivo) were determined. FCA mediated a significant increase in knee joint diameter and reduced weight-bearing (a surrogate for pain sensation) of the affected limb. Notably, these phenomena were substantially reduced when mice were prophylactically treated with losartan. Assessment of arterial relaxation and blood perfusion with acetylcholine stimulation revealed that FCA resulted in significant vascular dysfunction, which was resolved to naïve levels with losartan treatment. Through the actions of losartan, these findings indicate that the angiotensin II-type 1 receptor is a likely therapeutic target of importance in the development of the physical changes, pain sensation and vascular dysfunction found in inflammatory arthritis.

Cigarette smoke stimulates an inflammatory response and produces oxidants that cause oxidative stress in the lung, which promotes pathophysiological changes related to chronic obstructive pulmonary disease (COPD) (Kirkham, P. Pharmacol Ther 2006; 111: 476-94). Hydrogen peroxide (H2O2) is one of the oxidants detected in the breath condensate of COPD patients (Montuschi, P. Clin Chim Acta 2005; 35: 22-34). We aim to understand how chronic exposure to H2O2 alone or in combination with other inflammatory mediators influences epithelial cell responses relevant to COPD lung pathologenesis. BEAS-2B cells were exposed to H2O2 (2 h/d for 3 days) at different concentrations, alone or in combination with TGF-β (10 ng/ml) or LPS (500 ng/ml). Cell viability was assessed. IL-8 and IL-6 were measured by ELISA. Data was analysed using Multiple Comparison Test. Cells tolerated a repeated exposure of H2O2 (up to 15 μM) ± TGF-β or LPS without significant loss of viability. H2O2 stimulated modest release of IL-8 (mean ± SEM; 38±2 pg/ml) and IL-6 (84±13 pg/ml). However, repeated 15 μM H2O2 exposure significantly enhanced TGF-β induced IL-8 (TGF-β, 194±13 vs. TGF-β+ H2O2, 279±10 pg/ml; p<0.0001) but not IL-6 (TGF-β, 431±22 vs. TGF-β+ H2O2, 449±2 pg/ml). H2O2 enhanced LPS secretion of both IL-8 (LPS, 2487±21 vs. LPS+ H2O2, 2898±109 pg/ml; p<0.0001), and IL-6 (LPS, 2469±72 vs. LPS+ H2O2, 3277±62 pg/ml; p<0.0001). Repeated exposure of BEAS-2B cells to H2O2 induced minimal inflammatory response, but enhanced the effect of TGF-β and LPS on cytokine production. These data suggest such combined exposure models may be useful to study the effects of epithelial cell challenge relevant to COPD pathology.

Hyper-reactivity, inflammation and hyperplasia/hypertrophy of airway smooth muscle (ASM) limit airflow and are key features of chronic obstructive pulmonary disease (COPD). Proteinase activated receptor 2 (PAR2) is a critical modulator of inflammatory responses in respiratory disease such as asthma, yet is reported to promote ASM relaxation. However, the role of ASM PAR2 in COPD is not well understood (Sokolova & Reiser, Pharmacol Ther. 2007; 115(1):70-83). Our aim was to determine the presence and role of PAR2 in murine lung and ASM subjected to an oxidative stressor environment using both immunohistochemistry (IHC) and wire myography. PAR2 was detected using APR-32 antibody (Alomone, Israel) on both murine airway and lung tissue, with clean isotype. Data presented as mean ± SEM. An oxidative stressor environment increased trypsin-induced ASM relaxation in both bronchial (Oxidative 54.5±8.5 % vs. Control 35.1±3.7 %; n=6; p<0.05; paired t-test) and tracheal tissue (Oxidative 56.7±10.5 % vs. Control 30.3±2.9 %; n=6; p=0.052; paired t-test). This was PAR2 dependent, as relaxation to trypsin was virtually abolished in PAR2-/- compared with WT tracheal (WT 56.7±10.5 % vs. PAR2-/- 2.5±0.7 %; n=3-6; p<0.01; unpaired t-test) and bronchial tissue (WT 54.5±8.5 % vs. PAR2-/- 2.3± 0.9 %; n=3-6; p<0.01; unpaired t-test). Further confirmation of PAR2 dependent relaxation was achieved using a PAR2 specific peptide (2-FLIGRLO-NH2). In conclusion, this study confirms a functional PAR2 role in murine airway tissue; importantly, the role of PAR2 in mediating ASM relaxation appears to be enhanced in oxidative environments such as found in COPD. This may have important implications for future potential therapies.

Background Post-traumatic osteoarthritis (PTOA) is associated with articular cartilage damage and represents a major clinical challenge due to the poor regenerative capability of cartilage. We have recently developed a novel and robust dual injury murine model of PTOA, which combines destabilisation of the medial meniscus (DMM) and cartilage scratch1. This model results in accelerated OA-like symptoms including enhanced osteophyogenesis. Prior studies in the DMM model have demonstrated that absence of proteinase-activated receptor 2 (PAR2) in mice results in significant protection from early OA-like symptoms2,3. Objectives To investigate if the absence of PAR2 confers protection in a dual injury PTOA murine model. Methods PTOA was induced in both male C57BL/6 wild-type (WT) and PAR2-/- mice, via combined destabilisation of the medial meniscus and cartilage scratch (DCS). Twenty-eight days post-surgery, osteophytogenesis and bone changes were monitored using microcomputed tomography. Dynamic weight bearing was assessed as an indirect measurement of pain at day 14. Results Evaluation of the presence and number of osteophyte revealed no significant differences between WT and PAR2-/- mice at day 28. However, quantification of osteophytes revealed that PAR2-/- mice had significantly smaller osteophytes (p=0.006) with less mineralised bone (p=0.003). Moreover, analysis of metaphyseal trabecular bone on the operated leg showed a significant decrease in% bone volume/tissue volume (BV/TV) (p=0.025). Assessment of pain-related behaviour, using dynamic weight bearing at day 14, demonstrated that PAR2-/- mice exerted less load on their front paws. Conclusion The findings in this study show that PAR2 plays a role in accelerated OA-like symptoms (i.e., osteophyte formation) in a dual injury model of OA, where both destabilisation of the medial meniscus and cartilage damage drive disease pathology. Furthermore, the loss of PAR2 decreases pain behaviour suggesting that PAR2 is involved in pain sensing. Taken together, these results support the future exploration of PAR2 as a therapeutic target for PTOA. References [1] McCulloch K, Huesa C, Dunning L, van ‘t Hof R, Lockhart J, Goodyear CS. Accelerated Osteoarthritic-like Symptoms in a Novel Dual Injury Model Combining Destabilisation of the Medial Meniscus and Cartilage Damage. Journal of Bone and Mineral Research. 2018; 33:155 [2] Huesa C, Ortiz AC, Dunning L, McGavin L, Bennett L, McIntosh K, Crilly A, Kurowska-Stolarska M, Plevin R, van ‘t Hof RJ, et al. Proteinase-activated receptor 2 modulates OA-related pain, cartilage and bone pathology. Ann Rheum Dis. 2016;75:1989-97. [3] Ferrell WR, Kelso EB, Lockhart JC, Plevin R, and McInnes IB. Protease-activated receptor 2: a novel pathogenic pathway in a murine model of osteoarthritis. Annals of the Rheumatic Diseases. 2010;69:2051. Disclosure of Interests Kendal McCulloch: None declared, Carmen Huesa: None declared, Lynette Dunning: None declared, Rob van’t Hof Shareholder of: OsteoRx Ltd, John Lockhart: None declared, Carl Goodyear Grant/research support from: AstraZeneca, BMS, Celgene, Janssen, MedAnnex, Pfizer and UCB, Speakers bureau: Abbvie

JBMR PlusVolume 2, Issue S1 p. S1-S50 Society AbstractsOpen Access Bone Research Society Abstracts First published: 01 October 2018 https://doi.org/10.1002/jbm4.10073Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The Bone Research Society (BRS; www.boneresearchsociety.org), formerly the Bone and Tooth Society, was founded in 1950. The BRS is one of the largest national scientific societies in Europe dedicated to clinical and basic research into mineralized tissues and is the oldest such society in the world. Meetings are held annually, attracting a wide audience from throughout the UK and beyond. The presentations are traditionally balanced between clinical and laboratory studies. The participation of young scientists and clinicians is actively encouraged. The Annual Meeting this year was organized by a team from The University of Southampton, co-chaired by Dr Kate Ward and Professor Nicholas Harvey, and also including Dr Claire Clarkin, Professor Richard Oreffo, and Professor Cyrus Cooper. The program brought together world leaders in musculoskeletal clinical and basic science. A multidisciplinary faculty provided a breadth of education and research from the Developmental Origins of disease through tissue regeneration and biomechanical engineering. The sessions this year were: Rare bone diseases Muscle bone interactions Tissue engineering and regenerative medicine Cellular senescence Imaging: from mice to men Osteocytes Prevention of osteoporotic fracture through the lifecourse (Dent Award—Cyrus Cooper) Early-life origins of disease Ageing Osteo-angiogenic coupling More than 120 abstracts were submitted, and those that were accepted and presented at the meeting are listed here. Invited Speaker Abstracts I will describe the suite of tools and technologies developed within the Bradley group (typically with collaborators) that are focused on controlling or modulating cellular behavior—with a key vision being that the material itself should provide the cues and stimuli that drive cell fate and behavior. In my talk, I will describe a variety of high-throughput approaches developed in the group, from the use of combinatorial delivery strategies to microarray platforms that allow a deep analysis of how substrate morphology and structure drive cellular behavior. Citing Literature Biological processes leading to tissue formation during embryonic development are characterized by a large stability and reproducibility of events, typically referred to as “robustness.” Would regenerative medicine approaches be more repeatable and effective if they targeted the recapitulation of molecular pathways typical of tissue development? Within the exemplifying context of cartilage and bone repair, this lecture will introduce and discuss the challenges and opportunities of regenerative concepts based on mimicking developmental processes. Rather than engineering a tissue, the strategy would target the use of cells (eg, mesenchymal stromal cells) to engineer temporally staged processes, recapitulating events of development (eg, endochondral ossification for bone or joint cavitation for articular cartilage). The product would be a construct containing the necessary and sufficient cues to autonomously remodel into the target repair tissue upon grafting. In this perspective, however, cells in adults may strongly differ from multipotent embryonic cells, and typically reside in an environment, which is tightly regulated by post-natal mechanical conditioning or immune/inflammatory processes. Thus, shouldn't tissue regeneration strategies be inspired by development but adapted to be effective in a context, which is different from the embryo? This would require the redesign of the developmental machinery for regenerative purposes by establishing artificial events or conditions. Will the resulting approach of “developmental re-engineering” offer a chance for enhanced regeneration to those tissues with limited capacity to recover from injuries or within pathological settings, reducing the potential for endogenous repair? Citing Literature In recent years, skeletal stem and progenitor cell populations have been identified in bone marrow based on markers such as Nestin, Leptin receptor (LepR), and Gremlin1. Genetic lineage tracing models in mice have provided important insights in their roles in bone homeostasis, fracture repair, and hematopoiesis. More recently, the stem and progenitor cells that are resident in the synovial joint are beginning to be defined and their functions elucidated. Lineage tracing of progenitor cells in cartilage has provided important new insights into the process of articular cartilage formation and maintenance. Work in our lab has focused on characterizing stem and progenitor cells in the synovial membrane. During development, synovial joints form from a stripe of tissue in the limb bud that is characterized by expression of growth/differentiation factor 5 (Gdf5). Tracing of Gdf5-expressing cells showed that Gdf5-lineage cells persist in adult knee synovium up to at least 1 year of age. They express a range of mesenchymal stromal cell markers, such as Pdgfrα and Sca1, but show little overlap with cells expressing the skeletal stem/progenitor markers Nestin, LepR, and Gremlin1, suggesting they are a self-contained lineage within the skeletal system. FACS-sorted and culture-expanded Gdf5-lineage cells are highly chondrogenic but poorly osteogenic in vitro, and they promote cartilage repair upon orthotopic transplantation into cartilage defects of mice. Endogenously, Gdf5-lineage cells proliferate in synovium in response to traumatic cartilage injury, leading to synovial lining hyperplasia, and underpin spontaneous repair of cartilage defects. Both processes are dependent on the activity of the transcriptional co-factor Yes-associate protein (Yap) in these cells. In pathology, using the destabilization of the medial meniscus model of osteoarthritis, Gdf5-lineage cells were found to contribute to chondro-osteophyte formation and subchondral bone remodeling. In conclusion, recent findings are starting to unravel distinct mesenchymal stromal cell subsets in adult joint tissues, identifying the key players in joint pathophysiology and promising therapeutic targets for cartilage repair and treatment of osteoarthritis. Citing Literature With the aging of the population and projected increase in osteoporotic fractures, coupled with the declining use of osteoporosis medications, there is a compelling need for new approaches to treat osteoporosis. Given that age-related osteoporosis generally coexists with multiple other comorbidities (eg, atherosclerosis, diabetes, frailty), all sharing aging itself as the leading risk factor, there is growing interest in the “Geroscience Hypothesis,” which posits that manipulation of fundamental aging mechanisms will delay the appearance or severity of multiple chronic diseases because these diseases share the same underlying risk factor—age. In this context, one fundamental aging mechanism that has received considerable attention recently as contributing to multiple age-related morbidities is cellular senescence. There is now convincing evidence that senescent cells accumulate with age and drive age-related tissue dysfunction. Consistent with this, senescent cells have been shown to increase with aging in the bone microenvironment in mice and in humans. These cells produce a pro-inflammatory secretome that leads to increased bone resorption and decreased bone formation, and approaches that either eliminate senescent cells or impair the production of their pro-inflammatory secretome have been shown to prevent age-related bone loss in mice. Moreover, targeting senescent cells leads to a reduction in bone resorption and either a maintenance (trabecular bone) or increase (cortical bone) in bone formation, thus making this approach fundamentally different from conventional anti-resorptive therapy, which leads to a reduction in bone resorption and a coupled decrease in bone formation. Thus, targeting cellular senescence represents a novel therapeutic strategy to prevent not only bone loss but also potentially multiple age-related diseases simultaneously. Citing Literature In this talk, I would describe our work using label-free, non-invasive, and non-destructive techniques of Raman spectroscopy and multimodal non-linear microscopies such as coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) in the area of skeletal repair and regeneration for quantitative understanding and objective assessment of cell behavior and tissue development. Using Raman spectroscopy, we have studied osteogenesis in primary bone cells from mice and humans. We characterized signatures of early osteoblast behavior by quantifying changes in DNA, phosphate species, and collagen matrix during different stages of osteogenic commitment.1 It was found the Raman spectroscopy could detect changes in phosphates earlier than the alkaline phosphatase assay as well as distinguish different species. We have further aimed to quantitatively understand and objectively assess cell behavior and tissue development for applications in skeletal repair and regeneration using multimodal techniques. We combine CARS, SHG, and two-photon excited autofluorescence (TPEF) on a single platform for simultaneous interrogation. These techniques image the chemical and structural composition allowing us to study the differentiation of skeletal stem cells (SSCs) and visualization of temporal changes accompanying adipogenesis2 and chondrogenesis3 in a completely label-free, non-invasive, and non-destructive way. We also verified both visually as well as through gene expression analysis that the SSC-differentiated live tissue constructs remain viable and are unaffected by CARS and SHG imaging. Furthermore, elucidation of the architecture of the differentiated tissue is especially important for cartilage tissue engineering. The application of 3D in place of 2D imaging thus enabled us to obtain a comprehensive understanding of the collagen fiber network during the chondrogenic development of SSCs. Quantitation of the various molecular and structural readouts allowed us to develop assessment parameters to track the differentiation of SSCs and their in vitro tissue development. The non-invasive and non-destructive 3D imaging opens new avenues for real-time applications, while the label-free quantitation allows unprecedented insight and characterization of the development stages of skeletal “engineered-tissue” in the clinic for optimal use in therapy. References 1 Smith, SJ, Emery, R, Pitsillides, A, Clarkin, CE, Mahajan, S. Detection of early osteogenic commitment in primary cells using Raman spectroscopy. Analyst. 2017;142:1962–73. 2 Smus, JP, Costa, Moura C, McMorrow, E, Tare, RS, Oreffo, ROC, Mahajan, S. Tracking adipogenic differentiation of skeletal stem cells by label-free chemically selective imaging. Chem Sci. 2015;6:2282–6. 3 Moura, CC, Lanham, SA, Monfort, T, Bourdakos, KN, Tare, RS, Oreffo, ROC, Mahajan, S. Quantitative temporal interrogation in 3D of bioengineered human cartilage using multimodal label-free imaging [unpublished data]. Citing Literature It is well accepted that mechanics play an important role for the biological processes that occur during musculoskeletal regeneration and healing. The local mechanical environment is a result of a complex interaction between the soft and hard tissues at the organ, limb, and patient level of the musculoskeletal system as motion occurs. Identification of key parameters that influence and modify the internal loading conditions is therefore essential in order to better understand the mechanisms that govern and regulate musculoskeletal healing and regeneration. Computational models informed by imaging data provide an effective means for characterizing the mechanical environment. This talk will explore the fidelity of such computational methods in their application to human and animal models alike and discuss differences and similarities in their respective organ- and tissue-level loading conditions. Providing a quantitative understanding of the mechanical boundary conditions across animals will provide the essential basis for unravelling the detailed mechanisms governing the biology of musculoskeletal regeneration. Citing Literature Significant progress has been made over the last two decades to scrutinize bone porosity and particularly intracortical microstructure down to a cellular level, with important implications for our understanding of bone physiology, mechanics, and mechanobiology. However, this focus on bone porosity can divert the attention from the important interplay between angiogenesis and osteogenesis and the role of the vasculature for bone health and bone diseases for instance. On this account, recent advancements in high-resolution 3D X-ray and electron microscopy imaging will be presented here, which can be used to assess hard and soft tissues simultaneously, including mineralized bone tissue and soft tissues. The presentation will also shortly touch on our recent development of a novel micro-computed tomographic system that is optimized for soft tissues, an area traditionally considered suboptimal or unsuitable for X-ray imaging. Citing Literature It is not only osteoblasts and osteoclasts that are critical for bone remodeling but also the osteocyte, a central regulator of the activity of these two bone cell types in the growing, mature, and aging skeleton. The osteocyte is not only the bone mechanosensor but also a hormonally responsive cell that translates these two types of stimuli into signals that synchronize osteoblast and osteoclast activity. This synchronization is accomplished through both positive and negative signals sent by the osteocyte. For example, the osteocyte can send negative signals to osteoblasts such as sclerostin and DKK1 and positive signals of bone formation such as prostaglandin and wnts. Osteocytes can produce both M-CSF and RANKL for osteoclast formation. With menopause and aging, the osteocyte takes on more of its role as a negative regulator of skeletal mass. This long-lived cell becomes senescent, a state that is more likely to support resorption. The osteocyte can also function similarly to the osteoclast by producing factors such as TRAP and Cathepsin K to remove their perilacunar matrix under calcium-demanding conditions. Under physiological conditions, this perilacunar matrix is replaced, whereas under pathological conditions, this process continues and becomes detrimental with regard to bone mass. The osteocyte is also an endocrine cell that produces factors that target distant organs such as the kidney through factors such as FGF23. FGF23 is regulated by Phex and Dmp1, early osteocyte factors, and MEPE, a protein made by the mature osteocyte. Muscle function and myogenesis can be increased by factors made by osteocytes such as Wnt3a and prostaglandin; however, with aging, osteocytes produce unknown factors that decrease muscle mass. Ideally, manipulating the osteocyte so that it functions on the side of bone formation and/or maintenance of bone mass is a major goal. Recently, it has also been described that muscle factors such as irisin or BAIBA can also retain bone mass under conditions of unloading. Mechanical loading (exercise) maintains and extends healthy functions of osteocytes to retain bone mass. Loading of bone and factors made by contracted muscle may be a new avenue for designing therapeutics to maintain bone health. Citing Literature There is now substantial evidence from both human epidemiological studies and animal models that an adverse intrauterine environment induced by a variety of environmental and maternal factors such as diet, body composition, or endocrine factors can induce a phenotype in the offspring that is characterized by an increased risk of developing chronic non-communicable diseases in later life. The mechanism by which cues about nutrient availability in the postnatal environment are transmitted to the fetus and the process by which different, stable phenotypes are induced are beginning to be understood and involve the epigenetic regulation of specific genes. Epigenetic processes induce heritable change in gene expression without altering gene sequence. The major epigenetic mechanisms include DNA methylation, histone modification and non-coding RNAs. The epigenetic changes induced in response to nutritional cues from the mother may allow the fetus to adjust its developmental program in order to be better adapted to the future environment, while inappropriate adaptations may predispose an individual to increased risk of a range of non-communicable diseases. This talk will describe how both maternal and paternal diet can influence the health of the child through the altered epigenetic regulation of genes, how epigenetic changes in early life may be used as predictive markers of future disease risk, and how nutritional interventions in postnatal life may be able to reverse the epigenetic and phenotypic changes induced by an adverse early life environment. Citing Literature Non-communicable diseases (NCDs) pose an increasing threat to global health and economic sustainability in both high- and low-income countries, accounting for >70% of deaths globally. Fixed genetic variation accounts for only a small fraction of inherited NCD risk, and adult lifestyle interventions have had disappointing impact. Risk of NCDs is set partly during early life, when environmental influences including mother's (and to an extent father's) diet, body composition, exposure to stress and smoking or unhealthy alcohol intake affect development of the fetus and newborn, conditioning its responses to later environmental challenges such as an obesogenic lifestyle. If the cues that the baby detects are inaccurate, eg, as a result of unbalanced maternal diet or because lifestyle transition occurs between generations through migration or rapid economic development, its responses are mismatched to later environmental challenges, leading to greater NCD risk. Additionally, parents with obesity and NCDs such as diabetes can pass risk to their children, perpetuating the cycle across multiple generations. Gestational diabetes is also increasing and carries risk of later type 2 diabetes for both mother and child. Epidemiological, clinical, and basic science research has indicated underlying mechanisms, many of which involve epigenetic processes. These can serve as early markers of later risk, may be reversible, and could be used to monitor efficacy of interventions. Adopting a life course approach to the primary prevention of NCDs is now essential, starting in the preconception period by promoting healthy diets, body composition, and behavior among adolescents and young adults, not only for their later health but also for that of the next generation. While the problem of NCDs is global, sustainable solutions will have to be country—and culturally—specific. The life course approach to NCD prevention is now included in UN and WHO initiatives. Citing Literature Based on recent systematic reviews and meta-analyses, the risk of hip fracture is increased by at least twofold in patients with Parkinson's disease, recent stroke, dementia, HIV, heart failure, previously hospitalized with chronic obstructive pulmonary disease (COPD), end-stage renal disease, and type 1 (but not type 2) diabetes. Patients over age 65 years with Parkinson's disease also have more than a 10% per year risk of clinical fractures. In the United States, there are more patients with a high risk of fracture due to these conditions than patients with “osteoporosis” defined by a hip T-score ≤ −2.5. Besides the increased risk of fracture, patients with these comorbidities generally have poorer outcomes after hip fracture. For this reason, the “treatment threshold” probability of fracture that warrants drug treatment should be substantially lower for patients with these conditions than for other patients. Nevertheless, relatively few patients with these conditions receive treatment; for example, in the US, fewer than 5% of older patients with Parkinson's disease have received a prescription for an approved treatment for osteoporosis and only half of those received 2 prescriptions. Specialists who care for them have little experience with assessments and drug treatments to prevent fracture, and extra screening is barrier to many patients with comorbid conditions. Because these patients have generally been excluded from fracture prevention trials, there is no evidence that improving BMD would reduce their fracture risk. Compared with standard practice, treating older patients with these comorbid conditions without individual risk screening would reach many more patients at high risk of disabling fractures and treatment with zoledronic acid would overcome their poor persistence. Citing Literature A critical step during endochondral ossification in bone development as well as homeostatic bone renewal and fracture repair is the invasion of avascular cartilage by blood vessels and the recruitment of immature osteoprogenitor cells (OPCs) to sites of bone formation. The invasion of OPCs and blood vessels occurs in a tightly spatio-temporally synchronized manner, with some OPCs being wrapped around blood vessels as pericytes. This temporal and local synchronization of osteogenesis and angiogenesis is referred to as osteo-angiogenic coupling. Interestingly, many perivascular subpopulations in the bone marrow environment are thought to function as “reserve” skeletal progenitors or stem cells and as regulators of the local bone marrow microenvironment, including supporting roles for hematopoietic stem cell maintenance and functioning. Previous findings underline the significance of osteo-angiogenic coupling for skeletal physiology and hematopoietic integrity, and suggest an intense crosstalk between osteogenic cells and endothelial cells (ECs) that is conceivably involved in skeletal health and disease and could bear significant therapeutic value. However, the molecular mechanisms mediating the osteo-angiogenic crosstalk and the recruitment of OPCs to the vessels are far from completely characterized to date. Researchers at the KU Leuven SCEBP Lab aim to gain novel insights in the molecular control of skeletal cell functioning, with a focus on mesenchymal progenitors and osteoblast lineage cells and their interplay with the skeletal vasculature. The lab's research program is directed at understanding the mechanisms underlying bone formation in development, adult homeostasis, and fracture healing, but also in the significance of osteogenic cell biology in the broader physiological context of the organism, including hematopoiesis and global energy metabolism. Prime working models are genetically modified mice, including conditional and inducible knockout mice, in combination with fluorescent reporters and lineage tracing strategies. Citing Literature Oral Presentation Abstracts Social deprivation predicts a range of adverse health outcomes; however, its impact on outcomes after a hip fracture is not established. We examined the effect of area-level social deprivation on outcomes after hospital admission with a hip fracture in England. We used English Hospital Episodes Statistics linked by NHS Digital to the National Hip Fracture Database (04/2011–03/2015) and Office for National Statistics mortality database to identify patients admitted with hip fracture, aged 60+ years. Deprivation was measured using quintiles of the Index of Multiple Deprivation (Q1 = least deprived; Q5 =most deprived). Associations between deprivation and 30-day mortality and emergency 30-day readmission are described using odds ratios (ORs); logistic regression was used to adjust for age. Mean length of stay (LOS) in NHS acute and rehabilitation hospitals (“superspell”) was calculated; the association between deprivation and mean LOS was estimated using linear regression. Total NHS bed occupancy within 1 year post-fracture was also calculated. We identified 218,907 hospital admissions with an index hip fracture over 4 years. Median [IQR] was age 84 [78–89] years; 72.6% female. Overall 30-day mortality was 7.8% (n = 17,072/218,907). Among survivors, median superspell was 16 (10–28)days, and 12.4% were readmitted within 30 days (n = 19,497/157,303), median 1-year bed occupancy 21 (11–41) days. Greater deprivation was associated with higher 30-day mortality (Q5: 8.4% [n = 3,229/38,434] versus Q1: 7.2% [n = 3143/43,866]), age-adjusted OR 1.30 (95% CI [1.24, 1.37], p < 0.001), equating to on average 2697 excess deaths per year occurring among those who are deprived (quintiles 2–5 versus 1). Among survivors, age-adjusted mean superspell was longer in the most deprived versus least deprived quintile (Q5: 16.2 [15.3–17.1] days, Q1: 14.2 [13.6–14.7], p < 0.001). The 30-day readmission rate was higher in those most deprived 13.7% (Q5: n = 3708/27,001) compared with those least deprived 11.4% (Q1: n = 3693/32,353), age-adjusted OR 1.27 [1.21, 1.34], p < 0.001. A similar trend was observed when assessing mean 1-year NHS bed occupancy in the 71.9% who survive to 1 year (Q5: 24.7 [23.2–26.2] days; Q1: 20.8 [19.9–21.7], p < 0.001). Greater deprivation is associated with reduced 30-day survival and among those who do survive, longer hospital stays and a greater need to be readmitted to hospital once discharged. The extent to which the configuration of English hospital services, rather than patient case-mix, explains these apparent health inequalities remains to be determined. Citing Literature Osteoporosis is the commonest skeletal disorder, affecting millions and costing billions of pounds annually. Bone mineral density is highly heritable, but only 12% of the phenotype variance is currently accounted for. Treatments reduce fracture risk by only 50%, and there is urgent need to define new pathways that regulate bone turnover and strength. We hypothesized that rapid-throughput phenotyping of knockout mice would identify novel susceptibility alleles for bone and mineral disorders and provide in vivo models to elucidate their molecular basis. Translocation-associated membrane protein-2 knockout mice (Tram2-/-) were identified in this screen with reduced body weight, deafness, and spontaneous fractures, despite normal serum biochemistry. Detailed analysis (X-ray microradiography, micro-CT, backscattered-electron scanning-electron microscopy, biomechanical testing, n = 6 per sex, per genotype) demonstrated short stature (p < 0.001, ANOVA), grossly reduced bone mineral content and mineralization (p < 0.001, Kolmogorov-Smirnov test), profoundly reduced cortical (p < 0.001, ANOVA) and trabecular bone mass (p < 0.001, ANOVA), and decreased bone strength and stiffness (p < 0.001, ANOVA) in Tram2-/-mice. Tram2-/- primary osteoblasts had reduced proliferation (p < 0.001, t test) and mineralization, while osteoclasts had more nuclei (p < 0.001, t test) and increased resorption (p < 0.01, t test) compared with wild type. Tram2 lies downstream of BMP/Runx2 in osteoblasts and is associated with fracture in genomewide association studies. Tram2 is a component of the translocon responsible for the folding of type 1 collagen; however, we detected no abnormalities of type 1 collagen structure by electron microscopy or protein amount by Western blot in Tram2-/- mice. These data demonstrate that Tram2 is required for normal bone mineralization, structure, and strength. The abnormal skeletal phenotype in Tram2-/- mice likely results from impaired bone formation during growth, together with uncoupling of adult bone turnover, resulting in severe bone loss. Elucidation of the cellular and molecular mechanisms underlying this gross skeletal phenotype may identify novel tractable therapeutic targets for prevention and treatment of osteoporosis. Citing Literature Multiple myeloma is a plasma cell malignancy, which develops in the bone marrow and frequently leads to severe bone destruction. Current anti-resorptive therapies to treat the bone disease do little to repair damaged bone; therefore, new treatment strategies incorporating bone anabolic therapies are urgently required. We hypothesized that combination therapy using the standard-of-care anti-resorptive zoledronic acid (Zol) with a bone anabolic (anti-TGFβ/1D11) would be more effective at treating myeloma-induced bone disease than Zol therapy alone. JJN3 myeloma-bearing mice treated with combined Zol and 1D11 resulted in a 48% increase (p ≤ 0.001) in trabecular bone volume fraction compared with Zol alone and a 65% (p ≤ 0.0001) increase compared with 1D11 alone. The most significant finding was the substantial repair of U266-induced bone lesions with combination therapy, which resulted in a significant reduction in lesion area compared with vehicle (p ≤ 0.01) or Zol alone (p ≤ 0.01). These results reveal a novel finding and demonstrate that combined anti-resorptive and bone anabolic therapy are significantly more effective at treating established myeloma-induced bone disease than Zol alone. Th

Protease-activated receptor-2 (PAR2) is one member of a small family of transmembrane, G-protein-coupled receptors. These receptors are activated via cleavage of their N terminus by serine proteases (e.g., tryptase), unveiling an N terminus tethered ligand which binds to the second extracellular loop of the receptor. Increasing evidence has emerged identifying key pathophysiological roles for PAR2 in both rheumatoid arthritis (RA) and osteoarthritis (OA). Importantly, this includes both pro-inflammatory and destructive roles. For example, in murine models of RA, the associated synovitis, cartilage degradation, and subsequent bone erosion are all significantly reduced in the absence of PAR2. Similarly, in experimental models of OA, PAR2 disruption confers protection against cartilage degradation, subchondral bone osteosclerosis, and osteophyte formation. This review focuses on the role of PAR2 in rheumatic disease and its potential as an important therapeutic target for treating pain and joint degradation.

Osteoarthritis (OA) is no longer considered a cartilage-centric disease with remodelling of other joint tissues now recognized. While understudied, entheseal pathology is considered a secondary OA feature. A pivotal role for proteinase-activated receptor 2 (PAR2) in OA has been demonstrated previously in cartilage and subchondral bone at early time points, however the entheseal role of PAR2 has not been reported.OA was induced by destabilization of the medial meniscus (DMM) in wild type (WT) and PAR2 deficient (KO) animals. At 4 weeks and one year post surgery, knee joints were harvested for histological analysis. Medial collateral ligament (MCL) width was measured by 2D planimetry analysis. Immunohistochemistry was used to characterize the MCL and anterior cruciate ligament (ACL). Data were expressed as mean±SEM (n=4–6/group) and analysed using Student's t-test, with p<0.05 as the criterion of significance.MCL width increased between 4 weeks and 1 year in WT DMM (0.24 ±0.07 vs 0.40 ±0.008mm respectively,...

A role for endothelium-derived constricting factors (EDCF), and the angiotensin II type 1 receptor (AT1R) pathway, in the vascular impairment found in the rat Freund's complete adjuvant (FCA)-model of rheumatoid arthritis (RA) was examined. FCA arthritis was induced in rats±losartan. Vehicle-treated rats served as controls. Knee-joint swelling and red blood cell (RBC) aggregation were measured as indicators of inflammation and endothelium reactivity assessed by response to acetylcholine (ACh) on aortic rings. Results show that knee-joint swelling and RBC aggregation were elevated in the FCA+vehicle group and restored to control levels in the FCA+losartan-treated animals. ACh-induced relaxation of aortic rings taken from FCA+vehicle animals was significantly impaired compared to vehicle-controls and this vasoreactivity was restored to control levels in the FCA+losartan-treated group. Further examination of aorta from the FCA+vehicle animals revealed an EDCF that was reliant on cyclooxygenase-2 (but not cyclooxygenase-1), generation of superoxide anion generation (but not hydrogen peroxide) and activation of thromboxane-prostanoid receptor. Losartan administration in vivo or ex vivo (to aortic rings) prevented the generation of the EDCF. In summary, this is the first evidence of an EDCF in a model of RA and identifies this mechanism as potentially significant in the cardiovascular disorder associated with the disease.

Objective Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis.Methods OA was induced in wild-type (WT) and PAR2-deficient (PAR2(-/-)) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2(-/-) mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain.Results Osteophytes formed within 7 days post-DMM in WT mice but osteosclerosis was only evident from 14 days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2(-/-) mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2(-/-) mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2(-/-) mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone.Conclusions This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.

AimsRheumatoid arthritis (RA) is associated with high cardiovascular mortality. Impaired endothelial cell (EC) function and elevated angiotensin II levels may be central to the link between vascular dysfunction and RA. Here we investigated the action of angiotensin type 1 receptor (AT1R) blockade on endothelium-dependent relaxation of the isolated saphenous artery in a rat model of monoarthritis.Main MethodsAdjuvant arthritis was induced in rats with and without prophylactic losartan (AT1R antagonist) treatment. Vehicle-treated rats were used as controls. Wire myography was employed to investigate EC function of isolated rings of saphenous artery.Key FindingsEC-dependent relaxation in arteries from non-inflamed control rats was mediated by both nitric oxide (NO) and endothelium-derived hyperpolarising factor (EDHF) with the EDHF response dependent principally on functional myoendothelial gap junctions. While NO-dependent relaxation remained unaffected, the EDHF-mediated response was abolished in arteries from arthritic rats (P<0.001), however, substantial protection (approximately 50%) of the EDHF-relaxation was found in arthritic rats treated with losartan (P<0.01). Thus, the attenuated EDHF response found in the saphenous artery of arthritic rats was significantly reversed by AT1R blockade.SignificanceThese results suggest a key role for the angiotensin system in the EC dysfunction found in chronic joint inflammation and highlights AT1R as a potential therapeutic target to redress the vascular impairment and mortality associated with RA.