MMP-9 mediated Syndecan-4 shedding correlates with osteoarthritis severity.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disease inducing the degradation of the articular cartilage. Syndecan-4 (Sdc4) is a heparan sulfate proteoglycan, expressed under inflammatory conditions and by chondrocytes during OA. Little is known about Sdc4 shedding and its regulation in OA. Therefore, we investigated the regulation of Sdc4 shedding and underlying shedding mechanisms under OA conditions. DESIGN: Articular cartilage, serum, synovial fluid and synovial membrane from OA patients with different radiological severity were analyzed. ELISA, RT-qPCR and IHC for Sdc4, MMP-2 and -9 were performed. MMP inhibitors and siRNA were evaluated for their effect on Sdc4 shedding by ELISA and on IL-1 signaling by western blot (pERK/ERK). RESULTS: Shed Sdc4 was increased in synovial fluid of OA patients, but not in the serum and is a good predictor (AUC = 0.72) for OA severity with a sensitivity of 67.5% and specificity 65.2%. MMP-9, but not MMP-2, was increased in cartilage and synovial membrane at mRNA levels and in the synovial fluid at protein levels. Shed Sdc4 correlated with the amount of MMP-9 in synovial fluid. Further, the inhibition and knock-down of MMP-9 decreased the amount of shed Sdc4 in vitro. Increased Sdc4 shedding resulted in less phosphorylation of ERK upon IL-1ß stimulation. CONCLUSION: Shed Sdc4 might be a good prognostic biomarker for OA mediated cartilage degradation. MMP-9 seems to be the relevant sheddase for Sdc4 under OA conditions, desensitizing chondrocytes towards IL-1 signaling.

An altered heparan sulfate structure in the articular cartilage protects against osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a progressive degenerative disease of the articular cartilage caused by an unbalanced activity of proteases, cytokines and other secreted proteins. Since heparan sulfate (HS) determines the activity of many extracellular factors, we investigated its role in OA progression. METHODS: To analyze the role of the HS level, OA was induced by anterior cruciate ligament transection (ACLT) in transgenic mice carrying a loss-of-function allele of Ext1 in clones of chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl). To study the impact of the HS sulfation pattern, OA was surgically induced in mice with a heterozygous (Ndst1+/-) or chondrocyte-specific (Col2-Cre;Ndst1fl/fl) loss-of-function allele of the sulfotransferase Ndst1. OA progression was evaluated using the OARSI scoring system. To investigate expression and activity of cartilage degrading proteases, femoral head explants of Ndst1+/- mutants were analyzed by qRT-PCR, Western Blot and gelatin zymography. RESULTS: All investigated mouse strains showed reduced OA scores (Col2-rtTA-Cre;Ext1e2fl/e2fl: 0.83; 95% HDI 0.72-0.96; Ndst1+/-: 0.83, 95% HDI 0.74-0.9; Col2-Cre;Ndst1fl/fl: 0.87, 95% HDI 0.76-1). Using cartilage explant cultures of Ndst1 animals, we detected higher amounts of aggrecan degradation products in wildtype samples (NITEGE 4.24-fold, 95% HDI 1.05-18.55; VDIPEN 1.54-fold, 95% HDI 1.54-2.34). Accordingly, gelatin zymography revealed lower Mmp2 activity in mutant samples upon RA-treatment (0.77-fold, 95% HDI: 0.60-0.96). As expression of major proteases and their inhibitors was not altered, HS seems to regulate cartilage degeneration by affecting protease activity. CONCLUSION: A decreased HS content or a reduced sulfation level protect against OA progression by regulating protease activity rather than expression.

Targeting ß-catenin dependent Wnt signaling via peptidomimetic inhibitors in murine chondrocytes and OA cartilage.

OBJECTIVE: The canonical Wnt signaling pathway has been shown to be involved in regulating chondrocyte hypertrophic differentiation during Osteoarthritis (OA). The aim of this study was to test the therapeutic potential of two stapled peptide canonical Wnt inhibitors - SAH-Bcl9 and StAx-35R - in preventing Wnt induced cartilage changes in OA. METHODS: Primary neonatal murine chondrocytes and cartilage explants from OA patients undergoing total joint replacement for knee OA, were used for microscopy to determine matrix and cell penetrating capacity of fluorescein isothiocyanate FITC-tagged SAH-Bcl9 and StAx-35R peptides. T cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporter assays were used to monitor the inhibition of Wnt3a induced ß-catenin signaling by each peptide. Changes in chondrocyte phenotypic marker gene expression were analyzed by qRT PCR. RESULTS: Both peptides localized intercellular in primary murine chondrocytes and cartilage explants. They inhibited Wnt3a induced TCF/LEF promoter activity in primary murine chondrocytes. Both inhibitors did not rescue Wnt3a altered expression of chondrocyte phenotypic genes (Sox9, Col2a1, Acan) and hypertrophy marker gene (Col10a1) at high doses (100 ng/ml). Upon application of 10 ng/ml Wnt3a, StAx-35R partially reversed the Wnt effect on Sox9 and Col2a1 gene expression. Both peptides, however, reversed the downregulation of SOX9 and aggrecan (ACAN), and decrease of COL10A1 gene expression in preserved human OA cartilage explants. CONCLUSION: These data indicate that blockade of canonical Wnt signaling might be a therapeutic strategy to treat early OA cases and protect further cartilage degradation by preventing chondrocyte hypertrophic differentiation.

The role of stromal cells in inflammatory bone loss.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation, local and systemic bone loss and a lack of compensatory bone repair. Fibroblast-like synoviocytes (FLS) are the most abundant cells of the stroma and a key population in autoimmune diseases such as RA. An increasing body of evidence suggests that these cells play not only an important role in chronic inflammation and synovial hyperplasia, but also impact bone remodelling. Under inflammatory conditions FLS release inflammatory cytokines, regulate bone destruction and formation and communicate with immune cells to control bone homeostasis. Other stromal cells, such as osteoblasts and terminally differentiated osteoblasts, termed osteocytes, are also involved in the regulation of bone homeostasis and are dysregulated during inflammation. This review highlights our current understanding of how stromal cells influence the balance between bone formation and bone destruction. Increasing our understanding of these processes is critical to enable the development of novel therapeutic strategies with which to treat bone loss in RA.

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis.

The extracellular matrix (ECM) of articular cartilage is comprised of complex networks of proteins and glycoproteins, all of which are expressed by its resident cell, the chondrocyte. Cartilage is a unique tissue given its complexity and ability to resist repeated load and deformation. The mechanisms by which articular cartilage maintains its integrity throughout our lifetime is not fully understood, however there are numerous regulatory pathways known to govern ECM turnover in response to mechanical stimuli. To further our understanding of this field, we envision that proteomic analysis of the secretome will provide information on how the chondrocyte remodels the surrounding ECM in response to load, in addition to providing information on the metabolic state of the cell. In this review, we attempt to summarize the recent mass spectrometry-based proteomic discoveries in healthy and diseased cartilage and chondrocytes, to facilitate the discovery of novel biomarkers linked to degenerative pathologies, such as osteoarthritis (OA).

[Psoriasis vs. psoriatic arthritis : Similarities and differences in the pathophysiology]. / Psoriasis versus Psoriasisarthritis : Gemeinsamkeiten und Unterschiede in der Pathophysiologie.

Psoriasis is a chronic inflammatory skin disease with genetic and (auto)immunological backgrounds. Up to 30% of patients with psoriasis also develop a mostly oligoarticular arthritis with spinal involvement that is termed psoriatic arthritis (PsA) and shows a specific joint pattern which differs from that of rheumatoid arthritis (RA). Both Psa and psoriasis share a common main axis, the interleukin (IL) 23/IL17 pathway as well as major overlaps in the functions of tumor necrosis factor alpha (TNFalpha). Recently acquired knowledge supports the concept that in both diseases, similar genetic dispositions and molecular pathways lead to organ-specific disease patterns. In some types of PsA, genetic predisposition and the relevance of acute inflammatory reactions appear to be greater that in psoriasis, while in the latter exogenous factors and T­lymphocyte reactions in the skin seem to have a higher impact. A key difference between PsA and cutaneous psoriasis is the largely irreversible nature of inflammatory joint changes in PsA, whereas cutaneous plaques in psoriasis completely heal. The question of how interdependent both diseases are and whether immunologically primed T­lymphocytes from cutaneous lesions in PsA may transmit the disease to the synovial membranes and induce acute inflammation is not precisely known. A detailed analysis of these organ-specific differences may not only provide an explanation for the similar, but partly different efficacy of novel therapeutic strategies but may also lead to the development of personalized therapies that take into account the individually different manifestations of the diseases over time.

Articular cartilage calcification of the hip and knee is highly prevalent, independent of age but associated with histological osteoarthritis: evidence for a systemic disorder.

OBJECTIVES: Based on the concept of a systemic predisposition for articular cartilage calcification (CC), the aim of this study was to determine the prevalence and amount of bilateral CC of hip and knee joints in an unselected sample cohort by high-resolution digital contact radiography (DCR) and to analyze the association of CC with histological OA. METHODS: Both hip and knee joints of 87 donors (48 m and 39 f; mean age 62) were analyzed by DCR in this post-mortem study of an unselected cohort of donors. Histological OA (OARSI) of the main load bearing area of femoral heads and medial femoral condyles was determined. RESULTS: The prevalence of CC of the femoral head was 96.6%, of the knee 94.3%. Bilateral calcification was detected in 79.3% of hips and 86.2% of knees. Concomitant CC of all four joints was detected in 69.0% of donors. There was no difference between the amount of CC of hips and knees (P = 0.47). The amount of CC of any given hip or knee correlated with that of the contralateral hip (rs = 0.54, P < 0.001) or knee (rs = 0.50, P < 0.001). There was a correlation between the amount of CC and histological OA (hips rs = 0.48, P < 0.001, knees rs = 0.30, P = 0.004), but not between CC and age (hips rs = -0.09, P = 0.42; knees rs = 0.10, P = 0.34). CONCLUSIONS: These data support the concept that articular CC occurs as the result of a systemic disorder. CC appears to be an early element of hip and knee OA pathogenesis independent of age.

[Fibroblasts as pathogenic cells in rheumatic inflammation]. / Fibroblasten als pathogene Zellen in der rheumatischen Entzündung.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, synovial hyperplasia and progressive degeneration of affected joints. These processes are mediated by cells of the immune system as well as by synovial fibroblasts (RASF) originating from the lining layer of the synovium. In this scenario RASFs display an activated phenotype: they show an altered expression of adhesion molecules which allows attachment to articular cartilage and by synthesis of proteases they mediate progressive cartilage and bone destruction. Furthermore, they produce various cytokines and chemokines, which are essential for promoting the inflammatory response. In recent years it has become evident that RASFs not only passively respond to the proinflammatory milieu in the joints of RA patients but also actively contribute by the overproduction of several cytokines and chemokines. These proinflammatory cytokines trigger the transformation of RASFs into an aggressive and invasive phenotype. Additionally, the primarily altered genuine RASFs are actively involved in the recruitment and activation of immune cells. Taken together, they are key players in the development of the well-known chronic, destructive inflammatory response in joints affected by RA.

Phagocytic cell death leads to enhanced release of pro-inflammatory S100A12 in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF) is a prototypical autoinflammatory syndrome associated with phagocytic cell activation. Pyrin mutations are the genetic basis of this disease, and its expression has been shown in monocytes, granulocytes, dendritic cells, and synovial fibroblasts. Pyrin functions as a cytosolic pattern recognition receptor and forms a distinct pyrin inflammasome. The phagocyte-specific protein S100A12 is predominantly expressed in granulocytes and belongs to the group of damage associated molecular patterns (DAMP). S100A12 can be detected at massively elevated levels in the serum of FMF patients, even in clinically inactive disease. Whether this is crucial for FMF pathogenesis is as yet unknown, and we therefore investigated the mechanisms of S100A12 release from granulocytes of FMF patients presenting clinically inactive. RESULTS: We demonstrate that FMF neutrophils from patients in clinical inactive disease possess an intrinsic activity leading to cell death even in exogenously unstimulated neutrophils. Cell death resembles NETosis and is dependent on ROS and pore forming protein gasdermin D (GSDMD), as inhibitors for both are capable of completely block cell death and S100A12 release. When pyrin-activator TcdA (Clostridium difficile toxin A) is used to stimulate, neutrophilic cell death and S100A12 release are significantly enhanced in neutrophils from FMF patients compared to neutrophils from HC. CONCLUSIONS: We are able to demonstrate that activation threshold of neutrophils from inactive FMF patients is decreased, most likely by pre-activated pyrin. FMF neutrophils present with intrinsically higher ROS production, when cultured ex vivo. This higher baseline ROS activity leads to increased GSDMD cleavage and subsequent release of, e.g., S100A12, and to increased cell death with features of NETosis and pyroptosis. We show for the first time that cell death pathways in neutrophils of inactive FMF patients are easily triggered and lead to ROS- and GSDMD-dependent activation mechanisms and possibly pathology. This could be therapeutically addressed by blocking ROS or GSDMD cleavage to decrease inflammatory outbreaks when becoming highly active.

Decreased levels of nucleotide pyrophosphatase phosphodiesterase 1 are associated with cartilage calcification in osteoarthritis and trigger osteoarthritic changes in mice.

OBJECTIVE: To analyse the function of nucleotide pyrophosphatase phosphodiesterase (NPP1), a member of the pyrophosphate pathway, in osteoarthritis (OA). METHODS: mRNA expression of NPP1, ANK ankylosing protein and tissue non-specific alkaline phosphatase was assessed by quantitative PCR. NPP1 protein levels were analysed in mouse and human cartilage samples. Bone metabolism was analysed by F18-positron emission tomography-scanning and µCT in ttw/ttw mice. Ttw/ttw mice are mice carrying a loss-of-function mutation in NPP1. Calcification of articular cartilage was assessed using von Kossa staining and OA severity using the Mankin score. Cartilage remodelling was investigated by type X collagen immunohistochemistry. RESULTS: Expression of NPP1, but not the other members of this pathway, inversely correlated with cartilage calcification and OA severity in mouse and humans. Proinflammatory cytokines downregulated the expression of NPP1, demonstrating an influence of inflammation on matrix calcification. Ttw/ttw mutant mice, carrying a loss-of-function mutation in NPP1, exhibit increased bone formation process in joints compared with wild types. Ttw/ttw mice also developed spontaneous OA-like changes, evaluated by histological analysis and in vivo imaging. Ectopic calcifications were associated with increased expression of collagen X in the cartilage. CONCLUSION: The authors conclude that OA is characterised by the reactivation of molecular signalling cascades involving proinflammatory cytokines, thereby regulating the pyrophosphate pathway which consequently leads to cartilage ossification, at least in part resembling endochondral ossification.

[Osteoimmunology - IMMUNOBONE - Understanding the interaction between bone and immune system]. / Osteoimmunologie - IMMUNOBONE - Das Wechselspiel zwischen Knochen und Immunsystem verstehen. Schwerpunktprogramm 1468.

The special program "Osteoimmunology" under the leadership of the Universities of Erlangen, Jena, Gießen and Münster, is investigating in 27 projects clarification approaches on the causes of inflammatory rheumatic diseases, such as rheumatoid arthritis and ankylosing spondylitis with the aim of developing new forms of treatment. The molecular mechanisms involved in bone damage and the interaction between the immune system and bone and cartilage are topics of research.

Transforming growth factor beta1 and laminin-111 cooperate in the induction of interleukin-16 expression in synovial fibroblasts from patients with rheumatoid arthritis.

OBJECTIVES: In synovial tissues of patients with rheumatoid arthritis (RA), strong expression of laminins and integrins co-localises with increased expression of inflammatory cytokines. Synovial fibroblasts (SF) contribute to the pathogenesis of RA through increased expression of cytokines and chemoattractant factors, one of which is interleukin-16 (IL16). A study was undertaken to investigate the regulatory pathways of IL16 in SF from patients with RA (RA-SF) and osteoarthritis (OA-SF). METHODS: SF were seeded in laminin-coated flasks and activated by the addition of cytokines. The expression of IL16 was investigated by quantitative RT-PCR, immunoblotting and ELISA; its biological activity was determined by a cell migration assay. Cell-matrix interactions were investigated by cell binding and attachment assays. Relevant intracellular signalling pathways were studied by immunoblotting and with pharmacological blocking reagents. RESULTS: Stimulation of SF with transforming growth factor beta(1)(TGF-beta(1)) and growth on laminin-111 (LM-111) significantly increased the expression of IL16. Binding to LM-111 induced significantly more IL16 mRNA in RA-SF than in OA-SF (p<0.05). The IL16 cytokine was detected in supernatants of TGF-beta(1)-activated and in LM-111+TGF-beta(1)-activated RA-SF (38 to 62 pg/ml), but not in supernatants of OA-SF. This IL16 regulation involved p38MAPK, ERK1/2 and SMAD2 signalling, but not NFkappaB. CONCLUSIONS: Binding of RA-SF to LM-111 in the presence of TGF-beta(1) triggers a significant IL16 response and thus may contribute to the infiltration of CD4+ lymphocytes into synovial tissues. This mode of IL16 induction represents a novel pathway leading to IL16 production in RA-SF but not in OA-SF, which operates independently of NFkappaB signalling.

[Current trends in the design and development of monoclonal antibodies against inflammatory mediators for the treatment of rheumatoid arthritis]. / Aktuelle Trends in Design und Entwicklung monoklonaler Antikörper gegen Entzündungsmediatoren zur Therapie der rheumatoiden Arthritis.

As mediators of inflammation, cytokines contribute significantly to both the development and the extent of the inflammatory response in rheumatoid arthritis (RA). In addition, they regulate the differentiation of various cells involved in the pathogenesis of this disease. Tumour necrosis factor alpha (TNF alpha), interleukin (IL)-1 and IL-6 constitute prominent examples of such inflammatory cytokines and have been shown to play an important role in RA. As a consequence, the use of recombinant antibodies targeting these cytokines has revolutionized the treatment of RA. However, a considerable number of RA patients do not respond adequately to therapy with such biologics. Based on this notion, this article summarizes current trends in the design and development of monoclonal antibodies against inflammatory mediators. These include the identification of alterative target structures for anti-cytokine therapies, the specific modification of the antigen-binding CDR of therapeutic antibodies to reduce immunogenicity, alterations of the Fc part and the development of modified antibody fragments to improve the pharmacokinetics and to avoid non-specific immune reactions. Beyond that, efforts are undertaken to optimize the cost of these therapies.

Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-beta induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-kappaB.

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.

Nonlinear adsorption isotherm as a tool for understanding and characterizing molecularly imprinted polymers.

Molecularly imprinted polymers (MIPs) have frequently been characterized by quantities which are easily determined from experiments but have no theoretical foundation. This makes it difficult to compare different MIP preparations or to transfer MIP based methods to different experimental conditions. Since the adsorption isotherms of MIPs are markedly nonlinear, one can build a better characterization strategy on isotherms as shown by examples in this paper.

MATE1 regulates cellular uptake and sensitivity to imatinib in CML patients.

Although imatinib is highly effective in the treatment of chronic myeloid leukemia (CML), 25-30% patients do not respond or relapse after initial response. Imatinib uptake into targeted cells is crucial for its molecular response and clinical effectiveness. The organic cation transporter 1 (OCT1) has been proposed to be responsible for this process, but its relevance has been discussed controversially in recent times. Here we found that the multidrug and toxin extrusion protein 1 (MATE1) transports imatinib with a manifold higher affinity. MATE1 mainly mediates the cellular uptake of imatinib into targeted cells and thereby controls the intracellular effectiveness of imatinib. Importantly, MATE1 but not OCT1 expression is reduced in total bone marrow cells of imatinib-non-responding CML patients compared with imatinib-responding patients, indicating that MATE1 but not OCT1 determines the therapeutic success of imatinib. We thus propose that imatinib non-responders could be identified early before starting therapy by measuring MATE1 expression levels.