Trapped in a vicious loop: Toll-like receptors sustain the spontaneous cytokine production by rheumatoid synovium

Synovial tissue of patients with rheumatoid arthritis (RA) spontaneously produces several cytokines, of which a fundamental role in joint inflammation and destruction has been established. However, the factors sustaining this phenomenon remain poorly understood. In a recent report, blockade of Toll-like receptor 2 (TLR2) was found to inhibit the spontaneous release of inflammatory cytokines by intact RA synovial explant cultures. Adding to the recent evidence implicating other TLRs (in particular, TLR4), this observation highlights the potential of TLRs as therapeutic targets to suppress the local production of multiple cytokines and to control the chronic inflammatory loop in RA

Gene therapy in animal models of rheumatoid arthritis: are we ready for the patients?

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints, with progressive destruction of cartilage and bone. Anti-tumour necrosis factor-α therapies (e.g. soluble tumour necrosis factor receptors) ameliorate disease in 60–70% of patients with RA. However, the need for repeated systemic administration of relatively high doses in order to achieve constant therapeutic levels in the joints, and the reported side effects are downsides to this systemic approach. Several gene therapeutic approaches have been developed to ameliorate disease in animal models of arthritis either by restoring the cytokine balance or by genetic synovectomy. In this review we summarize strategies to improve transduction of synovial cells, to achieve stable transgene expression using integrating viruses such as adeno-associated viruses, and to achieve transcriptionally regulated expression so that drug release can meet the variable demands imposed by the intermittent course of RA. Evidence from animal models convincingly supports the application of gene therapy in RA, and the feasibility of gene therapy was recently demonstrated in phase I clinical trials

Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling

Bone morphogenetic protein-2 (BMP-2) has been proposed as a tool for cartilage repair and as a stimulant of chondrogenesis. In healthy cartilage, BMP-2 is hardly present, whereas it is highly expressed during osteoarthritis. To assess its function in cartilage, BMP-2 was overexpressed in healthy murine knee joints and the effects on proteoglycan (PG) synthesis and degradation were evaluated. Moreover, the contribution of BMP in repairing damage induced by interleukin-1 (IL-1) was investigated. Ad-BMP-2 was injected intra-articularly into murine knee joints, which were isolated 3, 7, and 21 days after injection for histology, immunohistochemistry, and autoradiography. In addition, patellar and tibial cartilage was isolated for RNA isolation or measurement of PG synthesis by means of 35SO4 2- incorporation. To investigate the role for BMP-2 in cartilage repair, cartilage damage was induced by intra-articular injection of IL-1. After 2 days, Ad-BMP-2, Ad-BMP-2 + Ad-gremlin, Ad-gremlin, or a control virus was injected. Whole knee joints were isolated for histology at day 4 or patellae were isolated to measure 35SO42- incorporation. BMP-2 stimulated PG synthesis in patellar cartilage on all days and in tibial cartilage on day 21. Aggrecan mRNA expression had increased on all days in patellar cartilage, with the highest increase on day 7. Collagen type II expression showed a similar expression pattern. In tibial cartilage, collagen type II and aggrecan mRNA expression had increased on days 7 and 21. BMP-2 overexpression also induced increased aggrecan degradation in cartilage. VDIPEN staining (indicating matrix metalloproteinase activity) was elevated on day 3 in tibial cartilage and on days 3 and 7 in patellar cartilage, but no longer was by day 21. Increased NITEGE staining (indicating aggrecanase activity) was found on days 7 and 21. In IL-1-damaged patellar cartilage, BMP-2 boosted PG synthesis. Blocking of BMP activity resulted in a decreased PG synthesis compared with IL-1 alone. This decreased PG synthesis was associated with PG depletion in the cartilage. These data show that BMP-2 boosts matrix turnover in intact and IL-damaged cartilage. Moreover, BMP contributes to the intrinsic repair capacity of damaged cartilage. Increased matrix turnover might be functional in replacing matrix molecules in the repair of a damaged cartilage matrix

NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox(-/- )mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox(-/- )mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox(-/- )mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox(-/- )mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox(-/- )mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Histological appearance of knee joints injected with an adenovirus overexpressing bone morphogenetic protein-2 (BMP-2)

<p><b>Copyright information:</b></p><p>Taken from "Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling"</p><p>http://arthritis-research.com/content/9/5/R102</p><p>Arthritis Research & Therapy 2007;9(5):R102-R102.</p><p>Published online 8 Oct 2007</p><p>PMCID:PMC2212581.</p><p></p> Right knee joints were injected intra-articularly with Ad-BMP-2 or a control virus. Mice were injected with SOprior to knee joint isolation for histology on days 3, 7, or 21. Paraffin sections were stained with safranin O/fast green , prepared for autoradiography , and stained immunohistochemically for VDIPEN or NITEGE . Controls displayed here are from day 3 . Cartilage of mice injected with Ad-BMP-2 appeared to have larger chondrocytes than controls . Proteoglycan synthesis had increased by stimulation with BMP-2 . BMP-2 stimulation also leads to increased VDIPEN staining and NITEGE staining . Arrows point to intense staining around chondrocytes. FastG, fast green; SafO, safranin O

Effect of bone morphogenetic protein-2 (BMP-2) overexpression on mRNA levels of extracellular matrix molecules and proteoglycan (PG) synthesis

<p><b>Copyright information:</b></p><p>Taken from "Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling"</p><p>http://arthritis-research.com/content/9/5/R102</p><p>Arthritis Research & Therapy 2007;9(5):R102-R102.</p><p>Published online 8 Oct 2007</p><p>PMCID:PMC2212581.</p><p></p> Relative expression of mRNA levels of extracellular matrix molecules. Cartilage of mice injected with either Ad-BMP-2 or Ad-luc was isolated after 3, 7, and 21 days. Cartilage was pooled per group per time point, and RNA was isolated. Cycle threshold values were first corrected for and then for the viral control, after which the fold increase/decrease was calculated. Decreases in mRNA levels compared with controls are on the negative scale. BMP-2 induced elevated levels of collagen type II and aggrecan. No changes in collagen type X expression were found. Effect of BMP-2 overexpression on PG synthesis. Murine knee joints were injected with either Ad-BMP-2 or a control virus. Cartilage was isolated 3, 7, or 21 days after viral injection and incubated with SO, after which the amount of incorporation was measured . To perform autoradiography, mice were injected with SOintraperitoneally prior to knee joint isolation, which was performed 3, 7, or 21 days after viral injection. These data show that BMP-2 stimulation of cartilage results in increased synthesis of PGs. Statistical analysis with a Student test. *< 0.05; **< 0.005; ***< 0.0005. N.D., not detectable

Role for bone morphogenetic protein (BMP) during natural reparative response to cartilage damage

<p><b>Copyright information:</b></p><p>Taken from "Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling"</p><p>http://arthritis-research.com/content/9/5/R102</p><p>Arthritis Research & Therapy 2007;9(5):R102-R102.</p><p>Published online 8 Oct 2007</p><p>PMCID:PMC2212581.</p><p></p> To test whether the newly synthesized gremlin adenovirus was efficient in blocking BMP, 3T3 cells were transfected with Ad-gremlin or a control virus and the 28-hour supernatant was incubated with a variety of known concentrations of BMP-2 protein and the BRE-luciferase cell line. This cell line contains a luciferase construct coupled to a BMP-responsive element. Luminescence was measured and showed that Ad-gremlin blocked BMP-2 efficiently. Mice were injected intra-articularly with interleukin-1 (IL-1)-beta to induce cartilage damage. After 2 days, an adenovirus expressing BMP-2, BMP-2 + gremlin, or a control virus was injected. After 4 days, patellae were isolated and incubated in medium with SOto assess proteoglycan (PG) synthesis , or whole knee joints were isolated to measure PG content of the cartilage . This showed that BMP-2 boosts PG synthesis and that blocking of BMP activity results in an abrogation of the natural reparative response after cartilage damage. Moreover, blocking of BMP activity with gremlin resulted in an overall outcome of PG depletion. Ad-gremlin injection alone, without IL-1, has no effect (data not shown). Statistical analysis with a Student test. *< 0.05; **< 0.005; ***< 0.0005. Bre-luc, bone morphogenetic protein-responsive element-luciferase