S.M. Brockbank

Characterisation of cell-penetrating peptide-mediated peptide delivery

1 Cell‐penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain–peptide conjugates, factors that will be critical in determining the most effective sequence. 2 In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1–3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell‐penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. 3 However, examination of cellular toxicity showed that antennapedia

The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.

OBJECTIVE To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function. METHODS Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion. CONCLUSIONS We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

Involvement of the Wnt signaling pathway in experimental and human osteoarthritis: Prominent role of Wnt‐induced signaling protein 1

OBJECTIVE Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA. METHODS Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/beta-catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression. RESULTS Wnt-induced signaling protein 1 (WISP-1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt-16 and Wnt-2B were also markedly up-regulated during the course of disease. Interestingly, increased WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP-1 resulted in interleukin-1-independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage. CONCLUSION This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) modulates key biological pathways associated with OA disease pathology

OBJECTIVE To examine the role of mitogen-activated protein kinase-activated protein kinase 2 (MK2) in mediating the cellular response to pro-inflammatory cytokines in human primary osteoarthritis (OA) chondrocytes. METHODS Delivery of a dominant negative MK2 was achieved in HeLa cells by adenoviral infection. Cellular heat shock protein (HSP27) activity was determined using a Bioplex assay. Primary OA chondrocytes were isolated by collagenase digestion of human articular cartilage. Phosphorylated MK2 was detected by immunoblotting and immunohistology. Transfection of primary chondrocytes with siRNA was achieved using cationic lipid and gene expression determined by real-time polymerase chain reaction. Production of prostaglandin E2 (PGE2) and matrixmetalloproteases (MMPs) was measured by enzyme-linked immunosorbent assay. RESULTS Over-expression of a dominant negative MK2 inhibited HSP27 phosphorylation and significantly reduced both interleukin 1 (IL-1)beta and tumour necrosis factor (TNF)-alpha mediated release of PGE2 in HeLa cells over a 24h period. Phosphorylated MK2 was detected in OA articular cartilage and in isolated primary OA chondrocytes, where it was induced by IL-1beta. Transfection of OA chondrocytes with MK2 siRNA antisense significantly reduced both basal and IL-1beta induced PGE2 release. siRNA mediated MK2 knockdown also significantly reduced both basal and IL-1beta induced MMP13 expression and MMP13 and MMP3 protein release but had no effect on MMP1. CONCLUSIONS Our data reveal that MK2 is active in OA human articular cartilage and in isolated primary human chondrocytes and that MK2 mediates the release of PGE2, MMP3 and MMP13. These findings suggest a role for MK2 in contributing to OA algesia and OA joint structural deterioration by mediating the downstream effects of p38 activation on PGE2 release and the expression and release of catabolic proteases.

Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment

Objective Aggrecan is a critical component of cartilage extracellular matrix. Several members of the ‘a disintegrin and metalloproteinase with thrombospondin motifs’ (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1β (IL-1β) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated. Methods MMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1β in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1β or meniscotibial ligament transaction was carried out in MMP17 null mice. Results The data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1β mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor. IAI of IL-1β into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17. Conclusion These data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.

Expression profiling of metalloproteinases and inhibitors in cartilage

Objective  To profile the expression of all known members of the matrix metalloproteinase (MMP), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and tissue inhibitor of metalloproteinases (TIMPs) gene families in normal cartilage and that from patients with osteoarthritis (OA).

218 CANONICAL WNT EXPRESSION IN THE SYNOVIUM INDUCES OA-LIKE CARTILAGE DEGENERATION

INTRODUCTION: In osteoarthritis (OA), cartilage damage is one of the main pathological features. There is, however, a significant involvement of the synovium in a large proportion of OA-patients. The mechanism through which the synovium contributes to OA pathology is not yet known. In cartilage, it is proposed that developmental processes important in embryonic development are reactivated. Associations have been identified of the occurrence of OA with polymorphisms of genes from the wnt/β-catenin pathway, a pathway that is involved in normal cartilage development. In the wnt/β-catenin pathway soluble wntproteins are secreted by cells and bind to Frizzled (Fzd) receptors, which sets into motion a signaling cascade leading to intra-cellular β-catenin accumulation and the transcription of a plethora of genes, like several proteases. The levels of β-catenin in the chondrocytes are crucial for the stability of chondrocyte phenotype. The aim of the present study is to investigate the contribution of the synovium to OA pathology via wnt signaling. METHODS: To demonstrate the presence of canonical wnt signaling in murine models for OA, murine knee joints isolated at several time point after induction of collagenase induced OA (CIOA), surgically induced OA and STR/ort mice were stained for the presence of β-catenin. CIOA was generated by intra-articular injection of collagenase, which induces joint instability. Surgical OA was induced by destabilization of the medial meniscus, by transecting the medial meniscotibial ligament. A longitudinal expression analysis was performed in 2 of the models, one with clear synovial involvement, CIOA, and the spontaneous OA model in STR/ort mice, which shows less synovial involvement. Synovial expression of components of the wnt signaling pathway was determined at several time points. From these results, targets were selected for the generation of adenoviral vectors to overexpress specific genes. To study in vivo effects, adenoviral vectors were injected intra-articularly in murine knee joints and joints were isolated and processed for histological examination at day 1, 3, and 7 after injection. To study the effect of these genes on chondrocytes, human chondrocytes were isolated from cartilage that was obtained from joint replacement surgery. One day after isolation, these cells were transfected with the adenoviral vectors, and incubated for 7 and 14 days. Hereafter Q-PCR was performed at the mRNA that was obtained from these cultures, to detect expression of aggrecan, collagens type I, II and X and several MMPs. Cartilage and synovial specimen were obtained at the time of joint replacement surgery after a signed informed consent was obtained. All animal experiments were approved by the institutional review board conforming to the local laws and regulations. RESULTS SECTION: In all experimental murine models for OA, β-catenin stained stronger in the cartilage compared to naïve mice, especially in the superficial cartilage layer (Figure 1). In the deeper, calcified layer cartilage stained positive in naïve mice as well. Also in synovium, β-catenin staining increased when OA developed (not shown). This indicates that during experimental OA, canonical wnt signaling occurs in the cartilage and synovium. Strong upregulation of the canonical wnts wnt16 (up to 256-fold) and wnt2b (up to 90-fold) was found in both models, although regulation was stronger in CIOA. Expression in the synovium was clearly higher compared to the cartilage. In the cartilage, no clear upregulation of canonical wnt-proteins was found. Clear intracellular accumulation of βcatenin was found in both synovium and cartilage, which indicates the activation of wnt/β-catenin in both tissues. This suggests that wntproteins that are expressed in the synovium, diffuse to the cartilage and induce wnt-signaling in chondrocytes. Wnt-1 induced signaling protein (WISP-1), a protein downstream canonical wnt signaling, was highly expressed in the synovium as well, again indicating activation of this pathway in the synovium. To determine whether canonical wnt expression in the synovium has the potency to cause cartilage damage, canonical wnt8a was overexpressed specifically in the synovium by intra-articular injection of an adenoviral vector. At day 1 and 3, no significant differences were observed in the cartilage from wnt8 overexpressing knee joints compared to joints transfected with control virus. Remarkably, at day 7, a strong induction of cartilage pathology was observed at the medial margin of the medial tibial plateau (Figure 2), a preferential site for the start of cartilage damage in our models. This shows that expression of canonical wnt in the synovium causes cartilage degeneration. In addition, overexpression of WISP-1, a mediator that is induced by canonical wnts induces MMPand aggrecanase mediated cartilage damage, already 4 days after transfection. Due to their size, wnt proteins and WISP-1 can reach the chondrocytes in the cartilage matrix and may alter the chondrocyte phenotype. This was underlined by increased β-catenin staining in cartilage of wnt8a overexpressing mice. Adenoviral overexpression of wnt8, wnt16 and WISP1 in human primary chondrocytes led to a significant increase within 14 days of Collagen type I, and a significant decrease of Collagen type II, suggesting loss of the articular chondrocyte phenotype. DISCUSSION: Canonical wnt expression and subsequent WISP-1 production is increased in the synovium during experimental OA. This synovial expression may lead to the degradation of cartilage as soon as 7 days after transfection, possibly by inducing changes in the articular chondrocyte phenotype. This identifies synovial wnt expression as a potential target for OA therapy. Future studies should focus on the inhibition of the canonical wnt16 and WISP-1, to underline its efficacy as a target for OA therapy. Paper No. 6 • 56th Annual Meeting of the Orthopaedic Research Society