Osteoarthritis (OA) is a widespread, debilitating joint disease associated with articular cartilage degradation. It is driven via mechano-inflammatory pathways, whereby catabolic genes in the cartilage-embedded chondrocytes are presumed up-regulated due to increased shear stress arising from friction at the cartilage surface as joints articulate. The enhanced expression of these cartilage-degrading and inflammatory genes leads to tissue degeneration. However, the nature of the stress, and how the cells within the joint respond to it, are poorly understood. Here we show, in a proof of concept study on a mouse model where surgical joint destabilisation has been carried out to induce OA, that the early up-regulation of the matrix metalloproteinase 3 (Mmp3) gene, a member of the matrix-degrading MMP family, and of the interleukin-1 beta (Il1b) gene, a key mediator of inflammatory response, are significantly suppressed when lipid-based lubricants are injected into the joints. We attribute this to the reduction in frictional stress on the chondrocytes due to the lubricant at the cartilage surface. At the same time, Timp1, a compression but not shear-stress sensitive gene, is unaffected by lubricant. Our results demonstrate that cartilage lubrication modulates catabolic gene regulation in OA, shed strong light on the nature of the chondrocytes' response to shear stress, and have clear implications for novel OA treatments. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) is a widespread, debilitating joint disease associated with degradation of the articular cartilage, the tissue that covers and protects the joint surfaces as they rotate. Such degradation is due to catabolic enzymes expressed by cartilage-embedded chondrocytes (the only cell type in cartilage) in response to mechanical stress. In this proof-of-concept study in a mouse OA model, we show that reduction of cartilage friction by liposome-based lubricants suppresses the production of the catabolic, OA-related genes in chondrocytes. Our findings provide direct evidence in an animal model that catabolic genes are induced in chondrocytes in a mechanosensitive manner, related to the friction at the cartilage surface, and identify putative novel OA treatments through efficient cartilage lubrication.

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2 , an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2 +/- bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2 +/- chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL-6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology.

Osteoarthritis (OA) is a widespread, debilitating joint disease associated with articular cartilage degradation. It is driven via mechano-inflammatory catabolic pathways, presumed up-regulated due to increased shear stress on the cartilage-embedded chondrocytes, that lead to tissue degeneration. Here we demonstrate that the up-regulation of the matrix metalloproteinase 3 (Mmp3) and interleukin-1beta (Il1b) genes upon surgical joint destabilization in a model of murine OA is completely suppressed when lipid-based lubricants are injected into the joints. At the same time, Timp1, a compression but not shear-stress sensitive gene, is unaffected by lubricant. Our results provide direct evidence that biolubrication couples to catabolic gene regulation in OA, shed strong light on the nature of the chondrocytes' response to shear stress, and have clear implications for novel OA treatments.

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.

More than 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease-modifying treatments for this disabling condition. Common polymorphic variants in ALDH1A2 , which encodes the key enzyme for synthesis of all-trans retinoic acid (atRA), are associated with severe hand OA. Here, we sought to elucidate the biological significance of this association. We first confirmed that ALDH1A2 risk variants were associated with hand OA in the U.K. Biobank. Articular cartilage was acquired from 33 individuals with hand OA at the time of routine hand OA surgery. After stratification by genotype, RNA sequencing was performed. A reciprocal relationship between ALDH1A2 mRNA and inflammatory genes was observed. Articular cartilage injury up-regulated similar inflammatory genes by a process that we have previously termed mechanoflammation, which we believe is a primary driver of OA. Cartilage injury was also associated with a concomitant drop in atRA-inducible genes, which were used as a surrogate measure of cellular atRA concentration. Both responses to injury were reversed using talarozole, a retinoic acid metabolism blocking agent (RAMBA). Suppression of mechanoflammation by talarozole was mediated by a peroxisome proliferator–activated receptor gamma (PPARγ)–dependent mechanism. Talarozole was able to suppress mechano-inflammatory genes in articular cartilage in vivo 6 hours after mouse knee joint destabilization and reduced cartilage degradation and osteophyte formation after 26 days. These data show that boosting atRA suppresses mechanoflammation in the articular cartilage in vitro and in vivo and identifies RAMBAs as potential disease-modifying drugs for OA.

OBJECTIVE:The adamalysin metalloproteinase 15 (ADAM15) has been shown to protect against development of osteoarthritis in mice. Here, we have investigated factors that control ADAM15 levels in cartilage. DESIGN:Secretomes from wild-type and Adam15 -/- chondrocytes were compared by label-free quantitative mass spectrometry. mRNA was isolated from murine knee joints, either with or without surgical induction of osteoarthritis on male C57BL/6 mice, and the expression of Adam15 and other related genes quantified by RT-qPCR. ADAM15 in human normal and osteoarthritic cartilage was investigated similarly and by fluorescent immunohistochemistry. Cultured HTB94 chondrosarcoma cells were treated with various anabolic and catabolic stimuli, and ADAM15 mRNA and protein levels evaluated. RESULTS:There were no significant differences in the secretomes of chondrocytes from WT and Adam15 -/- cartilage. Expression of ADAM15 was not altered in either human or murine osteoarthritic cartilage relative to disease-free controls. However, expression of ADAM15 was markedly reduced upon aging in both species, to the extent that expression in joints of 18-month-old mice was 45-fold lower than in that 4.5-month-old animals. IL-13 increased expression of ADAM15 in HTB94 ​cells by 2.5-fold, while modulators of senescence and autophagy pathways had no effect. Expression of Il13 in the joint was reduced with aging, suggesting this cytokine may control ADAM15 levels in the joint. CONCLUSION:Expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging human and murine joints, possibly due to a concomitant reduction in IL-13 expression. We thus propose IL-13 as a novel factor contributing to increased osteoarthritis risk upon aging.

ABSTRACT Complex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting the potential for these pathways to be targeted therapeutically. Primary cilia are subcellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1β, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviours, changing cytosolic NFκB translocation dynamics but leaving MAPK signalling unaffected. RNA-seq analysis indicates that IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf. Through microscopy, we find altered NFκB dynamics are independent of assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits inflammatory responses in the non-ciliated macrophage. We propose that ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme to tune cytoplasmic NFκB signalling and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation. This article has an associated First Person interview with the first author of the paper.

Objective Tumor necrosis factor α–stimulated gene 6 (TSG‐6) is an anti‐inflammatory protein highly expressed in osteoarthritis (OA), but its influence on the course of OA is unknown. Methods Cartilage injury was assessed by murine hip avulsion or by recutting rested explants. Forty‐two previously validated injury genes were quantified by real‐time polymerase chain reaction in whole joints following destabilization of the medial meniscus (DMM) (6 hours and 7 days). Joint pathology was assessed at 8 and 12 weeks following DMM in 10‐week‐old male and female fibroblast growth factor 2 (FGF2) −/− , TSG‐6 −/− , TSG‐6 tg (overexpressing), FGF2 −/− ;TSG‐6 tg (8 weeks only) mice, as well as strain‐matched, wild‐type controls. In vivo cartilage repair was assessed 8 weeks following focal cartilage injury in TSG‐6 tg and control mice. FGF2 release following cartilage injury was measured by enzyme‐linked immunosorbent assay. Results TSG‐6 messenger RNA upregulation was strongly FGF2‐dependent upon injury in vitro and in vivo. Fifteeen inflammatory genes were significantly increased in TSG‐6 −/− joints, including IL1 α, Ccl2 , and Adamts5 compared with wild type. Six genes were significantly suppressed in TSG‐6 −/− joints including Timp1, Inhibin β A , and podoplanin (known FGF2 target genes). FGF2 release upon cartilage injury was not influenced by levels of TSG‐6. Cartilage degradation was significantly increased at 12 weeks post‐DMM in male TSG‐6 −/− mice, with a nonsignificant 30% reduction in disease seen in TSG‐6 tg mice. No differences were observed in cartilage repair between genotypes. TSG‐6 overexpression was unable to prevent accelerated OA in FGF2 −/− mice. Conclusion TSG‐6 influences early gene regulation in the destabilized joint and exerts a modest late chondroprotective effect. Although strongly FGF2 dependent, TSG‐6 does not explain the strong chondroprotective effect of FGF2.

We review the establishment of computational biology in Greece and Cyprus from its inception to date and issue recommendations for future development.We compare output to other countries of similar geography, economy, and size-based on publication counts recorded in the literature-and predict future growth based on those counts as well as national priority areas.Our analysis may be pertinent to wider national or regional communities with challenges and opportunities emerging from the rapid expansion of the field and related industries.Our recommendations suggest a 2-fold growth margin for the 2 countries, as a realistic expectation for further expansion of the field and the development of a credible roadmap of national priorities, both in terms of research and infrastructure funding.

Purpose: Mechanical forces are critical for joint development, homeostasis and disease. During skeletal development mechanics contributes to formation of the joint, endochondral ossification during long bone elongation and growth plate closure. Physiological loading during adult life underpins articular cartilage health, continuity and thickness. Pathological loading of the joint leads to cartilage degradation and osteoarthritis (OA). However, we don't yet understand the relative contributions of the cartilage, bone and synovium or cells vs matrix in healthy or pathological mechanotransduction. In both chondrocytes and osteocytes, proteins critical to the assembly and function of the primary cilium have been shown to be important to the response to mechanics. Recently we have shown they regulate cartilage turnover. The cilium is a microtubule-based organelle most famously associated with transduction of extracellular cues including the hedgehog ligand and mechanics. As such mutations to ciliary proteins results in a group of human congenital diseases known as ciliopathies including skeletal disorders, further emphasising the cilium's critical role in musculoskeletal development. However, little is known about the cilium's post-natal roles within the joint. A core component of the cilium is IFT88, disruption of which alters mechanically induced matrix production and catabolism in chondrocytes. We hypothesise that the crucial role of the cilium and associated machinery persists into adulthood, is important for the joints response to physiological loading and by extension, cartilage health. Methods: We have created an adult, cartilage-specific, inducible deletion model of IFT88 (ACAN;IFT88fl/flCreERT2) and a pan tissue (ROSA26;IFT88fl/flCreERT2). These were validated by qPCR, western blot, in vitro and a ROSA26tdtomato reporter. Phenotyping of this mouse was conducted using histology, MicroCT and imageJ. The surgical destabilisation of the medial meniscus model (DMM) was used to challenge the joint with aberrant mechanics to induce OA followed by double-blinded OARSI scoring. Results: The reporter mouse reveals that aggrecan cre is driving in the articular surface, growth plate, menisci and some populations within the bone. qPCR indicates an approximate halving of IFT88 mRNA in the ACAN;IFT88fl/flCreERT2, termed cKO hereafter. Activation of the ROSA26;IFT88fl/flCreERT2 also results in a halving of IFT88 mRNA in multiple tissues and reduction of protein in cartilaginous tissue. Between 4 and 10 weeks of age in mice, endochondral ossification is nearing completion and growth plate closure begins. Deletion of IFT88 at 4 weeks of age results in a longer growth plate including enlarged bi-lateral growth plate cartilage at 6 weeks. Deletion at 6 weeks also results in a longer growth plate at 8 weeks; in each case the cKO growth plate remains at the length it was 2 weeks earlier prior to deletion. Between 8 and 10 weeks of age, cKO of IFT88 also results in longer growth plates as growth plate closure is inhibited. Between 6 and 10 weeks of age the articular surface thickens at a linear rate from ∼85μm to 115μm. In the cKO, this thickening over each 2 week period is inhibited. Two weeks after deletion, cartilage is ∼15μm thinner at every time point in the cKO. Further analysis of histology reveals a reduction in the calcified cartilage region, whereas no change was seen in the non-calcified region. At 12 weeks post-DMM there is a significant difference in OARSI score between with higher scores in the cKO. In older mice (6 months of age) there is a pronounced long bone phenotype with loss of trabeculae and total bone volume. Conclusions: We have successfully depleted chondrocytes of IFT88 using an aggrecan Cre system, however potentially only in a subset of the population. IFT88 maintains influence through adolescence and early adulthood. Our data suggests IFT88/the cilium continues to regulate growth plate dynamics during and beyond developmental stages, potentially regulating both the Hh-PTHrP-Hh feedback loop during endochondral ossification and growth plate closure mechanisms. We propose this is by dysregulation of upstream mechanotransduction. IFT88 maintains influence during articular cartilage maturation and in early adulthood. Upon knock out of IFT88, articular cartilage thickness is reduced and associated with changes to the calcified region. This is in some agreement with changes seen in small models of cartilage atrophy, where the mechanism is not yet understood. On the introduction of pathological mechanical load using DMM, IFT88 deletion leads to exacerbation of articular cartilage degradation, potentially owing to loss of physiological mechanotransduction prior to DMM challenge. On-going work is analysing the effects at earlier time points in disease progression (8 weeks post-DMM) and importantly the naive articular surface during adulthood. Collectively, with our previous in vitro work, we propose IFT88 regulates cartilage turnover and potentially ossification of deep zone articular cartilage. We are now testing these hypotheses, targeting other elements of the ciliome and exploring the molecular mechanisms downstream to IFT88 loss in mature and mechanically challenged cartilage to compare with changes seen in atrophy and OA.

Osteoarthritis (OA) is a common degenerative joint disease, characterized by cartilage loss and subchondral bone remodeling in response to abnormal mechanical load. Heparan sulfate (HS) proteoglycans bind to many proteins that regulate cartilage homeostasis, including growth factors, morphogens, proteases, and their inhibitors, and modulate their localization, retention, and biological activity. Changes in HS expression and structure may thus have important consequences for joint health. We analyzed normal and osteoarthritic human knee cartilage, and found HS biosynthesis was markedly disrupted in OA, with 45% of the 38 genes analyzed differentially regulated in diseased cartilage. The expression of several HS core proteins, biosynthesis, and modification enzymes was increased in OA cartilage, whereas the expression of the HS proteoglycans syndecan 4 and betaglycan was reduced. The structure of HS was also altered, with increased levels of 6-0-sulfation in osteoarthritic samples, which correlated with increased expression of HS6ST1, a 6-0-sulfotransferase, and GLCE, an epimerase that promotes 6-0-sulfation. siRNA silencing of HS6ST1 expression in primary OA chondrocytes inhibited extracellular signal-regulated kinase phosphorylation in response to fibroblast growth factor 2, showing that changes in 6-0-sulfation impact a key cartilage signaling pathway. Given the broad range of homeostatic and repair pathways that HS regulates, these changes in proteoglycan expression and HS structure are likely to have significant effects on joint health and progression of OA.

Purpose: A recent Genome Wide Association Study (GWAS) in hand OA has uncovered the association of two SNPs markers rs4238326[C] and rs3204689[C] with severe hand osteoarthritis. The two variants are all located within a single linkage disequilibrium block that contains one ALDH1A2 (aldehyde dehydrogenase 1 family member A2). This gene encodes the enzyme that irreversibly catalyzes the production of all-trans retinoic acid (atRA), and which regulates numerous physiological processes involved in embryonic limb development and in the maintenance of adult tissues. Trapeziectomy is a recognised surgical treatment for patients with intractable base of thumb OA. In this study, we developed a method to extract good quality RNA from the articular cartilage of the trapezium, the small bone at the base of the thumb. We investigated the correlation between the presence of variant ALDH1A2 alleles (snp3204689, and snp4238326) and mRNA levels of ALDH1A2 and a series of retinoic acid dependent genes and inflammatory response genes. Methods: We identified 26 patients, with base of thumb OA, scheduled to undergo trapeziectomy at the Nuffield Orthopaedic Centre, Oxford, UK. Following written informed consent, the removed intact trapezium was kept for research. Within one hour of collection, articular cartilage was dissected from the trapezium and snap frozen in liquid nitrogen. RNA was extracted using the RNeasy Micro Kit from QIAGEN. RNA yield and quality were tested using an Agilent Bioanalyzer and Nanodrop Spectrophotometer. The RNA integrity number (RIN) values were above 7 for all samples. Genomic DNA was extracted from the trapezium bone, and the allelic region of interest was amplified using specific primers. The purified PCR products sequenced and their genotype uncovered. The expression levels of selected genes including retinoic acid-dependent genes (e.g.,CYP19A1, CYP26a, CYP26b, CYP26c, retinoic acid receptor- RARα, β, and γ) as well as inflammatory genes (including ADAMTS4, ADAMTS5, CCL2, MMP13) were tested by RT-PCR using TaqMan Low Density Arrays (TLDA) microfluidic cards. Results: We detected a high prevalence of the two polymorphic variants (76.9%) in ALDH1A2 within the 26 patients. We classified the samples into five groups based on the number of variant alleles (see chart below). We found the mRNA level of ALDH1A2 was significantly lower in the Homozygous group compared to the wild type (p = 0.02, T-test). Among the retinoic acid dependent genes, the only one that correlated significantly with genotype was CYP19A1. We also found trends in the regulation of several other retinoic acid dependent and inflammatory response genes, but these were not statistically significant. Conclusions: Our data shows that polymorphic variants in ALDH1A2 are highly prevalent in our hand OA population and are associated with significantly lower levels of ALDH1A2 in hand OA cartilage. The lower expression level of ALDH1A2 in the variants is predicted to decrease cellular atRA levels, and at least one retinoic acid-dependent gene, CYP19A1, correlated with ALDH1A2. CYP19A1 encodes the enzyme that catalyzes the last step of the estrogen and testosterone biosynthesis pathway. This may potentially point to a role for retinoic acid in the regulation of sex hormone levels either systemically or in the tissue.

Purpose: A recent Genome Wide Association Study (GWAS) in hand OA has identified the association of hypomorphic variants in aldehyde dehydrogenase 1 family member A2 (ALDH1A2) with severe hand OA. This gene encodes the enzyme that irreversibly catalyzes the production of all-trans retinoic acid (atRA) from retinaldehyde. atRA has an essential role in embryonic limb development but its role in adult cartilage and in OA is not clear. Our group has demonstrated that endogenous retinoic acid is strongly suppressed by cartilage injury and this affects the regulation of inflammatory response genes. Maintaining cellular retinoic acid levels at time of injury, with a CYP 26 inhibitor, restrained inflammatory gene regulation. Previous work from our group has found that cartilage injury activates several signaling pathways involving release of transforming growth factor beta (TGFβ) and fibroblast growth factor (FGF2), and activation of TGFβ-activated kinase 1 (TAK1). Here we investigated the mechanism by which cartilage injury suppresses endogenous retinoic acid signaling. Methods: Articular cartilage was obtained from porcine metacarpophalangeal joints of 3-6 months old pigs. Cartilage injury was performed by explanting cartilage from the joint surface. To examine the role of specific injury pathways, cartilage injury was also performed on joints that had been injected with selective inhibitors (TGFβR (SB431542), FGF2R (SB402451) and TAK1 ((5Z)-7-oxozeanol) 1 hour prior to injury. RT-PCR was performed for known atRA-dependent genes. To identify injury-induced regulators of retinoic acid, a murine cartilage injury microarray was interrogated for molecules involved in the synthesis and degradation of atRA. Results: Cartilage injury led to rapid down-regulation of retinoic acid dependent genes (CYP26a, CYP26b and retinoic acid receptors: RARα, RARβ and RARγ) between 40 and 80% by 4h. For most genes this was sustained for up to 24h. This apparent fall in atRA dependent genes was not affected by blocking TGFβ or FGF2, but was inhibited by the TAK1 inhibitor, indicating that TAK1 controls atRA levels or signaling. Our cartilage injury microarray identified RDH12 as the most strongly regulated gene on the atRA pathway (12.5 fold, p=1.5x10-9). RDH12 is an important upstream enzyme, which converts retinaldehyde to retinol, and its overexpression will lead to reduction of cellular atRA level. ALDH1A2 protein was also suppressed 4 hours after porcine cartilage injury, predicting a further reduction of cellular atRA. Conclusions: Our study demonstrates that atRA dependent genes in chondrocytes are strongly suppressed by mechanical injury through TAK1 activation, indicating a reduction of endogenous atRA level. The strong induction of RDH12 and suppression of ALDH1A2 after injury may be responsible for driving these changes. We conclude that maintaining physiological levels of atRA is anti-inflammatory, and targeting the atRA pathway could reduce the catabolic response of cartilage to injury.

Purpose: Friction at cartilage surfaces, as joints articulate, can be damaging. On the one hand mechanical attrition may lead to direct tissue micro-damage. On the other hand friction-induced stresses at the cartilage surface are associated with shear of the near-surface cartilage-embedded chondrocytes, an effect which is known to upregulate expression of cartilage-degrading enzymes. A self-reinforcing cycle may then ensue in which enzymatic degradation results in progressively higher friction and higher shear stress. Continued joint use under these circumstances may lead to osteoarthritis. Suitable lubrication to reduce surface friction could slow this cycle down. In this study we describe recent developments both in creating novel lubrication vectors for cartilage, and preliminary findings on the effect of such vectors on the sliding of biological tissues and on gene regulation following joint destabilization in mice. Methods: We are developing a range of lubricating vectors based on phosphatidylcholine (PC) lipids, where friction reduction is provided by the hydration lubrication mechanism arising at their highly-hydrated phosphocholine headgroups. We have used such vectors a) in model nanotribological studies using a surface force balance, b) to modify tendon/sheath friction in a chicken model using a macroscopic tribometer, and c) injected into destabilized mice joints to examine their effect on subsequent gene regulation. Results: In model studies we find that small and large unilamellar vesicles (SUVs) of suitable PCs can complex with surface attached hyaluronan (HA) molecules and other surface-attached macromolecules to provide extremely good friction at physiologically high pressures (coefficients of friction down to 10−3–10−4); such lubrication vectors can also reduce tendon/sheath friction by over 70% in a very sustained manner. Preliminary results in a murine model where PC-SUV-based lubrication vectors were injected into destabilized joints revealed changes in regulation of genes that are associated both with compression and shear stress. Conclusions: Novel lipid-based vectors can act as extremely efficient boundary lubricants in physiological conditions, both on model surfaces and at naturally occurring interfaces such as in tendon/sheath gliding. Preliminary results indicate that they modulate mechano-sensitive gene regulation in murine joints providing mechanistic support for their putative chondroprotective role in vivo.

Matrix protease activity is fundamental to developmental tissue patterning and remains influential in adult homeostasis. In cartilage, the principal matrix proteoglycan is aggrecan, the protease-mediated catabolism of which defines arthritis; however, the pathophysiologic mechanisms that drive aberrant aggrecanolytic activity remain unclear. Human ciliopathies exhibit altered matrix, which has been proposed to be the result of dysregulated hedgehog signaling that is tuned within the primary cilium. Here, we report that disruption of intraflagellar transport protein 88 (IFT88), a core ciliary trafficking protein, increases chondrocyte aggrecanase activity in vitro. We find that the receptor for protease endocytosis in chondrocytes, LDL receptor-related protein 1 (LRP-1), is unevenly distributed over the cell membrane, often concentrated at the site of cilia assembly. Hypomorphic mutation of IFT88 disturbs this apparent hot spot for protease uptake, increases receptor shedding, and results in a reduced rate of protease clearance from the extracellular space. We propose that IFT88 and/or the cilium regulates the extracellular remodeling of matrix-independently of Hedgehog regulation-by enabling rapid LRP-1-mediated endocytosis of proteases, potentially by supporting the creation of a ciliary pocket. This result highlights new roles for the cilium's machinery in matrix turnover and LRP-1 function, with potential relevance in a range of diseases.-Coveney, C. R., Collins, I., Mc Fie, M., Chanalaris, A., Yamamoto, K., Wann, A. K. T. Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1-mediated endocytosis.

Background A Genome-Wide Association Study (GWAS) in hand OA has identified the association of hypomorphic variants within ALDH1A2 and severe hand OA (Styrkarsdottir U, et al. 2014). This gene encodes the enzyme catalysing the production of all-trans retinoic acid (atRA). atRA has an essential role in embryonic limb development, but its role in adult cartilage and in OA remains unclear. Previous work from our lab has shown that cartilage injury activates inflammatory signalling and regulates the expression of inflammatory genes. Trapeziectomy is a surgical treatment for patients with intractable base of thumb OA and is a good source of hand OA cartilage. Objectives The aim of this study was to investigate the expression of atRA-dependent and inflammatory genes in the cartilage of patients and to examine these with respect to the presence/absence of polymorphic ALDH1A2 variants. We also investigate the regulation of atRA upon cartilage injury and its effect on injury-induced inflammatory gene regulation. Methods We collected 26 trapeziectomy samples, and dissected the cartilage within one hour of collection. Genomic DNA was extracted for the identification of the two common variants (SNP rs4238326 and SNP rs3204689). Expression levels of atRA-dependent and inflammatory genes were tested by RT-PCR. Healthy cartilage was obtained from femoral heads of 5-week-old mice and porcine metacarpophalangeal joints of 3–6 months old pigs. Gene regulation upon cartilage injury was tested. To examine the role of specific injury-induced pathways, porcine joints were pre-injected with selective inhibitors to: TGFβR, FGF2R, TAK1 and CYP26, 1 hour prior to injury. Results Polymorphic variants in ALDH1A2 were common in this patient population and we identified 8 patients homozygous for both variants, and 5 patients who were wild type for both variants. mRNA levels of ALDH1A2 and atRA-dependent gene CYP19A were significantly lower in the homozygous group compared to wild type (figure 1). There were also trends in the regulation of several other atRA-dependent genes. Conversely, inflammatory genes such as HAS1, TSG6 and ADAMTS5 showed a general increase in homozygous patients. Cartilage injury in both porcine and murine tissue led to a rapid down-regulation of atRA-dependent genes (CYP26s and RARs). Injecting joints with a potent TAK1 inhibitor prevented the drop of atRA-dependent genes. Prior injection of the joint with a CYP26 (enzymes that normally break down atRA) inhibitor, restored levels of atRA-dependent genes after injury and suppressed injury-induced inflammation genes. Conclusions Polymorphic variants in ALDH1A2 are associated with significantly lower levels of ALDH1A2 and CYP19A1 mRNA in hand OA cartilage. Adult articular cartilage constitutively produces atRA, and this is strongly suppressed by mechanical injury through TAK1 activation. Preventing the drop in cellular atRA upon injury, by pre-incubating the joint with a CYP26 inhibitor, restores atRA levels and leads to a reduction in inflammatory gene regulation. These results indicate that atRA plays an important anti-inflammatory role in cartilage and provides a potential novel therapeutic strategy to treat hand OA. Disclosure of Interest None declared

INTRODUCTION:Matrix metalloproteinases (MMPs) and 'aggrecanase' a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are well established to play key roles in osteoarthritis (OA) through degradation of extracellular matrix (ECM) type II collagen and aggrecan, and are thus potential targets for development of OA therapies. OBJECTIVE:This paper aims to provide a comprehensive review of the expression and potential roles of other, lesser-known ADAMTSs and related adamalysins (or a disintegrin and metalloproteinases (ADAMs)) in cartilage, with a view to identifying potentially protective or homeostatic metalloproteinases in the joint and informing consequent selective inhibitor design. DESIGN:A comprehensive literature search was performed using PubMed terms 'osteoarthritis' and 'ADAMTS' or 'ADAM'. RESULTS:Several ADAMTSs and ADAMs were identified as having reportedly increased expression in OA. These include enzymes likely to play roles in cartilage matrix anabolism (e.g., the procollagen N-proteinases ADAMTS-2, ADAMTS-3 and ADAMTS-14), chondrocyte differentiation and proliferation (e.g., ADAM9, ADAM10, ADAM12), as well as enzymes contributing to cartilage catabolism (e.g., Cartilage oligomeric protein (COMP)-degrading ADAMTS-7 and ADAMTS-12). CONCLUSIONS:In addition to the well-characterised MMPs, ADAMTS-4 and ADAMTS-5, many other ADAMTSs and ADAMs are expressed in cartilage and several show significantly altered expression in OA. Studies aimed at elucidating the pathophysiological roles of these enzymes in cartilage will contribute to our understanding of OA pathogenesis and enable design of targeted inhibitors that effectively target metalloproteinase-mediated cartilage degradation while sparing cartilage repair pathways.

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor–related protein 1 (LRP1). We discovered that suramin (C51H40N6O23S6) bound to TIMP-3 with a KD value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis.