The Paget's disease of bone risk gene PML is a negative regulator of osteoclast differentiation and bone resorption.

Paget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml-/- mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml-/- was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml-/- mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml-/- mice and wild type, bone histomorphometry showed that Pml-/- mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper.

Ubiquitin-protein ligase Ubr5 cooperates with hedgehog signalling to promote skeletal tissue homeostasis.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.

Chondrocytes from osteoarthritic cartilage of obese patients show altered adiponectin receptors expression and response to adiponectin.

Obesity and osteoarthritis (OA) are well-known comorbidities and their precise molecular interactions are still unidentified. Adiponectin, a major adipokine, known to have an anti-inflammatory effect in atherosclerosis or Type 2 Diabetes Mellitus (T2DM), has also been postulated to be pro-inflammatory in OA. This dual role of adiponectin is still not explained. The precise mechanism by which adiponectin affects cartilage and chondrocytes remains to be elucidated. In the present observational study chondrocytes from 30 patients with OA (18 females and 12 males) undergoing total knee replacement (TKR) were isolated. Expression of adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2) was examined both at gene and protein levels in chondrocytes. The difference in adiponectin receptor expression between lean and obese patients with OA and the role of adiponectin in regulating pro-inflammatory genes (MCP-1, IL-6, and VCAM-1, MMP-1, MMP-2, and TIMP-1) has been investigated. We found that ADIPOR1 represented the most abundant adiponectin receptor in primary OA chondrocytes. ADIPOR1 and ADIPOR2 genes and ADIPOR1 protein were differently expressed in OA chondrocytes obtained from obese compared with lean patients with OA. Adiponectin induced gene expression of MCP-1, IL-6, and MMP-1 in all OA patients' chondrocytes. In contrast, VCAM-1 and MMP-2 were differently regulated by adiponectin depending on the patient's body mass index. This study suggests that adiponectin and ADIPOR1 may have important roles in the pathogenesis of cartilage degeneration in OA of obese subjects.

High phosphate induces skeletal muscle atrophy and suppresses myogenic differentiation by increasing oxidative stress and activating Nrf2 signaling.

Skeletal muscle wasting represents both a common phenotype of aging and a feature of pathological conditions such as chronic kidney disease (CKD). Although both clinical data and genetic experiments in mice suggest that hyperphosphatemia accelerates muscle wasting, the underlying mechanism remains unclear. Here, we showed that inorganic phosphate (Pi) dose-dependently decreases myotube size, fusion index, and myogenin expression in mouse C2C12 skeletal muscle cells. These changes were accompanied by increases in reactive oxygen species (ROS) production and Nrf2 and p62 expression, and reductions in mitochondrial membrane potential (MMP) and Keap1 expression. Inhibition of Pi entry, cytosolic ROS production, or Nrf2 activation reversed the effects of high Pi on Nrf2, p62, and myogenin expression. Overexpression of Nrf2 respectively increased and decreased the promoter activity of p62-Luc and myogenin-Luc reporters. Analysis of nuclear extracts from gastrocnemius muscles from mice fed a high-Pi (2% Pi) diet showed increased Nrf2 phosphorylation in sham-operated and 5/6 nephrectomized (CKD) mice, and both increased p62 phosphorylation and decreased myogenin expression in CKD mice. These data suggest that high Pi suppresses myogenic differentiation in vitro and promotes muscle atrophy in vivo through oxidative stress-mediated protein degradation and both canonical (ROS-mediated) and non-canonical (p62-mediated) activation of Nrf2 signaling.

Development of mouse models of angiosarcoma driven by p53.

Angiosarcomas are a rare group of tumours which have poor prognosis and limited treatment options. The development of new therapies has been hampered by a lack of good preclinical models. Here, we describe the development of an autochthonous mouse model of angiosarcoma driven by loss of p53 in VE-cadherin-expressing endothelial cells. Using Cdh5-Cre to drive recombination in adult endothelial cells, mice developed angiosarcomas with 100% penetrance upon homozygous deletion of Trp53 with a median lifespan of 325 days. In contrast, expression of the R172H mutant p53 resulted in formation of thymic lymphomas with a more rapid onset (median lifespan 151 days). We also used Pdgfrb-Cre-expressing mice, allowing us to target predominantly pericytes, as these have been reported as the cell of origin for a number of soft tissue sarcomas. Pdgfrb-Cre also results in low levels of recombination in venous blood endothelial cells in multiple tissues during development. Upon deletion of Trp53 in Pdgfrb-Cre-expressing mice (Pdgfrb-Cre,Trp53fl/fl mice), 65% developed lymphomas and 21% developed pleomorphic undifferentiated soft tissue sarcomas. None developed angiosarcomas. In contrast, 75% of Pdgfrb-Cre,Trp53R172H/R172H mice developed angiosarcomas, with 60% of these mice also developing lymphomas. The median lifespan of the Pdgfrb-Cre,Trp53R172H/R172H mice was 151 days. Re-implantation of angiosarcoma tumour fragments from Cdh5-Cre, Trp53fl/fl mice provided a more consistent and rapid model of angiosarcoma than the two spontaneous models. The ability to passage tumour fragments through the mouse provides a novel model which is amenable to preclinical studies and will help the development of potential new therapies for angiosarcoma.

Involvement of ADAM12 in Chondrocyte Differentiation by Regulation of TGF-ß1-Induced IGF-1 and RUNX-2 Expressions.

A disintegrin and metalloproteinase 12 (ADAM12) is known to be involved in chondrocyte proliferation and maturation; however, the mechanisms are not fully understood. In this study, expression and localization of ADAM12 during chondrocyte differentiation were examined in the mouse growth plate by immunohistochemistry. Adam12 expression during ATDC5 chondrogenic differentiation was examined by real-time PCR and compared with the expression pattern of type X collagen. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was used to generate Adam12-knockout (KO) ATDC5 cells. Adam12-KO and Adam12 overexpressing cells were used for analyses of ADAM12 expression with or without TGF-ß1 stimulation. ADAM12 was identified predominantly in chondrocytes of the proliferative zone in mouse growth plates by immunohistochemistry. Adam12 was upregulated prior to Col10a1 during chondrogenic differentiation in wild-type ATDC5 cells. In Adam12-KO ATDC5 cells, following initiation of chondrogenic differentiation, we observed a reduction in Igf-1 expression along with an upregulation of hypertrophy-associated Runx2, Col10a1, and type X collagen protein expressions. In ATDC5 wild-type cells, stimulation with TGF-ß1 upregulated the expressions of Adam12 and Igf-1 and downregulated the expression of Runx2. In contrast, in Adam12-KO ATDC5 cells, these TGF-ß1-induced changes were suppressed. Adam12 overexpression resulted in an upregulation of Igf-1 and downregulation of Runx2 expression in ATDC5 cells. The findings suggest that ADAM12 has important role in the regulation of chondrocyte differentiation, potentially by regulation of TGF-ß1-dependent signaling and that targeting of ADAM12 may have a role in management of abnormal chondrocyte differentiation.

The Potential of Intrinsically Magnetic Mesenchymal Stem Cells for Tissue Engineering.

The magnetization of mesenchymal stem cells (MSC) has the potential to aid tissue engineering approaches by allowing tracking, targeting, and local retention of cells at the site of tissue damage. Commonly used methods for magnetizing cells include optimizing uptake and retention of superparamagnetic iron oxide nanoparticles (SPIONs). These appear to have minimal detrimental effects on the use of MSC function as assessed by in vitro assays. The cellular content of magnetic nanoparticles (MNPs) will, however, decrease with cell proliferation and the longer-term effects on MSC function are not entirely clear. An alternative approach to magnetizing MSCs involves genetic modification by transfection with one or more genes derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesizes single-magnetic domain crystals which are incorporated into magnetosomes. MSCs with either or mms6 and mmsF genes are followed by bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by magnetic resonance (MR) and which have no deleterious effects on MSC proliferation, migration, or differentiation. The stable transfection of magnetosome-associated genes in MSCs promotes assimilation of magnetic nanoparticle synthesis into mammalian cells with the potential to allow MR-based cell tracking and, through external or internal magnetic targeting approaches, enhanced site-specific retention of cells for tissue engineering.

Dexamethasone Down-regulates Osteocalcin in Bone Cells through Leptin Pathway.

Glucocorticoid therapy, especially at higher doses, is associated with significant adverse side effects including osteoporosis. Leptin, secreted from adipose tissue, has diverse effects on bone tissue regulation. As glucocorticoids stimulate leptin synthesis and secretion directly in adipose tissue we hypothesised that dexamethasone (DEX) induced osteoporosis may, in part, be mediated by an osteoblast dependent leptin-leptin receptor pathway. Human bone cells expressed leptin and leptin receptors (Ob-Ra and Ob-Rb). DEX increased leptin, Ob-Ra and Ob-Rb expression in a dose-dependent manner while decreasing expression of osteocalcin. In the presence of leptin, Cbfa1 and osteonectin expression showed no significant change, whereas osteocalcin expression was decreased. Recombinant human quadruple antagonist leptin suppressed DEX-induced osteocalcin downregulation. The signaling pathway involved up-regulation of JAK2. In conclusion, upregulation of leptin and Ob-Rb in human bone cells by DEX is associated with down-regulation of osteocalcin expression. The down regulation of osteocalcin by DEX was partially through a leptin autocrine/paracrine loop. Adverse effects of DEX on the skeleton may be modified by targeting leptin signaling pathways.

Kindlin-1 Promotes Pulmonary Breast Cancer Metastasis.

In breast cancer, increased expression of the cytoskeletal adaptor protein Kindlin-1 has been linked to increased risks of lung metastasis, but the functional basis is unknown. Here, we show that in a mouse model of polyomavirus middle T antigen-induced mammary tumorigenesis, loss of Kindlin-1 reduced early pulmonary arrest and later development of lung metastasis. This phenotype relied on the ability of Kindlin-1 to bind and activate ß integrin heterodimers. Kindlin-1 loss reduced α4 integrin-mediated adhesion of mammary tumor cells to the adhesion molecule VCAM-1 on endothelial cells. Treating mice with an anti-VCAM-1 blocking antibody prevented early pulmonary arrest. Kindlin-1 loss also resulted in reduced secretion of several factors linked to metastatic spread, including the lung metastasis regulator tenascin-C, showing that Kindlin-1 regulated metastatic dissemination by an additional mechanism in the tumor microenvironment. Overall, our results show that Kindlin-1 contributes functionally to early pulmonary metastasis of breast cancer.Significance: These findings provide a mechanistic proof in mice that Kindin-1, an integrin-binding adaptor protein, is a critical mediator of early lung metastasis of breast cancer. Cancer Res; 78(6); 1484-96. ©2018 AACR.

Cellular pharmacodynamic effects of Pycnogenol® in patients with severe osteoarthritis: a randomized controlled pilot study.

BACKGROUND: The standardized maritime pine bark extract (Pycnogenol®) has previously shown symptom alleviating effects in patients suffering from moderate forms of knee osteoarthritis (OA). The cellular mechanisms for this positive impact are so far unknown. The purpose of the present randomized pilot controlled study was to span the knowledge gap between the reported clinical effects of Pycnogenol® and its in vivo mechanism of action in OA patients. METHODS: Thirty three patients with severe OA scheduled for a knee arthroplasty either received 100 mg of Pycnogenol® twice daily or no treatment (control group) three weeks before surgery. Cartilage, synovial fluid and serum samples were collected during surgical intervention. Relative gene expression of cartilage homeostasis markers were analyzed in the patients' chondrocytes. Inflammatory and cartilage metabolism mediators were investigated in serum and synovial fluid samples. RESULTS: The oral intake of Pycnogenol® downregulated the gene expression of various cartilage degradation markers in the patients' chondrocytes, the decrease of MMP3, MMP13 and the pro-inflammatory cytokine IL1B were statistically significant (p ≤ 0.05). Additionally, protein concentrations of ADAMTS-5 in serum were reduced significantly (p ≤ 0.05) after three weeks intake of the pine bark extract. CONCLUSIONS: This is the first report about positive cellular effects of a dietary supplement on key catabolic and inflammatory markers in patients with severe OA. The results provide a rational basis for understanding previously reported clinical effects of Pycnogenol® on symptom scores of patients suffering from OA. TRIAL REGISTRATION: ISRCTN10754119 . Retrospectively registered 08/10/2015.

Macrophage 11ß-HSD-1 deficiency promotes inflammatory angiogenesis.

11ß-Hydroxysteroid dehydrogenase-1 (11ß-HSD1) predominantly converts inert glucocorticoids into active forms, thereby contributing to intracellular glucocorticoid levels. 11ß-HSD1 is dynamically regulated during inflammation, including in macrophages where it regulates phagocytic capacity. The resolution of inflammation in some disease models including inflammatory arthritis is impaired by 11ß-HSD1 deficiency or inhibition. However, 11ß-HSD1 deficiency/inhibition also promotes angiogenesis, which is beneficial in mouse models of surgical wound healing, myocardial infarction or obesity. The cell types responsible for the anti-inflammatory and anti-angiogenic roles of 11ß-HSD1 have not been characterised. Here, we generated Hsd11b1MKO mice with LysM-Cre mediated deletion of Hsd11b1 to investigate whether 11ß-HSD1 deficiency in myeloid phagocytes is pro-angiogenic and/or affects the resolution of inflammation. Resolution of inflammatory K/BxN-induced arthritis was impaired in Hsd11b1MKO mice to a similar extent as in mice globally deficient in 11ß-HSD1. This was associated with >2-fold elevation in levels of the endothelial marker Cdh5 mRNA, suggesting increased angiogenesis in joints of Hsd11b1MKO mice following arthritis. A pro-angiogenic phenotype was confirmed by measuring angiogenesis in subcutaneously implanted polyurethane sponges, in which Hsd11b1MKO mice showed 20% greater vessel density than their littermate controls, associated with higher expression of Cdh5 Thus, 11ß-HSD1 deficiency in myeloid phagocytes promotes angiogenesis. Targeting 11ß-HSD1 in macrophages may be beneficial in tissue repair.

Regional Differences Between Perisynovial and Infrapatellar Adipose Tissue Depots and Their Response to Class II and Class III Obesity in Patients With Osteoarthritis.

OBJECTIVE: Obesity is associated with an increased risk of developing osteoarthritis (OA), which is postulated to be secondary to adipose tissue-dependent inflammation. Periarticular adipose tissue depots are present in synovial joints, but the association of this tissue with OA has not been extensively explored. The aim of this study was to investigate differences in local adipose tissue depots in knees with OA and characterize the changes related to class II and class III obesity in patients with end-stage knee OA. METHODS: Synovium and the infrapatellar fat pad (IPFP) were collected during total knee replacement from 69 patients with end-stage OA. Histologic changes, changes in gene and protein expression of adiponectin, peroxisome proliferator-activated receptor γ (PPARγ), and Toll-like receptor 4 (TLR-4), and immune cell infiltration into the adipose tissue were investigated. RESULTS: IPFP and synovium adipose tissue depots differed significantly and were influenced by the patient's body mass index. Compared to adipocytes from the IPFP and synovium of lean patients, adipocytes from the IPFP of obese patients were significantly larger and the synovium of obese patients displayed marked fibrosis, increased macrophage infiltration, and higher levels of TLR4 gene expression. The adipose-related markers PPARγ in the IPFP and adiponectin and PPARγ in the synovium were expressed at lower levels in obese patients compared to lean patients. Furthermore, there were increased numbers of CD45+ hematopoietic cells, CD45+CD14+ total macrophages, and CD14+CD206+ M2-type macrophages in both the IPFP and synovial tissue of obese patients. CONCLUSION: These differences suggest that IPFP and synovium may contain 2 different white adipose tissue depots and support the theory of inflammation-induced OA in patients with class II or III obesity. These findings warrant further investigation as a potentially reversible, or at least suppressible, cause of OA in obese patients.

Adult-onset idiopathic chondrolysis of the hip.

We report the case of a 23-year-old man diagnosed with adult-onset idiopathic chondrolysis of the hip. Chondrolysis of the hip is a disorder most frequently seen in children who have suffered with slipped capital femoral epiphyses. Idiopathic chondrolysis of the hip is extremely rare and to our knowledge, its onset has never been documented in adults aged over 20. With reference to the available medical literature, we summarise the current clinical management of this unusual but important cause of young adult hip pain.

Biosynthesis of magnetic nanoparticles by human mesenchymal stem cells following transfection with the magnetotactic bacterial gene mms6.

The use of stem cells to support tissue repair is facilitated by loading of the therapeutic cells with magnetic nanoparticles (MNPs) enabling magnetic tracking and targeting. Current methods for magnetizing cells use artificial MNPs and have disadvantages of variable uptake, cellular cytotoxicity and loss of nanoparticles on cell division. Here we demonstrate a transgenic approach to magnetize human mesenchymal stem cells (MSCs). MSCs are genetically modified by transfection with the mms6 gene derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesises single-magnetic domain crystals which are incorporated into magnetosomes. Following transfection of MSCs with the mms6 gene there is bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by MR and which have no deleterious effects on cell proliferation, migration or differentiation. The assimilation of magnetic nanoparticle synthesis into mammalian cells creates a real and compelling, cytocompatible, alternative to exogenous administration of MNPs.

Intranodal Palisaded Myofibroblastoma Masquerading as N2 Non-Small Cell Lung Carcinoma.

Intranodal palisaded myofibroblastoma is a rare and benign tumor that usually presents in the inguinal region. We report the case of a 68-year-old woman with a right paratracheal mass and right upper lobe non-small cell lung carcinoma initially staged as T1b N2 M0. After mediastinal staging, the right paratracheal mass was found to be an intranodal palisaded myofibroblastoma, which had caused erroneous upstaging of the lung carcinoma to N2 disease. This had the potential of leading to suboptimal treatment of the primary lung carcinoma if formal mediastinal staging had not been performed. To the best of our knowledge, this is the first report in the English literature of an intranodal palisaded myofibroblastoma occurring concurrently with lung cancer. This case highlights the importance of mediastinal staging in lung cancer. Mediastinoscopy remains the gold standard.

Functional roles of CSPG4/NG2 in chondrosarcoma.

CSPG4/NG2 is a multifunctional transmembrane protein with limited distribution in adult tissues including articular cartilage. The purpose of this study was to investigate the possible roles of CSPG4/NG2 in chondrosarcomas and to establish whether this molecule may have potential for targeted therapy. Stable knock-down of CSPG4/NG2 in the JJ012 chondrosarcoma cell line by shRNA resulted in decreased cell proliferation and migration as well as a decrease in gene expression of the MMP (matrix metalloproteinase) 3 protease and ADAMTS4 (aggrecanase). Chondrosarcoma cells in which CSPG4/NG2 was knocked down were more sensitive to doxorubicin than wild-type cells. The results indicate that CSPG4/NG2 has roles in regulating chondrosarcoma cell function in relation to growth, spread and resistance to chemotherapy and that anti-CSPG4/NG2 therapies may have potential in the treatment of surgically unresectable chondrosarcoma.

Mapping Chondrocyte Viability, Matrix Glycosaminoglycan, and Water Content on the Surface of a Bovine Metatarsophalangeal Joint.

OBJECTIVE: The purpose of this study was to determine if there were variations in chondrocyte viability, matrix glycosaminoglycan (GAG), and water content between different areas of the articular surface of a bovine metatarsophalangeal joint, a common and reliable source of articular cartilage for experimental study, which may compromise the validity of using multiple samples from different sites within the joint. METHODS: Nine fresh cadaveric bovine metatarsophalangeal joints were obtained. From each joint, 16 osteochondral explants were taken from 4 facets, yielding a total of 144 cartilage specimens for evaluation of chondrocyte viability, matrix GAG, and water content. A less invasive method for harvesting osteochondral explants and for processing the biopsy for the assessment of chondrocyte viability was developed, which maintained maximal viability within each cartilage explant. RESULTS: There was no significant difference between the 16 biopsy sites from the different areas of the joint surface with respect to chondrocyte viability, matrix GAG and water content. Pooled data of all samples from each joint established the baseline values of chondrocyte viability to be 89.4% ± 3.8%, 94.4% ± 2.2%, and 77.9% ± 7.8%, in the superficial quarter, central half, and deep quarter (with regard to depth from the articular surface), respectively. The matrix GAG content of bovine articular cartilage was 6.06 ± 0.41 µg/mg cartilage, and the cartilage water content was 72.4% ± 1.5%. There were also no significant differences of these 3 variables between the different joints. CONCLUSION: It is thus reasonable to compare biopsies obtained from different sites, as a biopsy from one site would be considered representative of the whole joint.

Sympathetic Nervous System Control of Carbon Tetrachloride-Induced Oxidative Stress in Liver through α-Adrenergic Signaling.

In addition to being the primary organ involved in redox cycling, the liver is one of the most highly innervated tissues in mammals. The interaction between hepatocytes and sympathetic, parasympathetic, and peptidergic nerve fibers through a variety of neurotransmitters and signaling pathways is recognized as being important in the regulation of hepatocyte function, liver regeneration, and hepatic fibrosis. However, less is known regarding the role of the sympathetic nervous system (SNS) in modulating the hepatic response to oxidative stress. Our aim was to investigate the role of the SNS in healthy and oxidatively stressed liver parenchyma. Mice treated with 6-hydroxydopamine hydrobromide were used to realize chemical sympathectomy. Carbon tetrachloride (CCl4) injection was used to induce oxidative liver injury. Sympathectomized animals were protected from CCl4 induced hepatic lipid peroxidation-mediated cytotoxicity and genotoxicity as assessed by 4-hydroxy-2-nonenal levels, morphological features of cell damage, and DNA oxidative damage. Furthermore, sympathectomy modulated hepatic inflammatory response induced by CCl4-mediated lipid peroxidation. CCl4 induced lipid peroxidation and hepatotoxicity were suppressed by administration of an α-adrenergic antagonist. We conclude that the SNS provides a permissive microenvironment for hepatic oxidative stress indicating the possibility that targeting the hepatic α-adrenergic signaling could be a viable strategy for improving outcomes in patients with acute hepatic injury.