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Background and objectives The LIM-and-SH3-domain-protein-1 (Lasp-1) is an actin-associated protein and is localised at focal adhesion sites where it is involved in organisation of actin polymerization and focal adhesion turnover prozesses. We investigated its role in regulating synovial fibroblast (SF) interaction with components of the extracellular matrix (ECM) and in establishing cell-cell contacts during RA. Materials and methods Lasp-1 expression was analysed in tissue from RA patients and in the hind paws of arthritic hTNFtg mice by Western blot, immunofluorescence and immunohistochemistry. Furthermore, Lasp-1-/- mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Migration characteristics of SF derived from wild type (wt), Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice were analysed in a modified scratch assay and by live cell imaging. Cell-matrix interactions and cell-cell contacts of isolated SF from all different genotypes were investigated using fibronectin coating in an electrical cell/substrate impedance sensing assay (ECIS). Additionally, we used an in vitro three-dimensional organ culture system for functional analyes. Results Lasp-1 expression levels were increased in human RA tissue and hTNFtg mice in comparison to healthy controls. Evaluation of Lasp-1-/-/hTNFtg mice revealed milder arthritis score compared to hTNFtg mice,which was confirmed by immunohistochemistry. Results of the scratch assays demonstrated a significantly reduced migration rate of Lasp-1-/- SF (-43,7% vs wt SF) and Lasp-1-/-/hTNFtg SF (-69,11% vs hTNFtg SF). Furthermore, live cell imaging studies demonstrated striking differences in the migration velocity and in migration edge formation of Lasp-1-/-/hTNFtg SF compared to hTNFtg SF. ECIS analysis demonstrated an increased cell-cell contact formation in Lasp1-/- compared to wt SF (+22% versus wt SF) and prolonged cell-cell interaction remodelling of Lasp-1-/-/hTNFtg SF in comparison to hTNFtg SF. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusion Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis by controlling the dynamics of cell-matrix and cell-cell contacts.

Background and objectives Lasp-1 (LIM-and-SH3-domain-protein-1) is an actin-binding protein that modifies cytoskeleton organisation and is localised at cell-matrix interaction sites where it is involved in cell adhesion, migration and metastatic invasion. Its role in regulating synovial fibroblast (SF) interaction with components of extracellular matrix (ECM) and cell-cell contacts during RA is unknown. Furthermore, involved signalling pathways have to be elucidated. Materials and methods Lasp-1 expression in RA tissue and hind paws of arthritic hTNFtg mice was analysed via Western blot and immunohistochemistry. Furthermore, Lasp-1 ko mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Cell migration properties of SF derived from wild type (wt), Lasp-1 -/- , hTNFtg and Lasp1 -/- /hTNFtg mice were measured using a modified scratch assay and by live cell imaging. Extracellular matrix structure and organisation from wt, Lasp-1 -/- , hTNFtg and Lasp-1 -/- /hTNFtg SF were analysed by electron microscopy (EM). Cell-matrix interactions as well as cell-cell contacts of isolated SF from mice of all genotypes were investigated using fibronectin and self-purified collagen matrix in an electrical cell/substrate impedance sensing assay (ECIS). Via pull down assays and EM components of integrin mediated cell-matrix interaction complexes were examined. In addition, src-phosphorylation levels were evaluated by Western blot. Results Lasp-1 expression levels were increased in human RA and in hTNFtg mice compared to healthy controls. Lasp-1 -/- /hTNFtg mice displayed milder clinical symptoms confirmed by immunohistochemistry. Migration assays presented a significantly reduced migration rate of Lasp-1 -/- and Lasp-1 -/- /hTNFtg SF compared to SF from wt and hTNFtg mice. Analyses of SF ECM showed thickened collagen fibrils with less crosslinking of Lasp-1 -/- SF in comparison to wt controls. In ECIS, Lasp-1 -/- SF adhered to both fibronectin and self purified ECM faster than other genotypes (-50% vs. hTNFtg and wt SF). Additionally, Lasp-1 -/- SF formed a higher number of cell-cell contacts than wt and hTNFtg SF (-17% vs. hTNFtg and -22% wt vs. SF). Pull down assays identified cortactin, uPar and vinculin as components of cell-matrix interaction complexes with increased levels of vinculin found in complexes from Lasp-1 -/- compared to wtSF. Finally, both Lasp-1 -/- and Lasp-1 -/- /hTNFtg SF showed reduced src-phosphorylation compared to wt and hTNFtg SF. Conclusion The loss of Lasp-1 leads to altered src-phosphorylation and changes in cell-matrix interaction complexes as well as altered extracellular matrix organisation. Furthermore, Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and to decreased cartilage degradation in hTNFtg mice in vivo .

Background and Objectives Lasp-1 is an important actin-crosslinking protein. It’s localises at focal adhesions, along stress fibres and leading edges and is involved in cellular migration and metastatic dissemination of different cancers. Rheumatoid arthritis synovial fibroblasts (RASF) are also able to enter the bloodstream from sites of cartilage destruction to new locations where they re-initiate the destructive processes. The underlying mechanisms of this process are of special interest, but currently unclear. Therefore we have investigated the role of Lasp-1 in synovial fibroblast migration during RA. Materials and Methods Lasp-1 expression and its subcellular location was analysed using Western blotting and immunofluorescence microscopy of cells seeded on various coatings, including fibronectin, laminin and a self-purified collagen matrix. Furthermore, Lasp-1 deficient mice were generated, which were interbred with hTNFtg mice, to form a model of inflammatory arthritis. Mice of all genotypes were analysed using a clinical score, measuring weight, grip strength and paw swelling over a time course of 14 weeks. Hind paws from each genotype were isolated, paraffin-embedded and toluidine blue stained in order to assess synovial inflammation, cartilage degradation and SF attachment. Cell migration properties of SFs derived from WT, Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg were measured using a modified scratch assay and by live cell imaging. Results Lasp-1 expression was up regulated in RASF and in SF from hTNFtg mice compared to healthy controls. In immunofluorescence, Lasp-1 was co-localised with cortactin, a marker for structures of cell adhesion and invasion, particularly when cultured on a purified collagen matrix. Lasp1-/-/hTNFtg mice showed milder clinical symptoms in comparison to hTNFtg mice. Notably, histopathological analyses revealed less cartilage destruction (0.4 mm vs. 1.6 mm, p Conclusion Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and results in a decreased cartilage degradation and destruction and less SF attachment to articular cartilage in hTNFtg mice in vivo. Therefore, Lasp-1 is an important target for therapeutic strategies aimed to reduce the invasive and migratory behaviour of synovial fibroblast in rheumatoid arthritis.

Background Recent studies demonstrated the potential of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to emigrate from affected joints and migrate via the bloodstream toward distant healthy cartilage in the SCID mouse model of disease. While the mechanisms of breaking endothelial barriers by RA-FLS are largely unclear, the junctional adhesion molecule C (JAM-C) has become of interest in this context due to its involvement in trans-endothelial migration of leukocytes. Using the human TNF transgenic (hTNFtg) mouse as a model for human RA, we studied the role of JAM-C in the transmigration of FLS derived from these mice. Methods The expression pattern of JAM-C on wild-type and hTNFtg FLS was analyzed by Western-blot and immunocytochemistry. We investigated the transmigratory capacity of these cells in a transmigration assay using murine endothelioma cells (bEnd.5) as an endothelial barrier, IL-1alpha treated murine cartilage tissue served as chemoattractant stimulus within this setting. Functional analyses included the knock down of JAM-C expression by siRNA against murine JAM-C as well as its neutralization by blocking antibodies. Results The expression of JAM-C could be detected on the surface of both wild-type and hTNFtg FLS and it was primarly located on sites of cell-cell interactions. Additionally, Western blot data showed an elevated expression of JAM-C in FLS from hTNFtg mice. Our transmigration experiments demonstrated a markedly higher potential of hTNFtg FLS to migrate through the endothelial monolayer than FLS from wild-type mice (+40%, p≤0.05), and cartilage explants pre-treated with IL-1alpha as chemoattractant strengthened the migratory capacity of hTNFtg FLS. Interestingly, knock down of JAM-C expression on hTNFtg FLS mediated by siRNA decreased the number of transmigrated cells of about 40% comparing with mock control (p≤0.01). Equally, the neutralization of JAM-C by blocking antibodies against murine JAM-C resulted in a reduction of transmigration on a similar level. Conclusions The inflammatory environment characteristic for joints of hTNFtg mice induces an up-regulation of JAM-C on FLS similar to that of human FLS during RA. Moreover, the differentiation of a trans-migrating phenotype of FLS, capable to break through endothelial barriers is supported by this environment. In this context, JAM-C seems to be contributed to this process of transmigration and, thus, to the extravasation of FLS and, therefore targeting JAM-C may be a promising therapeutic strategy for RA. Disclosure of Interest None Declared

Background and Objectives RA synovial fibroblasts (SF) have been suggested to contribute to the spreading of disease through their ability to leave cartilage destruction sites, migrate via the bloodstream and re-initiate the destructive process at distant articular cartilage surfaces. In this context, the actin-crosslinking protein Lasp-1 is of interest, because it is localised at leading edges of migrating cells and regulates metastatic dissemination of different tumours. Therefore, it is particularly important to investigate the role of Lasp-1 in SF migration and its effects on RA. Materials and Methods To identify different Lasp-1 expression levels in the hind paws of wt and hTNFtg mice, an established model for human RA, Western- blot analyses were performed. In parallel, Lasp-1 expression and its sub-cellular distribution was investigated in SF from wt and hTNFtg mice by Western-blot analyses and immunofluorescence. The migratory capacity of SFs derived from wild-type, Lasp-1 -/- , hTNFtg and Lasp1 -/- /hTNFtg mice was studied in a modified scratch assay as well as in live cell imaging studies. Furthermore, a transmigration assay using SF from all four genotypes and murine endothelioma cells (bEnd.5) as an endothelial barrier was carried out. For more detailed information, SF transmigration was evaluated when endothelial cells were also pre-treated with TNF-alpha, mimicking inflammatory conditions. Results Lasp-1 expression is upregulated in SF from hTNFtg mice and localises to structures of cell adhesion and invasion. In the scratch assay, a significantly reduced migration rate was detected in Lasp-1 -/- SFs after 24 hrs (–43.7% versus wt, p -/- /hTNFtg, respectively (–69.11% versus hTNFtg, p -/- /hTNFtg compared to hTNFtg SF. Furthermore, analyses showed a significant reduction of transmigration of Lasp1 -/- /hTNFtg compared to hTNFtg SF that was even enhanced by TNF-alpha stimulation of the endothelial cells. Interestingly, interbred Lasp1 -/- /hTNFtg mice presented milder clinical symptoms and analyses of histopathology revealed less cartilage degradation and less attachment of synovial tissue to the cartilage than hTNFtg mice at an age of 14 weeks. Conclusions Our data provide that the migratory capacity of SF is regulated by Lasp-1 and influences the severity of arthritis in hTNFtg mice. SF – when activated – migrate through the formation of invasive and adhesive membrane structures such as invadopodia, where Lasp-1 is prominently localised. Thus, targeting Lasp-1 may be a promising strategy to reduce the invasive and migratory behaviour of synovial fibroblasts in RA.

Background and objectives Recent studies demonstrated the potential of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to migrate long distances via the bloodstream and invade distant cartilage in the SCID mouse model of disease.While the mechanisms of trans-endothelial migration of RA-FLS are largely unclear the junctional adhesion molecule C (JAM-C) has become of interest due to its involvement in trans-endothelial migration of leucocytes. Here, the authors used the hTNFtg mouse as a model for human RA and studied the role of JAM-C in the transmigration of FLS derived from these mice. Materials and methods The expression of JAM-C on wild-type and hTNFtg FLS was investigated by western-blot analysis and immunocytochemistry. The transmigratory capacity of these cells was studied in a transmigration assay using murine endothelioma cells (bEnd.5) as an endothelial barrier and IL-1alpha treated murine cartilage tissue as chemoattractant stimulus. Functional analyses included the knock down of JAM-C expression by siRNA against murine JAM-C as well as its neutralisation by blocking antibodies. Results The authors found the expression of JAM-C on the surface of both wild-type and hTNFtg FLS which is prominently located on sites of cell-cell interactions. Moreover, Western blot data revealed an elevated expression of JAM-C in FLS from hTNFtg mice. Transmigration experiments demonstrated a significantly higher potential of hTNFtg FLS to migrate through the endothelial monolayer than FLS from wild-type mice (+40%, p≤0,05), and cartilage explants pretreated with IL-1α enhanced the migratory capacity of hTNFtg FLS. Interestingly, siRNA-mediated knock down of JAM-C expression on hTNFtg FLS resulted in a reduction of transmigration of about 40% compared to mock control (p≤0,01). Likewise, the neutralisation of JAM-C by blocking antibodies against murine JAM-C reduced the number of transmigrated hTNFtg FLS on a similar level. Conclusion Our data demonstrate that the inflammatory environment within the joints of hTNFtg mice induces an up-regulation of JAM-C on FLS, which is characteristic for human RA-FLS. Moreover, they indicate that this environment supports the development of a trans-migrating phenotype of FLS able to get through endothelial barriers. JAM-C seems to be involved functionally in the transmigration and, thus, in extravasation of FLS and, therefore targeting JAM-C may be a promising therapeutic strategy for RA.