MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

Objective To investigate the function of microRNA‐138 (miR‐138) in human articular chondrocytes (HACs). Methods The expression of miR‐138 in intact cartilage and cultured chondrocytes and the effects of miR‐138 overexpression on chondrocyte marker genes were investigated. Targets of miR‐138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays. Results Expression levels of miR‐138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR‐138 is able to repress the expression of COL2A1 by directly targeting Sp‐1 and hypoxia‐inducible factor 2α (HIF‐2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR‐138 in the RNA‐induced silencing complex and confirmed binding of Sp‐1 to the COL2A1 promoter region in HACs. Conclusion We propose that an evolutionary pressure helps to suppress expression levels of miR‐138 in human cartilage, thus enabling expression of appropriate tissue‐specific matrix genes. Inhibition of miR‐138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.

HO-1 (haem oxygenase-1) catalyses the degradation of haem and possesses anti-inflammatory and cytoprotective properties. The role of inflammatory mediators in the pathogenesis of OA (osteoarthritis) is becoming increasingly appreciated. In the present study, we investigated the effects of HO-1 induction in OA and healthy HACs (human articular chondrocytes) in response to inflammatory cytokine IL-1 β (interleukin-1β) under hypoxic conditions. Hypoxia was investigated as it is a more physiological condition of the avascular cartilage. Hypoxic signalling is mediated by HIFs (hypoxia-inducible factors), of which there are two main isoforms, HIF-1α and HIF-2α. Normal and OA chondrocytes were stimulated with IL-1β. This cytokine suppresses HO-1 expression and exerts both catabolic and anti-anabolic effects, while increasing HIF-1α and suppressing HIF-2α protein levels in OA chondrocytes in hypoxia. Induction of HO-1 by CoPP (cobalt protoporphyrin IX) reversed these IL-1β actions. The hypoxia-induced anabolic pathway involving HIF-2α, SOX9 [SRY (sex determining region Y)-box 9] and COL2A1 (collagen type II α1) was suppressed by IL-1β, but importantly, levels were restored by HO-1 induction, which down-regulated TNFα (tumour necrosis factor α), MMP (matrix metalloproteinase) activity and MMP-13 protein levels. Depletion of HO-1 using siRNA (small interfering RNA) abolished the CoPP effects, further demonstrating that these were due to HO-1. The results of the present study reveal the different mechanisms by which HO-1 exerts protective effects on chondrocytes in physiological levels of hypoxia.

OBJECTIVE:To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia-inducible factors (HIFs) in these responses.METHODS:Normal human articular cartilage from a range of donors was obtained at the time of above-the-knee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin-1α. Cell transfections were performed on isolated human chondrocytes.RESULTS:Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF-2α to a specific site in the master-regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF-1α. Manipulation of the hypoxia-sensing pathway through depletion of HIF-targeting prolyl hydroxylase-containing protein 2 (PHD-2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissue-specific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage.CONCLUSION:We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue-specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF-specific hydroxylase PHD-2 may represent a relevant target for cartilage repair.

Aggrecan is a major matrix component of articular cartilage, and its degradation is a crucial event in the development of osteoarthritis (OA). Adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) is a major aggrecan-degrading enzyme in cartilage, but there is no clear correlation between ADAMTS-5 mRNA levels and OA progression. Here, we report that post-translational endocytosis of ADAMTS-5 by chondrocytes regulates its extracellular activity. We found 2- to 3-fold reduced aggrecanase activity when ADAMTS-5 was incubated with live porcine cartilage, resulting from its rapid endocytic clearance. Studies using receptor-associated protein (RAP), a ligand-binding antagonist for the low-density lipoprotein receptor-related proteins (LRPs), and siRNA-mediated gene silencing revealed that the receptor responsible for ADAMTS-5 clearance is LRP-1. Domain-deletion mutagenesis of ADAMTS-5 identified that the noncatalytic first thrombospondin and spacer domains mediate its endocytosis. The addition of RAP to porcine cartilage explants in culture increased the basal level of aggrecan degradation, as well as ADAMTS-5-induced aggrecan degradation. Notably, LRP-1-mediated endocytosis of ADAMTS-5 is impaired in chondrocytes of OA cartilage, with ∼90% reduction in protein levels of LRP-1 without changes in its mRNA levels. Thus, LRP-1 dictates physiological and pathological catabolism of aggrecan in cartilage as a key modulator of the extracellular activity of ADAMTS-5.

Purpose: Articular chondrocytes are adapted to live in conditions of low O2. Such hypoxic conditions in the avascular cartilage play an important role in extracellular matrix synthesis and survival of chondrocytes. In previous work we have shown anti-inflammatory and chondroprotective effects of heme oxygenase-1 (HO-1) on osteoarthritic (OA) condrocytes in primary cultures in 20% O2. However, the influence of O2 tension on HO-1 function in healthy and OA chondrocytes remains unknown. Methods: Human chondrocytes were obtained from healthy donors and patients with diagnosis of advanced OA undergoing total knee joint replacement. The chondrocytes were isolated by digestion with collagenase and used in primary culture. Healthy and OA chondrocyes were cultured in 20% or 1% O2 tension, and in 5% CO2. Cells were stimulated with IL-1β (10 ng/ml) for 48h. HO-1 was induced by incubation with 10 μM cobalt protoporphyrin IX (CoPP). Protein expression was assessed by Western blot, ELISA and immunocytochemistry. Nitrite production and matrix metalloproteinase (MMP) activity were evaluated by fluorometric methods. HO-1 gene silencing was achieved by using a gene-specific siRNA. Results: HO-1 protein was expressed in both healthy and OA chondrocytes in hypoxia (1% O2) and normoxia (20% O2). IL-1β down-regulated HO-1 expression in all conditions whereas CoPP treatment counteracted this effect. CoPP treatment was able to reduce the levels of TNFα and MMP activity after IL-1β stimulation. CoPP also decreased the production of nitrite induced by IL-1β in healthy and OA cells at both O2 concentrations. This effect was accompanied by a reduction in iNOS expression at 24h. In hypoxic conditions HIF-2α and SOX9 expression was decreased by IL-1β in both healthy and OA chondrocytes. However, HO-1 induction was able to reverse this effect and prevented the decrease in type collagen II. In addition, IL-1β induced HIF-1α expression irrespective of O2 tension whereas HO-1 induction by CoPP down-regulated HIF-1α expression in OA chondrocytes only. Conclusions: HO-1 induction in primary chondrocytes cultured in hypoxic conditions resulted in stronger anti-inflammatory and chondroprotective effects compared with normoxia. These results suggest that HO-1 could be a physiologically important chondroprotective factor.

Articular cartilage functions by providing a specialized mechanically competent extracellular matrix to withstand load bearing, thus protecting the underlying bones and allowing their near friction-free articulation in the joints. The resident chondrocytes maintain this function throughout life and do so by retaining a stable phenotype that resists hypertrophy and vascular invasion from the bone. This articular phenotype is distinct from the chondrocytes that drive endochondral ossification, which undergo hypertrophy and apoptosis followed by vascularization and bone formation (1). Unfortunately, stem cells used for cartilage repair seem to have a similar fate, making the repair tissue inadequate for normal joint function (2). How the articular chondrocyte avoids this and maintains its specialized phenotype is one of the fundamentally important issues in skeletal biology, yet the mechanisms remain unclear. Recent studies, however, have begun to provide some much needed insights. Although different growth factors (including transforming growth factor family members, bone morphogenetic proteins, and fibroblast growth factor family members) are important in skeletal development, there is relatively little evidence that these molecules are endogenously produced in physiologically significant amounts by adult articular cartilage. However, Klinger and colleagues, whose article appears elsewhere in this issue of Arthritis & Rheumatism (3), have identified a critical role for secreted matrix protein chondromodulin 1 in stabilizing the chondrocyte phenotype and inhibiting vascular invasion and endochondral ossification in stem cell–mediated articular cartilage repair (3). In their study, using a miniature pig model of cartilage repair, chondromodulin 1 was overexpressed in osteochondral progenitor cells (cells infected with adeno-associated virus vectors carrying chondromodulin 1 complementary DNA [AAV-Chm-1]), or AAV-Chm-1 vectors were directly administered to cartilage defects undergoing microfracture to induce repair. In both cases of chondromodulin 1 treatment, elaboration of a type II collagen–rich matrix was seen at 6 weeks, and most importantly, the tissue resisted calcification and vascular invasion over an extended experimental period (6 months). When progenitor cells were administered to cartilage defects with intact subchondral bone, although little calcification was observed, the repair tissue was fibrocartilaginous, with strong staining for type I collagen and less staining for type II collagen compared to the equivalent chondromodulin 1 treatment. Chondromodulin 1 has been shown to have antiangiogenic properties (4), but its mechanism of action is far from fully elucidated. Since Klinger and colleagues found no effect of chondromodulin 1 overexpression on VEGF mRNA levels, it appears not to inhibit this key angiogenic factor, at least in vitro. However, the situation in vivo is liable to be far more complex, and it will be of great interest to assess the levels of VEGF in chondromodulin 1–treated cartilage repair tissue. Klinger and colleagues also suggest that chondromodulin 1 prevents chondrocyte hypertrophy through suppression of type X collagen in cultured osteochondral progenitor cells. This in vitro finding must now be investigated in vivo, and it will also be of great interest to investigate if runt-related transcription factor 2 (RUNX-2) is down-regulated by chondromodulin 1, since this transcription factor plays an important role in endochondral ossification through regulation of type X collagen and induction of VEGFA (5,6). The role of SOX9 in this process should also be investigated further. SOX9 has been shown to be essential for early events in cartilage differentiation (7) and for expression of the cartilage-specific matrix genes in human articular chondrocytes (8). Moreover, mutations in SOX9 cause the severe skeletal abnormality of campomelic dysplasia (9). Interestingly, however, SOX9 is greatly down-regulated as chondrocytes undergo hyperChris L. Murphy, PhD: The Kennedy Institute and Imperial College London, London, UK. Address correspondence to Chris L. Murphy, PhD, The Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, London W6 8LH, UK. E-mail: c.murphy@imperial.ac.uk. Submitted for publication February 4, 2011; accepted in revised form March 1, 2011.

Meta-epidemiology evolves from epidemiology and meta-analysis.It is a subject which attempts to describe distribution of research evidence for a specific question, examine heterogeneity and associated risk factors, identify and control bias across studies and summarise research evidence as appropriate.Unlike classic epidemiology, the observational subjects of meta-epidemiology are papers not patients.It is therefore an observational study in research evidence with a unique statistical method -meta-analysis.The term "meta-epidemiology" first appeared in literature in 1997 in David Naylor's editorial for meta-analysis in clinical research [1].It was defined by Sterne and his colleagues in 2002 as a statistical method to identify and quantify the influence of study level characteristics such as allocation concealment in randomised controlled trials [2].Since then, several papers have been published with regard to "meta-epidemiology" [3,4].The concept, however, had not been generalised until 2007 when the Osteoarthritis Research Society International (OARSI) had its first systematic review of 51 treatments in osteoarthritis (OA) where research evidence including clinical effectiveness and cost-effectiveness were analysed, compared and summarised across treatments [5].Meta-epidemiology is no longer a statistical method, but instead translational research to close the gap between bench work and bedside.The concept is further reinforced by the OARSI recent update on research evidence in OA therapy [6].A full picture of metaepidemiology starts fading into OA therapy with more epidemiological aspects including distribution of evidence, change of evidence, associated risk factors, bias of evidence, summary and clinical application of evidence.Meta-epidemiology is not only a subject in the therapeutic area.It spreads very quickly, for example into other OA areas, from genetic [7] to clinical practice [8], from treatment effect [9] to contextual therapy [10], from randomised control trials [11] to observational studies [12].We anticipate that in the next 5 years, this subject will further evolve to improve the evidence based clinical practice.

Human articular cartilage is an avascular tissue, and therefore it functions in a hypoxic environment. Cartilage cells, the chondrocytes, have adapted to this and actually use hypoxia to drive tissue-specific functions. We have previously shown that human chondrocytes enhance cartilage matrix synthesis in response to hypoxia specifically through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated up-regulation of master regulator transcription factor SOX9, which in turn drives expression of the main cartilage-specific extracellular matrix genes. HIF-alpha isoforms are themselves regulated by specific prolyl hydroxylase domain-containing proteins, which target them for proteosomal degradation. In fact, prolyl hydroxylase domains are the direct oxygen sensors because they require molecular oxygen as a co-substrate. Here, we have identified PHD2 as the dominant isoenzyme regulating HIF-2alpha stability in human chondrocytes. Moreover, specific inhibition of PHD2 using RNA interference-mediated depletion caused an up-regulation of SOX9 and enhanced extracellular matrix protein production. Depletion of PHD2 resulted in greater HIF-2alpha levels and therefore enhanced SOX9-induced cartilage matrix production compared with the levels normally found in hypoxia (1% oxygen) implying that PHD2 inhibition offers a novel means to enhance cartilage repair in vivo. The need for HIF-specific hydroxylase inhibition was highlighted because treatment with the 2-oxoglutarate analogue dimethyloxalylglycine (which also inhibits the collagen prolyl 4-hydroxylases) prevented secretion of type II collagen, a critical cartilage matrix component.

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.

Mesenchymal stem cells (MSCs) have great potential for cell-based therapies. However, lack of cell-specific markers thwarts full realization of this as it prevents their identification in vivo, and subsequent purification. In the present study, to ensure cell purity multiple individual clones were derived from the bone marrow of BALB/b and BALB/c mice, and subsequently defined as MSCs by demonstrating their multipotentiality and self-renewal ability. In an effort to define the molecular signature of such MSCs and identify potentially cell-specific markers, an extensive genome-wide microarray analysis was performed comparing eight individual undifferentiated MSC clones to four different controls-corresponding differentiated MSC clones, bone marrow adherent cells, freshly isolated bone marrow cells, and embryonic fibroblasts. Strikingly, all MSC clones expressed differentially high levels of six-transmembrane epithelial antigen of the prostate (STEAP1 and STEAP2). Further, both STEAP members showed an extremely similar expression profile to stem cell antigen-1 (Sca-1) as demonstrated by two-dimensional hierarchical cluster analysis. Most importantly, differentially high levels of STEAP1 and STEAP2 proteins were also detected in human multipotent bone marrow adherent cultures. Thus, STEAPs may represent novel markers of MSCs in man as well as mice. Depletion of STEAP1 in human MSCs using RNAi resulted in decreased cell adhesion to tissue culture plastic. Further work is now needed to fully uncover its function in these cells, and to explore its potential as a marker of MSCs.