Matthias Geyer

Actual status of antiinterleukin-1 therapies in rheumatic diseases

Purpose of reviewSeveral studies have evaluated the efficacy and safety of novel therapeutic options targeting interleukin-1 (IL-1), which not only plays a significant role in rheumatoid arthritis, but also in other rheumatic diseases, for which only limited therapeutical options exist. Recent findingsThree different strategies have been pursued and evaluated in the past years: preventing IL-1 binding by occupying IL-1 receptors with anakinra, an imitation of the naturally occurring IL-1 receptor antagonist, anakinra; development of the fully human monoclonal anti-IL-1β antibody canakinumab; and synthesis of the dimeric fusion protein rilonacept, consisting of the ligand-binding domain of interleukin-1 receptor type I and its accessory protein, bound to human IgG1. Each of these three anti-IL-1 agents proved efficacy in distinct clinical situations and disease entities. SummaryOwing to the observation that IL-1 is not only involved in signaling processes resulting in autoimmune and crystal-induced joint destruction but also in several hereditary autoinflammatory syndromes, its value both in pathophysiology as well as for novel advances in medication has significantly improved in the past years leading to an enrichment of the current therapeutic armamentarium for the affected patients.

The Pathogenesis of Systemic Sclerosis Revisited

Although infectiological stimuli, environmental factors and genotypic features are known to contribute to the initiation and perpetuation of systemic sclerosis (SSc), its etiology still remains to be enigmatic, and less elusive insights are to be achieved by ongoing and future investigations. Being characterized, however, as chronic autoimmune disease with excessive collagen accumulation in skin, synovia and visceral organs such as lung, heart, and digestive tract along with obliterating angiopathy, the pathophysiology of SSc can be summarized as being based on imbalances of the cellular and humoral immune system, vascular dysfunction and activation of resident connective tissue cells. A complex interplay between these major components manages to establish and maintain the inability of the vasculature to adequately react to the need for dilatation, constriction and growth of new vessels, to cause the increased deposition of extracellular matrix constituents as well as to facilitate immunological disarrangement. Despite parallels to the chicken and egg causality dilemma, all of these account for what later clinicians observe in patients suffering from Raynaud’s phenomenon, digital ulcers, sclerodactyly, rigidity of the face, microstomia, sicca syndrome, dyspnea, dry cough, pulmonary hypertension, palpitations, syncopes, renal insufficiency, dysphagia, gastroesophageal reflux, dyspepsia, generalized arthralgias, but also dyspareunia, or erectile dysfunction.

Migratory potential of rheumatoid arthritis synovial fibroblasts: Additional perspectives

Cell migration is a central part of physiological and pathophysiological processes including wound healing, immune defense, matrix remodeling and organ homeostasis. Different cell types have migratory potential including cells of the immune system and cells required in wound healing and tissue repair. These cells migrate locally through the tissue to the site of damage. The fibroblast is a central cell type of wound healing. In rheumatoid arthritis (RA), activated synovial fibroblasts (SFs) have the ability to invade joint cartilage, actively contributing to joint destruction in RA. Recently, RASFs have been shown to be able to migrate to non-affected areas and joints through the blood stream and to invade distant cartilage. RASFs most likely use similar mechanisms comparable to lymphocytes and tumor cells for long-distance and vascular trans-migration. Future experiments will address the goal to keep the transformed-appearing fibroblasts in the affected joints using therapeutical strategies that inhibit the pathophysiological changes of transformed-appearing RASFs but do not interfere with the physiological processes of ‘normal’ fibroblasts.

Endogenous regeneration after collagenase-induced knee joint damage in the adult newt Notophthalmus viridescens

Objectives To determine whether adult newts (Notophthalmus viridescens) are able to repair experimentally-induced joint damage in order to generate a model system for the study of endogenous joint regeneration. Methods Joint instability and articular cartilage lesions of the knee joint of adult newts (N viridescens) were induced by intra-articular injection of collagenase. The changes over time were analysed clinically, by MRI, histologically and by reverse transcription PCR to detect selected relevant markers. Results After rapid onset of disease with joint luxation, loss of proteoglycans and cartilage volume, the signs ameliorated continuously by regeneration of the affected joint compartments. The majority of joints were morphologically intact and functionally operative after 10 weeks. Upregulation of chondrogenic key genes, homogenous expression levels of factors implicated in cartilage homeostasis and limb regeneration as well as the distribution of the blastemal marker 22/18 in both treated and untreated knees suggest that joint regeneration in adult newts only partially invokes pathways of embryological organogenesis. Conclusions Newts are able to regenerate articular cartilage injuries and to restore tissue integrity and function after induction of damage using a procedure known to induce experimental osteoarthritis in murine models. Further analysis of the underlying molecular mechanisms may contribute to the development of novel treatment approaches in joint failure.

OP0073 RNA-SEQ Based Identification of Factors Involved in Knee Joint Regeneration after Induced Osteoarthritis in Notophthalmus Viridescens

Background While tissue damage in mammals is mostly accompanied by fibrotic scar formation, many urodele amphibians including the red-spotted newt Notophthalmus viridescens are able to restore damaged organs or even lost appendages. To study endogenous knee joint regeneration in adult vertebrates, the newt has been established as a model organism in our working group. Like in murine models, osteoarthritis (OA) like symptoms can either be induced by surgical damage to articular cartilage or by intra-articular injection of enzymes, e.g. collagenase. In contrast to murine OA models, newts display symptoms including joint instability and luxation, but joint function is restored in these animals within 3 months. Objectives To evaluate the molecular pathways involved in knee joint regeneration. Methods To induce OA, animals were either treated by intra-articular injection of collagenase, mono-iodoacetate or by surgical manipulation of articular cartilage. To study gene expression changes on the transcriptional level during regeneration in the newt, RNA from knee joints was isolated and pooled. Subsequently, a quantitative transcriptome analysis using Illumina® next-generation sequencing (NGS) was performed. Relevant molecular pathways guiding regeneration were analyzed in all OA models tested. Candidate genes using public available databases have been selected, and pathway analyses of the gained data have been performed. Verification of selected candidate genes on the mRNA level has been done by Real Time PCR analyses. Adhesion of newt-derived cell lines and primary newt cells to Tenascin-C (TN-C) was analyzed in vitro. To compare molecular pathways guiding wound healing in amphibians and mice, the expression pattern of selected candidate genes (TN-C and cysteine-rich protein 61 (Cyr61/CCN1)) was also analyzed in murine OA models on the mRNA (Real Time PCR) and protein level (immunohistochemistry). Results During regeneration, several matricellular proteins including TN-C, CCN family members and their related pathways were found to be differentially regulated. In contrast to the murine OA models, Cyr61/CCN1 mRNA expression levels peaked at day 5 (5.9±0.72 in the collagenase model) and ceased afterwards, while mRNA expression levels in collagenase (day 10 0.6±0.07 to 2.5±0.61 at day 60) and surgically treated newts (day 10 3.39±0.64 to 1.6±0.2 at day 60) showed higher expression levels during later time points. On the protein level, CCN1 expression was detectable in chondrocytes and osteophytes in the collagenase induced murine OA model. In adhesion experiments using the newt-derived cell line B1H1 TN-C coating had anti-adhesive properties (TN-C vs. Control 42% reduction). Conclusions Extracellular matrix proteins are important during regenerative and repair processes, since they may alter cellular adhesion and guide migration. Identification of novel proteins and evaluation of their impact on cellular behaviour can help to understand the pathways involved. Future experiments on cells from OA patients might help in the development of new treatment options for human OA. Disclosure of Interest None declared

The Elicitation of Rheumatic Symptoms at Vascular Sites

The use of pharmacological agents is a key issue of therapeutic medicine. For more than a decade, TNF-α inhibitors have been prescribed for a growing number of conditions in different disciplines cove

THU0460 Tenascin-C in Joint Regeneration After Induced Osteoarthritis in the Newt Notophthalmus Viridescens

Background In mammals tissue destruction often results in fibrotic scar tissue formation, which significantly impairs organ function. In contrast, many urodele amphibians have a remarkable regenerative capacity: Damaged tissues or even lost extremities are almost perfectly regenerated, both in function and size. We established the red-spotted newt Notophthalmus viridescens as a model organism to study endogenous knee joint regeneration in adult vertebrates. In this model, osteoarthritis (OA)-like symptoms can be induced by intra-articular injection of collagenase or surgical removal of articular cartilage. Treated animals primarily display joint instability and luxation, but joint functionality is completely restored after approximately 3 months. Objectives To identify key players involved in the underlying mechanisms driving knee joint regeneration using a high resolution gene analysis approach. Methods A cDNA array was performed after surgically and collagenase-induced knee damage in newts, and dysregulated candidate genes were selected. Microarray results were verified on the mRNA level by real-time PCR. Protein expression levels of the dysregulated candidates were analysed by immunohistochemistry (IHC). Additionally, in vitro experiments in newt-derived cell lines and primary newt cells (chondrocytes/fibroblasts) with tenascin-C (TN-C) knockdown were performed. Results Several matricellular proteins including TN-C were found to be upregulated during the regenerative process (at day 10 TN-C vs. control 22.4-fold in collagenase-induced OA model, 15.6-fold in surgically induced OA model). IHC revealed high TN-C expression in the periosteum in untreated newt legs. During the early phase of regeneration (at day 10) additional expression at the injury site (surgically induced OA) was observed. At later stages (20 and 40 days after injury) TN-C expression was detectable in regenerating tissues including articular cartilage. In adhesion experiments using the newt-derived cell line B1H1 TN-C coating had anti-adhesive properties (TN-C vs. control: 42% reduction). However, TN-C knock-down in newt cell lines and primary newt cells did not alter cellular adhesive behaviour. Conclusions TN-C plays an important role during knee joint regeneration. Further detailed studies will facilitate to identify additional molecular pathways guiding these regenerative processes unique for the newt and might allow the transfer to the development of new treatment options for human OA. Disclosure of Interest None declared

AB0100 The Role of Delta/Notch like Egf-Related Receptor in Chondrogenesis of Human Mesenchymal Stem Cells

Background The Delta/Notch like EGF-related receptor (DNER) is a single-pass transmembrane protein with characteristic EGF-like repeats in the extracellular domain, similar to those of the Notch receptor and its ligand Delta. DNER is an activator of the Notch signaling pathway, which plays a major role in cell fate determination and differentiation. In our previous study, an overexpression of DNER was observed in lesional areas of human osteoarthritic articular cartilage when compared to unaffected zones of the same tissue. Objectives Based on these findings we analyzed the role of DNER in the chondrogenic differentiation of human mesenchymal stem cells (hMSC). Methods HMSCs were nucleofected with an overexpression vector for DNER and a control vector. Chondrogenic differentiation of the hMSCs was performed in a pellet mass culture system for 28 days. The culture medium was supplemented with transforming growth factor beta 3 (TGF-β3) and dexamethasone. Overexpression of DNER and the effect on specific chondrogenic markers like aggrecan, collagen type 1 and 2, Sox9, collagen type 10 were analyzed with immunohistochemical staining and semi-quantitative real-time PCR. Chondrogenesis of the hMSCs was monitored using alcian blue staining. Results Alcian blue staining increased over time assuming chondrogenic differentiation of the hMSCs. Histologically, an overexpression of DNER was detected 7 days after nucleofection and remained stable during chondrogenesis, whereas no staining of DNER could be detected in the mock-transfected cells (ctr cells). Collagen 1 expression was higher in the ctr cells at the beginning of chondrogenesis compared to the pDNER-nucleofected cells (pDNER cells) and decreased in both over time. Collagen 2 was slightly expressed from day 21 in the ctr and pDNER cells. Sox9 was greater expressed in the ctr cells at the beginning of chondrogenesis and decreased in a time-dependent manner. In pDNER cells the Sox9 expression increased at day 14 and remained stable over time. The collagen 10 level is constantly higher in the pDNER nucleofected cells compared to the ctr cells. In both, the expression increased in a time-dependent manner. Aggrecan expression increased during chondrogenesis in the ctr and pDNER cells but the expression was higher in the pDNER nucleofected cells on day 28. The semi-quantitative real-time PCR data confirmed the overexpression of DNER 2 days after nucleofection and the effect, that collagen 1 expression is downregulated and collagen 2 expression upregulated in the pDNER transfected cells compared to control until day 14. Conclusions Since DNER overexpression led to a downregulation of collagen type 1 in the hMSCs, while the expression of collagen type 2 and aggrecan were upregulated, our results support the idea that DNER might positively influence chondrogenesis and exert a reparative role during OA pathophysiology. This promotion might be regulated via an intermediate overexpression of collagen type 10. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5616

A9.2 Tenascin-C expression during knee joint regeneration in the red-spotted NEWT notophthalmus viridescens

Background In mammals, tissue or organ damage often results in fibrotic scar formation, which in turn may impair proper organ function. Many urodele amphibians are capable of regenerating different tissues including lenses, heart, and spinal cord or even entire body parts like limbs or tails without any functional impairment. We previously established the red-spotted newt Notophthalmus viridescens as a model organism to study endogenous knee joint regeneration in adult vertebrates. After surgical removal of articular cartilage from the femur or after intra-articular collagenase injection, newts displayed osteoarthritis (OA)-like symptoms, including joint instability and luxation. However, after approximately 3 months the joint function was completely restored in the treated animals. To evaluate the underlying molecular mechanisms guiding this regenerative process, a cDNA approach was used in this study. Methods A cDNA array derived from regenerating newt myocardium containing 100000 normalised expressed sequence tags (ESTs) was carried out after surgically and collagenase-induced knee damage in newts to identify key players involved in knee joint regeneration. Several ESTs were found to be regulated during the regenerative process. By using public databases, several deregulated candidate genes were identified and selected for further analysis on mRNA (Real‑Time PCR) and protein level (immunohistochemistry). Results In the cDNA array, a number of genes was found to be deregulated during the course of knee joint regeneration, including several matricellular proteins like tenascin-C (TN-C), periostin and osteonectin/SPARC. Among them, TN-C showed the strongest upregulation during the regenerative process and was selected for further studies. On the mRNA level, a strong upregulation of TN-C mRNA was found in both models: While in the collagenase model TN-C was upregulated at 10 days and 40 days after OA induction, TN-C expression ceased in the surgically-induced OA model at day 40. In immunohistochemical analyses, TN-C expression was restricted to the periosteum 10 days after damage. As knee joint regeneration progressed, TN-C was also found in regenerating cartilage (40 days after damage). Conclusions TN-C is involved in the regenerative process after OA induction in the newt. In vitro siRNA knockdown of TN-C might help to identify the molecular pathways involved. Analysis of the selected candidate genes and identification of underlying pathways guiding regeneration might ease the development of new treatment modalities of OA in humans.