Synovial fibroblasts and articular tissue remodelling: Role and mechanisms.

Synovial joints are unique functional elements of the body and provide the ability for locomotion and for physical interaction with the environment. They are composed of different connective tissue structures, of which the synovial membrane is one central component. It shows a number of peculiarities that makes it different from other membranes in our body, while several lines of evidence suggest that synovial fibroblasts, also termed fibroblast-like synoviocytes (FLS) critically contribute to these peculiarities. This becomes evident particularly under disease conditions such as in rheumatoid arthritis and osteoarthritis, where the synovium is a key pathophysiological component. Therefore, an in-depth knowledge of FLS biology is not only important for understanding key features of articular function but also provides explanations for important characteristics of both degenerative and inflammatory joint diseases. This article reviews the structure, biochemical composition and functions of the synovial membrane and by focusing on the role of synovial fibroblasts explains key features of articular tissue remodelling particularly under disease conditions.

Synovial fibroblasts as potential drug targets in rheumatoid arthritis, where do we stand and where shall we go?

Fibroblast-like synoviocytes or synovial fibroblasts (FLS) are important cellular components of the inner layer of the joint capsule, referred to as the synovial membrane. They can be found in both layers of this synovial membrane and contribute to normal joint function by producing extracellular matrix components and lubricants. However, under inflammatory conditions like in rheumatoid arthritis (RA), they may start to proliferate, undergo phenotypical changes and become central elements in the perpetuation of inflammation through their direct and indirect destructive functions. Their importance in autoimmune joint disorders makes them attractive cellular targets, and as mesenchymal-derived cells, their inhibition may be carried out without immunosuppressive consequences. Here, we aim to give an overview of our current understanding of the target potential of these cells in RA.

Deletion of activin A in mesenchymal but not myeloid cells ameliorates disease severity in experimental arthritis.

OBJECTIVE: The aim of this study was to assess the extent and the mechanism by which activin A contributes to progressive joint destruction in experimental arthritis and which activin A-expressing cell type is important for disease progression. METHODS: Levels of activin A in synovial tissues were evaluated by immunohistochemistry, cell-specific expression and secretion by PCR and ELISA, respectively. Osteoclast (OC) formation was assessed by tartrat-resistant acid phosphatase (TRAP) staining and activity by resorption assay. Quantitative assessment of joint inflammation and bone destruction was performed by histological and micro-CT analysis. Immunoblotting was applied for evaluation of signalling pathways. RESULTS: In this study, we demonstrate that fibroblast-like synoviocytes (FLS) are the main producers of activin A in arthritic joints. Most significantly, we show for the first time that deficiency of activin A in arthritic FLS (ActßAd/d ColVI-Cre) but not in myeloid cells (ActßAd/d LysM-Cre) reduces OC development in vitro, indicating that activin A promotes osteoclastogenesis in a paracrine manner. Mechanistically, activin A enhanced OC formation and activity by promoting the interaction of activated Smad2 with NFATc1, the key transcription factor of osteoclastogenesis. Consistently, ActßAd/d LysM-Cre hTNFtg mice did not show reduced disease severity, whereas deficiency of activin A in ColVI-Cre-expressing cells such as FLS highly diminished joint destruction reflected by less inflammation and less bone destruction. CONCLUSIONS: The results highly suggest that FLS-derived activin A plays a crucial paracrine role in inflammatory joint destruction and may be a promising target for treating inflammatory disorders associated with OC formation and bone destruction like rheumatoid arthritis.

TNFR2 is critical for TNF-induced rheumatoid arthritis fibroblast-like synoviocyte inflammation.

OBJECTIVES: TNF-induced activation of fibroblast-like synoviocytes (FLS) is a critical determinant for synovial inflammation and joint destruction in RA. The detrimental role of TNF-receptor 1 (TNFR1) has thoroughly been characterized. The contributions of TNFR2, however, are largely unknown. This study was performed to delineate the role of TNFR2 in human FLS activation. METHODS: TNFR2 expression in synovial tissue samples was determined by immunohistochemistry. Expression of TNFR2 was silenced using RNAi or CRISPR/Cas9 technologies. Global transcriptional changes were determined by RNA-seq. QPCR, ELISA and immunoblotting were used to validate RNA-seq results and to uncover pathways operating downstream of TNFR2 in FLS. RESULTS: TNFR2 expression was increased in RA when compared with OA synovial tissues. In particular, RA-FLS demonstrated higher levels of TNFR2 when compared with OA-FLS. TNFR2 expression in RA-FLS correlated with RA disease activity, synovial T- and B-cell infiltration. TNF and IL1ß were identified as inflammatory mediators that upregulate TNFR2 in RA-FLS. Silencing of TNFR2 in RA-FLS markedly diminished the TNF-induced expression of inflammatory cytokines and chemokines, including CXCR3-binding chemokines and the B-cell activating factor TNFSF13B. Immunobiochemical analyses revealed that TNFR2-mediated expression of inflammatory mediators critically depends on STAT1. CONCLUSION: Our results define a critical role for TNFR2 in FLS-driven inflammation and unfold its participation in the unresolved course of synovial inflammation in RA.

Antibody-mediated inhibition of syndecan-4 dimerisation reduces interleukin (IL)-1 receptor trafficking and signalling.

OBJECTIVE: Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown. METHODS: Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse. RESULTS: We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA. CONCLUSION: Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

BCP crystals promote chondrocyte hypertrophic differentiation in OA cartilage by sequestering Wnt3a.

OBJECTIVE: Calcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be associated to hypertrophic differentiation of chondrocytes. One morphogen regulating hypertrophic chondrocyte differentiation is Wnt3a. METHODS: Calcification and sulfation of extracellular matrix of the cartilage was analysed over a time course from 6 to 22 weeks in mice and different OA grades of human cartilage. Wnt3a and ß-catenin was stained in human and murine cartilage. Expression of sulfation modulating enzymes (HS2St1, HS6St1) was analysed using quantitative reverse transcription PCR (RT-PCR). The influence of BCP crystals on the chondrocyte phenotype was investigated using quantitative RT-PCR for the marker genes Axin2, Sox9, Col2, MMP13, ColX and Aggrecan. Using western blot for ß-catenin and pLRP6 we investigated the activation of Wnt signalling. The binding capacity of BCP for Wnt3a was analysed using immunohistochemical staining and western blot. RESULTS: Here, we report that pericellular matrix sulfation is increased in human and murine OA. Wnt3a co-localised with heparan sulfate proteoglycans in the pericellular matrix of chondrocytes in OA cartilage, in which canonical Wnt signalling was activated. In vitro, BCP crystals physically bound to Wnt3a. Interestingly, BCP crystals were sufficient to induce canonical Wnt signalling as assessed by phosphorylation of LRP6 and stabilisation of ß-catenin, and to induce a hypertrophic shift of the chondrocyte phenotype. CONCLUSION: Consequently, our data identify BCP crystals as a concentrating factor for Wnt3a in the pericellular matrix and an inducer of chondrocyte hypertrophy.

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.

OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

Modulation of Transient Receptor Potential Channels 3 and 6 Regulates Osteoclast Function with Impact on Trabecular Bone Loss.

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss.

Cartilage Trauma Induces Necroptotic Chondrocyte Death and Expulsion of Cellular Contents.

Necroptotic cell death is characterized by an activation of RIPK3 and MLKL that leads to plasma membrane permeabilization and the release of immunostimulatory cellular contents. High levels of chondrocyte death occur following intra-articular trauma, which frequently leads to post-traumatic osteoarthritis development. The aim of this study is to assess necroptosis levels in cartilage post-trauma and to examine whether chondrocyte necroptotic mechanisms may be investigated and modified in vitro. Fractured human and murine cartilage, analysed immunohistochemically for necroptosis marker expression, demonstrated significantly higher levels of RIPK3 and phospho-MLKL than uninjured controls. Primary murine chondrocytes stimulated in vitro with the TNFα and AKT-inhibitor alongside the pan-caspase inhibitor Z-VAD-fmk exhibited a significant loss of metabolic activity and viability, accompanied by an increase in MLKL phosphorylation, which was rescued by further treatment of chondrocytes with necrostatin-1. Transmission electron microscopy demonstrated morphological features of necroptosis in chondrocytes following TNFα and Z-VAD-fmk treatment. Release of dsDNA from necroptotic chondrocytes was found to be significantly increased compared to controls. This study demonstrates that cartilage trauma leads to a high prevalence of necroptotic chondrocyte death, which can be induced and inhibited in vitro, indicating that both necroptosis and its consequential release of immunostimulatory cellular contents are potential therapeutic targets in post-traumatic arthritis treatment.

FOXO3 is involved in the tumor necrosis factor-driven inflammatory response in fibroblast-like synoviocytes.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα.

Apolipoprotein B binds to enolase-1 and aggravates inflammation in rheumatoid arthritis.

OBJECTIVE: Immune cells from patients with rheumatoid arthritis (RA) express more enolase-1 (ENO1) on their surface than those from healthy subjects, and they elicit an enhanced inflammatory response. This study is aimed to identify the ligands of ENO1 that could promote inflammatory loops in vitro and enhance the arthritis severity in vivo. METHODS: ENO1-binding proteins in RA synovial fluid were identified by mass spectromety, and affinity to ENO1 was evaluated by means of a ligand blotting and binding assay, surface plasmon resonance and confocal microscopy. Proinflammatory response by the interaction between ENO1 and apolipoprotein B (apoB) was tested in vitro and in vivo using peripheral blood mononuclear cells and a K/BxN serum transfer arthritis model and low-density lipoproteins receptor (LDLR) knockout mice. RESULTS: ApoB in the synovid fluid of patients with RA was identified as a specific ligand to ENO1 with a higher affinity than plasminogen, a known ENO1 ligand. ApoB binding to ENO1 on monocytes elicited the production of tumour necrosis factor-α, interleukins (IL)-1ß and IL-6 through both p38 mitogen-activated protein kinase and NF-κB pathways. In the K/BxN serum transfer arthritis model, administration of apoB increased the production of proinflammatory cytokines and exaggerated arthritis severity. The severity of K/BxN serum transfer arthritis in LDLR knockout mice was comparable with wild-type mice. CONCLUSIONS: A key component of atherogenic lipids, apoB, aggravated arthritis by potentiating the inflammatory response via its interaction with ENO1 expressed on the surface of immune cells. This suggests a novel mechanism by which lipid metabolism regulates chronic inflammation in RA.

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes.

Objectives: The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods: To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results: Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion: Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

Forced exercise-induced osteoarthritis is attenuated in mice lacking the small leucine-rich proteoglycan decorin.

OBJECTIVE: Interterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor ß (TGFß), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFß in decorin-deficient (Dcn-/-) cartilage leads to changes in biomechanical properties and resistance to osteoarthritis (OA). METHODS: Unchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor ß-1 (TGFß1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFß1 treatment. In addition, OA was induced in Dcn-/- and wild-type (WT) mice via forced exercise on a treadmill. RESULTS: AFM analysis revealed a strikingly higher compressive stiffness in Dcn-/- than in WT cartilage. This was accompanied by increased negative charge and enhanced sulfation of GAG chains, but not by alterations in the levels of collagens or proteoglycan core proteins. In addition, decorin-deficient chondrocytes were shown to release more active TGFß1. Increased TGFß signalling led to enhanced Chst11 sulfotransferase expression inducing an increased negative charge density of cartilage matrix. These negative charges might attract more water resulting in augmented compressive stiffness of the tissue. Therefore, decorin-deficient mice developed significantly less OA after forced exercise than WT mice. CONCLUSIONS: Our study demonstrates that the disruption of decorin-restricted TGFß signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.

Dose-dependent effect of parathyroid hormone on fracture healing and bone formation in mice.

BACKGROUND: Parathyroid hormone (PTH) is the only clinically approved osteoanabolic drug for osteoporosis treatment. However, PTH is not established for the treatment of fracture healing, and doses of PTH diverge significantly between different studies. We hypothesized that the effect of PTH on promoting fracture healing and bone formation is dose dependent. MATERIALS AND METHODS: In vivo, mice were treated with PTH (10, 40, and 200 µg/kg) in a closed femoral fracture model. Fracture healing was analyzed after 4 weeks. The fourth lumbar vertebra was analyzed to assess systemic effects. In addition, osteoblasts from calvaria of mice were treated in vitro with PTH doses of 10-5-50 nM, and their differentiation was analyzed after 26 days. RESULTS: In vivo, PTH dose-dependently stimulated bone formation in the fracture callus and the vertebral body. However, PTH treatment did not increase biomechanical stiffness of the fractured femora in a dose-dependent manner. The increased bone formation in the 200 µg/kg group was associated with a depletion of osteoclasts, indicating diminished bone remodeling. Of interest, in vitro, we observed diminished mineralization with the highest doses of PTH in osteoblast cultures. CONCLUSIONS: PTH dose-dependently stimulates bone formation in vivo. However, during fracture healing, this did not result in a dose-dependent increase of the mechanical stiffness of the fracture callus. Taken together, our in vivo and in vitro data indicate that the dose-dependent effects of PTH during fracture healing are based on the actions on multiple cell types, thereby influencing not only bone formation but also osteoclastic callus remodeling.

Combined chondroitin sulfate and glucosamine for painful knee osteoarthritis: a multicentre, randomised, double-blind, non-inferiority trial versus celecoxib.

OBJECTIVES: To compare the efficacy and safety of chondroitin sulfate plus glucosamine hydrochloride (CS+GH) versus celecoxib in patients with knee osteoarthritis and severe pain. METHODS: Double-blind Multicentre Osteoarthritis interVEntion trial with SYSADOA (MOVES) conducted in France, Germany, Poland and Spain evaluating treatment with CS+GH versus celecoxib in 606 patients with Kellgren and Lawrence grades 2-3 knee osteoarthritis and moderate-to-severe pain (Western Ontario and McMaster osteoarthritis index (WOMAC) score ≥301; 0-500 scale). Patients were randomised to receive 400 mg CS plus 500 mg GH three times a day or 200 mg celecoxib every day for 6 months. The primary outcome was the mean decrease in WOMAC pain from baseline to 6 months. Secondary outcomes included WOMAC function and stiffness, visual analogue scale for pain, presence of joint swelling/effusion, rescue medication consumption, Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT-OARSI) criteria and EuroQoL-5D. RESULTS: The adjusted mean change (95% CI) in WOMAC pain was -185.7 (-200.3 to -171.1) (50.1% decrease) with CS+GH and -186.8 (-201.7 to -171.9) (50.2% decrease) with celecoxib, meeting the non-inferiority margin of -40: -1.11 (-22.0 to 19.8; p=0.92). All sensitivity analyses were consistent with that result. At 6 months, 79.7% of patients in the combination group and 79.2% in the celecoxib group fulfilled OMERACT-OARSI criteria. Both groups elicited a reduction >50% in the presence of joint swelling; a similar reduction was seen for effusion. No differences were observed for the other secondary outcomes. Adverse events were low and similarly distributed between groups. CONCLUSIONS: CS+GH has comparable efficacy to celecoxib in reducing pain, stiffness, functional limitation and joint swelling/effusion after 6 months in patients with painful knee osteoarthritis, with a good safety profile. TRIAL REGISTRATION NUMBER: NCT01425853.

Stable activation of fibroblasts in rheumatic arthritis-causes and consequences.

The progressive destruction of articular cartilage is a hallmark of RA, a systemic autoimmune disease predominantly affecting synovial joints that often results in severe disability. Fibroblast-like synoviocytes (FLSs) have been demonstrated to play a key role in both the initiation and perpetuation of the disease. During RA pathogenesis, FLSs acquire a permanently aggressive, tumour-like phenotype that mediates cartilage destruction both directly and indirectly. This short review summarizes the recent advances in the understanding of FLS cellular transformation during RA, as well as the consequences for disease progression and for novel treatment strategies.

OCT angiography in the mouse: A novel evaluation method for vascular pathologies of the mouse retina.

PURPOSE: To investigate the application of optical coherence tomography (OCT) angiography in the retinas of healthy mice and to evaluate choroidal neovascularization (CNV) in a mouse model of laser-induced CNV. METHODS: C57BL/6J mice aged 18-25 weeks were examined using the spectral-domain optical coherence tomography device RTVue XR Avanti (Optovue, Inc, Fremont, California, USA). Blood flow in different retinal layers was detected using the split-spectrum amplitude-decorrelation angiography algorithm. Fluorescein angiography (FA) images were obtained using the Heidelberg Spectralis device (Heidelberg, Germany). RESULTS: Using the RTVue XR Avanti, we were able to obtain high-quality OCT angiography images of normal vasculature in the superficial, deep capillary and choriocapillary layers in laser-treated mice and untreated controls. Whereas no blood flow was detectable in the outer retina of untreated mice, blood flow and hence neovascular vessels were found in laser-treated mice. CONCLUSIONS: OCT angiography can clearly visualize the normal vascular plexus in the different retinal layers in the mouse retina and choroid. With OCT angiography, it is possible to verify the choroidal neovascularization induced by laser treatment. Thus, OCT angiography is a helpful imaging tool for non-invasive, in vivo evaluation of laser-induced CNV in the mouse.

A homeostatic function of CXCR2 signalling in articular cartilage.

OBJECTIVE: ELR+ CXC chemokines are heparin-binding cytokines signalling through the CXCR1 and CXCR2 receptors. ELR+ CXC chemokines have been associated with inflammatory arthritis due to their capacity to attract inflammatory cells. Here, we describe an unsuspected physiological function of these molecules in articular cartilage homeostasis. METHODS: Chemokine receptors and ligands were detected by immunohistochemistry, western blotting and RT-PCR. Osteoarthritis was induced in wild-type and CXCR2(-/-) mice by destabilisation of the medial meniscus (DMM). CXCR1/2 signalling was inhibited in vitro using blocking antibodies or siRNA. Chondrocyte phenotype was analysed using Alcian blue staining, RT-PCR and western blotting. AKT phosphorylation and SOX9 expression were upregulated using constitutively active AKT or SOX9 plasmids. Apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. RESULTS: CXCL6 was expressed in healthy cartilage and was retained through binding to heparan sulfate proteoglycans. CXCR2(-/-) mice developed more severe osteoarthritis than wild types following DMM, with increased chondrocyte apoptosis. Disruption of CXCR1/2 in human and CXCR2 signalling in mouse chondrocytes led to a decrease in extracellular matrix production, reduced expression of chondrocyte differentiation markers and increased chondrocyte apoptosis. CXCR2-dependent chondrocyte homeostasis was mediated by AKT signalling since forced expression of constitutively active AKT rescued the expression of phenotypic markers and the apoptosis induced by CXCR2 blockade. CONCLUSIONS: Our study demonstrates an important physiological role for CXCR1/2 signalling in maintaining cartilage homeostasis and suggests that the loss of ELR+ CXC chemokines during cartilage breakdown in osteoarthritis contributes to the characteristic loss of chondrocyte phenotypic stability.

FHL2 regulates the resolution of tissue damage in chronic inflammatory arthritis.

OBJECTIVE: We analysed the role of the adaptor molecule four-and-a-half Lin11, Isl-1 & Mec-3 (LIM) domain protein 2 (FHL2) in the activation of fibroblast-like synoviocytes in human rheumatoid arthritis (RA) and tumour necrosis factor α (TNFα)-dependent animal models of the disease. METHODS: Synovial tissues of patients with RA and osteoarthritis (OA) as well as hind paw sections from arthritic human TNFα transgenic (hTNFtg) mice and synovial fibroblasts from these were analysed. The effects of cytokines on the expression of FHL2 and disease-relevant matrixmetalloproteases (MMPs) were determined. Analyses of human tissue specimens from patients treated with anti-TNFα as well as anti-TNFα treatment of hTNFtg mice were performed to substantiate the TNFα effects on FHL2 levels. FHL2(-/-) mice and hTNFtg mice (with constitutive or inducible transgene expression) were crossbred to generate TNFα overexpressing FHL2-deficient animals. Signalling pathways were analysed in cells from these mice and in human cells after knock down of FHL2 by western blot. RESULTS: FHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)ß-induced FHL2 expression, TNFα suppressed FHL2. In vivo, anti-TNFα treatment led to higher FHL2 levels both in RA patients and hTNFtg mice. The loss of FHL2 increased joint destruction in hTNFtg mice, which was accompanied by elevated MMP-13. In vitro, TNFα-mediated MMP-13 was significantly higher in FHL2(-/-) cells and after knock down of FHL2, which was caused by prolonged p38 MAPK activation. CONCLUSIONS: These data suggest that FHL2 serves as a protective factor and that, rather than promoting the pathology, the upregulation of FHL2 in RA occurs in frame of a regenerative attempt.