Use of oxygen-loaded nanobubbles to improve tissue oxygenation: Bone-relevant mechanisms of action and effects on osteoclast differentiation.

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation.

Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin.

Introduction: For decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone. Methods: Primary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation. Results: The organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells. Discussion: This 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups.

Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them prime therapeutic targets. Osteoclast research is limited by the heterogeneity of osteoclast populations generated in vitro, where the mixture of undifferentiated monocytes, binuclear pre-osteoclasts and multinucleated osteoclasts has by necessity been considered a single osteoclast population. This study describes the differentiation of primary human CD14+ monocyte-derived osteoclasts in 3D collagen gels. These osteoclasts remained small (>95% with ≤5 nuclei) but were viable and active; when released from the gel with collagenase, they fused rapidly when reseeded onto solid substrates and resorbed dentine for 2-3 weeks. 3D-generated osteoclasts expressed cell surface markers of osteoclast differentiation (e.g., CD9, RANK, OSCAR, CD63, CD51/61) which, with their small size, enabled live cell sorting of highly enriched viable subpopulations of human osteoclasts that retained full functional resorption capacity. Low-yield osteoclast preparations were strongly enriched to remove undifferentiated cells (e.g., 13.3% CD51/61+ to 84.2% CD51/61+), and subpopulations of CD9+CD51/61- early osteoclasts and CD9+CD51/61+ mature cells were distinguished. This novel approach allows the study of selected populations of differentiating osteoclasts in vitro and opens the door to in-depth transcriptomic and proteomic analysis of these cells, increasing our ability to study human osteoclast molecular mechanisms relevant to development, aging and disease.

Aberrant paracrine signalling for bone remodelling underlies the mutant histone-driven giant cell tumour of bone.

Oncohistones represent compelling evidence for a causative role of epigenetic perturbations in cancer. Giant cell tumours of bone (GCTs) are characterised by a mutated histone H3.3 as the sole genetic driver present in bone-forming osteoprogenitor cells but absent from abnormally large bone-resorbing osteoclasts which represent the hallmark of these neoplasms. While these striking features imply a pathogenic interaction between mesenchymal and myelomonocytic lineages during GCT development, the underlying mechanisms remain unknown. We show that the changes in the transcriptome and epigenome in the mesenchymal cells caused by the H3.3-G34W mutation contribute to increase osteoclast recruitment in part via reduced expression of the TGFß-like soluble factor, SCUBE3. Transcriptional changes in SCUBE3 are associated with altered histone marks and H3.3G34W enrichment at its enhancer regions. In turn, osteoclasts secrete unregulated amounts of SEMA4D which enhances proliferation of mutated osteoprogenitors arresting their maturation. These findings provide a mechanism by which GCTs undergo differentiation in response to denosumab, a drug that depletes the tumour of osteoclasts. In contrast, hTERT alterations, commonly found in malignant GCT, result in the histone-mutated neoplastic cells being independent of osteoclasts for their proliferation, predicting unresponsiveness to denosumab. We provide a mechanism for the initiation of GCT, the basis of which is dysfunctional cross-talk between bone-forming and bone-resorbing cells. The findings highlight the role of tumour/microenvironment bidirectional interactions in tumorigenesis and how this is exploited in the treatment of GCT.

Loss of mutual protection between human osteoclasts and chondrocytes in damaged joints initiates osteoclast-mediated cartilage degradation by MMPs.

Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.

Distinct roles for the hypoxia-inducible transcription factors HIF-1α and HIF-2α in human osteoclast formation and function.

Bone homeostasis is maintained by a balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Hypoxia modulates this relationship partially via direct and indirect effects of the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor on osteoclast formation and bone resorption. Little data is available on the role(s) of the HIF-2α isoform of HIF in osteoclast biology. Here we describe induction of HIF-1α and HIF-2α during the differentiation of human CD14+ monocytes into osteoclasts. Knockdown of HIF-1α did not affect osteoclast differentiation but prevented the increase in bone resorption that occurs under hypoxic conditions. HIF-2α knockdown did not affect bone resorption but moderately inhibited osteoclast formation. Growth of osteoclasts in 3D gels reversed the effect of HIF-2α knockdown; HIF-2α siRNA increasing osteoclast formation in 3D. Glycolysis is the main HIF-regulated pathway that drives bone resorption. HIF knockdown only affected glucose uptake and bone resorption in hypoxic conditions. Inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) reduced osteoclast formation and activity under both basal and hypoxic conditions, emphasising the importance of glycolytic metabolism in osteoclast biology. In summary, HIF-1α and HIF-2α play different but overlapping roles in osteoclast biology, highlighting the importance of the HIF pathway as a potential therapeutic target in osteolytic disease.

Osteoblast-Osteoclast Coculture Amplifies Inhibitory Effects of FG-4592 on Human Osteoclastogenesis and Reduces Bone Resorption.

The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcription factor, HIF, which drives both osteogenesis and angiogenesis. The recent clinical approval of PHD enzyme inhibitors, which stabilize HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis, and skeletal fracture and nonunion. However, bone-resorbing osteoclasts also play a central role in bone remodeling and pathological osteolysis, and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the result of PHD enzyme inhibition in vivo would be mediated by a balance between increased bone formation and increased bone resorption. It is essential that we improve our understanding of the effects of HIF on osteoclast formation and function and consider the potential contribution of inhibitory interactions with other musculoskeletal cells. The PHD enzyme inhibitor FG-4592 stabilized HIF protein and stimulated osteoclast-mediated bone resorption, but inhibited differentiation of human CD14+ monocytes into osteoclasts. Formation of osteoclasts in a more physiologically relevant 3D collagen gel did not affect the sensitivity of osteoclastogenesis to FG-4592, but increased sensitivity to reduced concentrations of RANKL. Coculture with osteoblasts amplified inhibition of osteoclastogenesis by FG-4592, whether the osteoblasts were proliferating, differentiating, or in the presence of exogenous M-CSF and RANKL. Osteoblast coculture dampened the ability of high concentrations of FG-4592 to increase bone resorption. These data provide support for the therapeutic use of PHD enzyme inhibitors to improve bone formation and/or reduce bone loss for the treatment of osteolytic pathologies and indicate that FG-4592 might act in vivo to inhibit the formation and activity of the osteoclasts that drive osteolysis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Transcriptomic profiling of the myeloma bone-lining niche reveals BMP signalling inhibition to improve bone disease.

Multiple myeloma is an incurable, bone marrow-dwelling malignancy that disrupts bone homeostasis causing skeletal damage and pain. Mechanisms underlying myeloma-induced bone destruction are poorly understood and current therapies do not restore lost bone mass. Using transcriptomic profiling of isolated bone lining cell subtypes from a murine myeloma model, we find that bone morphogenetic protein (BMP) signalling is upregulated in stromal progenitor cells. BMP signalling has not previously been reported to be dysregulated in myeloma bone disease. Inhibition of BMP signalling in vivo using either a small molecule BMP receptor antagonist or a solubilized BMPR1a-FC receptor ligand trap prevents trabecular and cortical bone volume loss caused by myeloma, without increasing tumour burden. BMP inhibition directly reduces osteoclastogenesis, increases osteoblasts and bone formation, and suppresses bone marrow sclerostin levels. In summary we describe a novel role for the BMP pathway in myeloma-induced bone disease that can be therapeutically targeted.

The Adenosine A2B Receptor Drives Osteoclast-Mediated Bone Resorption in Hypoxic Microenvironments.

Osteoclast-mediated bone destruction is amplified in the hypoxic synovial microenvironment of rheumatoid arthritis (RA). This increased bone resorption is driven by the hypoxia-inducible transcription factor HIF. We identified hypoxic induction of the HIF-regulated adenosine A2B receptor in primary human osteoclasts (mRNA, 3.8-fold increase, p < 0.01) and sought to identify the role(s) of purinergic signaling via this receptor in the bone resorption process. Primary human osteoclasts were differentiated from CD14+ monocytes and exposed to hypoxia (2% O2) and A2B receptor inhibitors (MRS1754, PSB603). The hypoxic increase in bone resorption was prevented by the inhibition of the A2B receptor, at least partly by the attenuation of glycolytic and mitochondrial metabolism via inhibition of HIF. A2B receptor inhibition also reduced osteoclastogenesis in hypoxia by inhibiting early cell fusion (day 3-4, p < 0.05). The A2B receptor is only functional in hypoxic or inflammatory environments when the extracellular concentrations of adenosine (1.6-fold increase, p < 0.05) are sufficient to activate the receptor. Inhibition of the A2B receptor under normoxic conditions therefore did not affect any parameter tested. Reciprocal positive regulation of HIF and the A2B receptor in a hypoxic microenvironment thus enhances glycolytic and mitochondrial metabolism in osteoclasts to drive increased bone resorption. A2B receptor inhibition could potentially prevent the pathological osteolysis associated with hypoxic diseases such as rheumatoid arthritis.

Factors influencing variation in participation in the National Diabetes Audit and the impact on the Quality and Outcomes Framework indicators of diabetes care management.

OBJECTIVE: Participation in the National Diabetes Audit (NDA) has become a contractual requirement for all general practices in England and is used as part of the assessment framework for sustainability and transformation partnership (STP) footprints. The study aimed to investigate general practice-related factors which may influence participation in the NDA, and the impact that participation in the NDA may have on diabetes management and patient care. RESEARCH DESIGN: A cross-sectional analysis of routine primary care data from 45 725 646 patients aged 17+ years registered across 7779 general practices in England was performed using logistic regression. The main outcome measures included general practice voluntary participation in the NDA, general practice-related factors (practice size, deprivation, diabetes prevalence, geographic area, practice population age) and diabetes management outcomes (cholesterol, blood pressure, hemoglobin A1c (HbA1c)). RESULTS: Participation in the NDA differed significantly according to practice size (t(7653)=-9.93, p=0.001), level of deprivation (χ2(9)=36.17, p<0.0001), diabetes prevalence (p<0.0001), practice population age (p<0.0001), and geographic area (χ2(26)=676.9, p<0.0001). In addition, the Quality and Outcomes Framework diabetes indicator HbA1c (OR 1.01, CI 1.0 to 1.01, p=0.0001) but not cholesterol (p=0.055) or blood pressure (p=0.76) was independently associated with NDA participation when controlling for practice-related factors. CONCLUSION: Variation in NDA participation exists. It is suggested that some practices may need additional support when submitting data to the NDA and that NDA participation may have an impact on diabetes outcomes. However, the use of NDA outcomes as a measure of progress with diabetes care by STPs is still unclear and further investigation is needed.

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export.

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Does laryngeal reinnervation or type I thyroplasty give better voice results for patients with unilateral vocal fold paralysis (VOCALIST): study protocol for a feasibility randomised controlled trial.

INTRODUCTION: A functioning voice is essential for normal human communication. A good voice requires two moving vocal folds; if one fold is paralysed (unilateral vocal fold paralysis (UVFP)) people suffer from a breathy, weak voice that tires easily and is unable to function normally. UVFP can also result in choking and breathlessness. Current treatment for adults with UVFP is speech therapy to stimulate recovery of vocal fold (VF) motion or function and/or injection of the paralysed VF with a material to move it into a more favourable position for the functioning VF to close against. When these therapies are unsuccessful, or only provide temporary relief, surgery is offered. Two available surgical techniques are: (1) surgical medialisation; placing an implant near the paralysed VF to move it to the middle (thyroplasty) and/or repositioning the cartilage (arytenoid adduction) or (2) restoring the nerve supply to the VF (laryngeal reinnervation). Currently there is limited evidence to determine which surgery should be offered to adults with UVFP. METHODS AND ANALYSIS: A feasibility study to test the practicality of running a multicentre, randomised clinical trial of surgery for UVFP, including: (1) a qualitative study to understand the recruitment process and how it operates in clinical centres and (2) a small randomised trial of 30 participants recruited at 3 UK sites comparing non-selective laryngeal reinnervation to type I thyroplasty. Participants will be followed up for 12 months. The primary outcome focuses on recruitment and retention, with secondary outcomes covering voice, swallowing and quality of life. ETHICS AND DISSEMINATION: Ethical approval was received from National Research Ethics Service-Committee Bromley (reference 11/LO/0583). In addition to dissemination of results through presentation and publication of peer-reviewed articles, results will be shared with key clinician and patient groups required to develop the future large-scale randomised controlled trial. TRIAL REGISTRATION NUMBER: ISRCTN90201732; 16 December 2015.

Multiple Roles of Angiopoietin-Like 4 in Osteolytic Disease.

Hypoxia and the hypoxia-inducible factor (HIF) transcription factor drive pathological bone loss in conditions including rheumatoid arthritis (RA), osteoarthritis, osteoporosis, primary bone tumours, and bone metastatic cancer. There is therefore considerable interest in determining the function(s) of HIF-induced genes in these pathologies. Angiopoietin-like 4 (ANGPTL4) is an adipose-derived, HIF-1α- and PPARγ-induced gene that was originally discovered as an endocrine and autocrine/paracrine regulator of lipid metabolism. Given the inverse relationship between bone adiposity and fracture risk, ANGPTL4 might be considered a good candidate for mediating the downstream effects of HIF-1α relevant to osteolytic disease. This review will consider the possible roles of ANGPTL4 in regulation of osteoclast-mediated bone resorption, cartilage degradation, angiogenesis, and inflammation, focusing on results obtained in the study of RA. Possible roles in other musculoskeletal pathologies will also be discussed. This will highlight ANGPTL4 as a regulator of multiple disease processes, which could represent a novel therapeutic target in osteolytic musculoskeletal disease.

Hypoxia-inducible factor 1-alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl-4-hydroxylase 2.

Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, provoking interest in HIF activation as a therapeutic strategy to improve osteoblast mineralization and treat pathological osteolysis. However, HIF also enhances the bone-resorbing activity of mature osteoclasts. It is therefore essential to determine the full effect(s) of HIF on both the formation and the bone-resorbing function of osteoclasts in order to understand how they might respond to such a strategy. Expression of HIF-1α mRNA and protein increased during osteoclast differentiation from CD14+ monocytic precursors, additionally inducing expression of the HIF-regulated glycolytic enzymes. However, HIF-1α siRNA only moderately affected osteoclast differentiation, accelerating fusion of precursor cells. HIF induction by inhibition of the regulatory prolyl-4-hydroxylase (PHD) enzymes reduced osteoclastogenesis, but was confirmed to enhance bone resorption by mature osteoclasts. Phd2+/- murine osteoclasts also exhibited enhanced bone resorption, associated with increased expression of resorption-associated Acp5, in comparison with wild-type cells from littermate controls. Phd3-/- bone marrow precursors displayed accelerated early fusion, mirroring results with HIF-1α siRNA. In vivo, Phd2+/- and Phd3-/- mice exhibited reduced trabecular bone mass, associated with reduced mineralization by Phd2+/- osteoblasts. These data indicate that HIF predominantly functions as a regulator of osteoclast-mediated bone resorption, with little effect on osteoclast differentiation. Inhibition of HIF might therefore represent an alternative strategy to treat diseases characterized by pathological levels of osteolysis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Hypoxia-Induced Fibroblast Growth Factor 11 Stimulates Osteoclast-Mediated Resorption of Bone.

Over-activation of osteoclasts is directly responsible for pathological bone loss in conditions such as rheumatoid arthritis and cancer metastasis to bone. Hypoxia is a common feature of these conditions, associated with poor prognosis, which also stimulates osteoclast-mediated bone resorption via induction of the hypoxia-inducible transcription factor HIF-1α. Here, we investigate the effects of fibroblast growth factor 11 (FGF11) on osteoclast function. FGF11 is an intracellular FGF that was induced both by hypoxia (2% O2, p < 0.01) and by inhibition of the HIF-regulating prolyl hydroxylase enzymes (CoCl2, p < 0.001) in osteoclasts. Isoform-specific siRNA demonstrated that the induction of Fgf11 mRNA expression by hypoxia is HIF-1α-dependent (p < 0.01). Hypoxic stimulation of bone resorption was inhibited in osteoclasts treated with siRNA targeting FGF11 (p < 0.05). This was at least partially due to reduced secretion of an unidentified pro-resorptive factor downstream of FGF11. FGF11 expression within hypoxic, resorbing osteoclasts co-localised with microtubule-associated alpha-tubulin. FGF11 was also abundantly expressed in osteoclasts within the rheumatoid synovium and in giant cell tumour of bone. This study suggests FGF11 as a novel factor driving pathological bone resorption in osteolytic disease and as a potential target for the development of new anti-resorptive therapeutic agents.

Hypoxic regulation of osteoclast differentiation and bone resorption activity.

Bone integrity is maintained throughout life via the homeostatic actions of bone cells, namely, osteoclasts, which resorb bone, and osteoblasts, which produce bone. Disruption of this balance in favor of osteoclast activation results in pathological bone loss, which occurs in conditions including osteoporosis, rheumatoid arthritis, primary bone cancer, and cancer metastasis to bone. Hypoxia also plays a major role in these conditions, where it is associated with disease progression and poor prognosis. In recent years, considerable interest has arisen in the mechanisms whereby hypoxia and the hypoxia-inducible transcription factors, HIF-1α and HIF-2α, affect bone remodeling and bone pathologies. This review summarizes the current evidence for hypoxia-mediated regulation of osteoclast differentiation and bone resorption activity. Role(s) of HIF and HIF target genes in the formation of multinucleated osteoclasts from cells of the monocyte-macrophage lineage and in the activation of bone resorption by mature osteoclasts will be discussed. Specific attention will be paid to hypoxic metabolism and generation of ATP by osteoclasts. Hypoxia-driven increases in both glycolytic flux and mitochondrial metabolic activity, along with consequent generation of mitochondrial reactive oxygen species, have been found to be essential for osteoclast formation and resorption activity. Finally, evidence for the use of HIF inhibitors as potential therapeutic agents targeting bone resorption in osteolytic disease will be discussed.

Angiopoietin-like 4 is over-expressed in rheumatoid arthritis patients: association with pathological bone resorption.

INTRODUCTION: Osteoclasts are responsible for the bone loss associated with rheumatoid arthritis (RA). The secreted adipokine angiopoietin-like 4 (ANGPTL4) specifically increases osteoclast-mediated bone resorption. We have investigated expression of ANGPTL4 and its regulatory transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α), in osteoclasts and other cells within rheumatoid synovium. We have also examined whether circulating levels of ANGPTL4 differ in RA patients compared with that in normal controls or patients with osteoarthritis (OA). RESULTS: Immunohistochemical analysis revealed that bone-apposing osteoclasts within the rheumatoid synovium express both ANGPTL4 and HIF-1α. ANGPTL4 was also strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68+ macrophages and plasma cells within RA synovium. Little ANGPTL4 was evident in normal synovial tissue. This reflected the over-expression of HIF-1α in rheumatoid versus normal synovial tissue. The concentration of ANGPTL4 was higher in both the serum and the synovial fluid of RA patients than in patients with OA or normal controls. High serum ANGPTL4 associated with elevated levels of the serum marker of bone resorption, receptor activator for nuclear factor κB ligand (RANKL). CONCLUSIONS: Over-expression of ANGPTL4 in multiple cell types within the rheumatoid synovium potentially provides a local pool of ANGPTL4 to stimulate osteoclast-mediated bone resorption in RA. Additionally, correlation of high serum ANGPTL4 with circulating RANKL suggests that ANGPTL4 may represent a novel marker for bone destruction in RA.

The art of midwifery: can creative images of birth enhance holistic care?

Art related to birth stimulates debate, particularly if it is perceived to be taboo and challenging popular images of mother and child. Birth traditionally has been in a woman's sphere of experience, thus it has been left unexplored on a wider level. The Birth Rites Collection was originally developed to enable partnerships with artists and childbirth professionals. The other important reason for the Birth Rites project was to begin to make contemporary cutting edge art around childbirth because there has been a real lack of work which explores this subject. Student midwives have been able to engage with these and other artworks related to childbirth and now produce their own original art which is attracting acclaim. The Art of Midwifery student midwife project aims to promote more aesthetic and creative ways of learning to enhance midwifery students' self-awareness and thus promote holistic, woman-centred and sensitive care. Students have visited art exhibitions to interact with artworks related to curriculum themes and explore models and philosophies of birth. This paper reports some of their activities, summarises their responses and evaluates the collaboration.

Differential regulation of HIF-mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts.

Inappropriate osteoclast activity instigates pathological bone loss in rheumatoid arthritis. We have investigated how osteoclasts generate sufficient ATP for the energy-intensive process of bone resorption in the hypoxic microenvironment associated with this rheumatic condition. We show that in human osteoclasts differentiated from CD14(+) monocytes, hypoxia (24 h, 2% O2 ): (a) increases ATP production and mitochondrial electron transport chain activity (Alamar blue, O2 consumption); (b) increases glycolytic flux (glucose consumption, lactate production); and (c) increases glutamine consumption. We demonstrate that glucose, rather than glutamine, is necessary for the hypoxic increase in ATP production and also for cell survival in hypoxia. Using siRNA targeting specific isoforms of the hypoxia-inducible transcription factor HIF (HIF-1α, HIF-2α), we show that employment of selected components of the HIF-1α-mediated metabolic switch to anaerobic respiration enables osteoclasts to rapidly increase ATP production in hypoxia, while at the same time compromising long-term survival. We propose this atypical HIF-driven metabolic pathway to be an adaptive mechanism to permit rapid bone resorption in the short term while ensuring curtailment of the process in the absence of re-oxygenation.