Fibroblast-like cells in the synovial lining (type B lining cells), stroma and pannus tissue are targeted by many signals, such as the following: ligands binding to cell surface receptors; lipid soluble, small molecular weight mediators (eg nitric oxide [NO], prostaglandins, carbon monoxide); extracellular matrix (ECM)-cell interactions; and direct cell-cell contacts, including gap junctional intercellular communication. Joints are subjected to cyclic mechanical loading and shear forces. Adherence and mechanical forces affect fibroblasts via the ECM (including the hyaluronan fluid phase matrix) and the pericellular matrix (eg extracellular matrix metalloproteinase inducer [EMMPRIN]) matrices, thus modulating fibroblast migration, adherence, proliferation, programmed cell death (including anoikis), synthesis or degradation of ECM, and production of various cytokines and other mediators [1]. Aggressive, transformed or transfected mesenchymal cells containing proto-oncogenes can act in the absence of lymphocytes, but whether these cells represent regressed fibroblasts, chondrocytes or bone marrow stem cells is unclear.

The aim of this study was to explore the molecular profile of proliferating rheumatoid arthritis synovial fibroblasts (RA-SF). Total RNA was extracted from two cultures of RA-SF (low-density [LD] proliferating cells and high-density [HD] nonproliferating cells) and suppression subtractive hybridization was performed to compare differential gene expression of these two cultures. Subtracted cDNA was subcloned, and nucleotide sequences were analyzed to identify each clone. Differential expression of distinct clones was confirmed by semiquantitative RT-PCR. The expression of certain genes in synovial tissues was examined by in situ hybridization. In both LD and HD cells, 44 clones were upregulated. Of the 88 total clones, 46 were identical to sequences that have previously been characterized. Twenty-nine clones were identical to cDNAs that have been identified, but with unknown functions so far, and 13 clones did not show any significant homology to sequences in GenBank (NCBI). Differential expression of distinct clones was confirmed by RT-PCR. In situ hybridization showed that certain genes, such as S100A4, NFAT5, unr and Fbx3, were also expressed predominantly in synovial tissues from patients with RA but not from normal individuals. The expression of distinct genes in proliferating RA-SF could also be found in RA synovium, suggesting that these molecules are involved in synovial activation in RA. Most importantly, the data indicate that the expression of certain genes in RA-SF depends on the stage of proliferation; therefore, the stage needs to be considered in any analysis of differential gene expression in SF.

We have analyzed the pattern of procoagulant and fibrinolytic gene expression in affected joints during the course of arthritis in two murine models. In both models, we found an increased expression of tissue factor, tissue factor pathway inhibitor, urokinase plasminogen activator, and plasminogen activator inhibitor 1, as well as thrombin receptor. The observed pattern of gene expression tended to favor procoagulant activity, and this pattern was confirmed by functional assays. These alterations would account for persistence of fibrin within the inflamed joint, as is seen in rheumatoid arthritis.

Background The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C). Results Cells are able to adhere to and spread on III1-C coated on a dish. Both β1 integrins and cell surface heparan sulfate proteoglycans serve as receptors for III1-C. For example, cell attachment to III1-C is partially inhibited by agents that block β1 integrins or by heparin. Complete inhibition of cell attachment is seen only when integrin blocking agents are combined with heparin. Affinity chromatography revealed the binding of proteins that likely represent the integrin β1 and α5 submits to a III1-C column. Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents. In addition, cell adhesion to III1-C and ERK1/2 activation by III1-C are both inhibited by heparan sulfate but not by chondroitin sulfate. Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation. Affinity chromatography experiments demonstrated that 35SO4-labeled cell surface heparan sulfate proteoglycans bound specifically to III1-C. Conclusions The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs. Cells interact with this domain through cell surface heparan sulfate proteoglycans and integrins, and both classes of receptors are required for optimal cell adhesion and ERK1/2 activation.

To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture.

Leukocyte ingress into the synovium is a key process in the pathogenesis of rheumatoid arthritis and other inflammatory conditions. In this review, the role of endothelial cells in leukocyte extravasation will be discussed, including the role of the most relevant cellular adhesion molecules. These molecules play an important role in mediating leukocyte-endothelial interactions. It is likely that different adhesive pathways are involved in different steps of leukocyte adhesion to and migration through endothelia. Targeting of pathological endothelial function, including leukocyte-endothelial adhesion, may be useful for the future management of inflammatory arthritis.

Fibroblast biology: Synovial fibroblasts in rheumatoid arthritis - leading role or chorus line?

The synovium lines the noncartilaginous surfaces of the diarthrodial joints, and synovial tissue is also found in tendon sheaths and bursae [1]. Several rheumatic diseases are characterized by synovial inflammation. In these conditions, descriptive studies of synovial biopsy specimens may contribute to an understanding of the events that take placein vivo, and they complement experimental animal studies as well as in-vitro studies. Examination of synovial tissue is generally more relevant than synovial fluid analysis, except, for example, the analysis of neutrophils and platelets, and studies of soluble mediators. Recently, there has been an enormous upsurge in investigations of the pathological changes in the synovium [2] because of the availability of new methods to obtain synovial biopsy samples [3,4] and because of the development of immunohistological methods, in-situ hybridization, and the polymerase chain reaction. Moreover, the complementary DNA microarray technology may hold great promise for synovial tissue analysis in the future [5].

Paired synovial tissue samples were obtained from both clinically uninvolved (CU) and clinically involved (CI) knee joints of eight rheumatoid arthritis (RA) patients. In addition, biopsies were taken from five control subjects. We observed the expression of the chemokines CXCL8, CXCL9, CXCL10, CCL2 and CCL4 in CI and CU joints of RA patients. In particular, CXCL8 protein levels were specifically increased in CI joints compared with CU joints, which was confirmed by immunohistochemistry and in situ hybridization.

Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunohistochemical analysis was used to determine the expression of Ang-1, Ang-2, Tie1 and Tie2 in synovial tissue of normal subjects and those with RA and OA. Ang-1, Ang-2, Tie1 and Tie2 mRNA and protein expression were quantified in synovial tissues and RA synovial tissue fibroblasts with real-time reverse transcription polymerase chain reaction and western blot analysis. In RA, Ang-1 positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than in OA and normal synovial tissue. The expression pattern of Ang-2 in synovial tissue was similar in RA and OA, whereas the Ang-2 expression was low in normal tissue. Synovial tissue from subjects with RA and OA showed a significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to that from normal subjects. Tie2 was significantly upregulated in the RA and OA synovial tissue lining cells, macrophages and smooth muscle cells compared to normal synovial tissue. Generally Ang-1, Ang-2, Tie1 and Tie2 mRNA levels were higher in RA synovial tissue compared to normal and OA synovial tissues, and RA synovial tissue fibroblasts. Western blot analysis also demonstrated greater Tie1 and Tie2 protein expression in RA and OA synovial tissue compared to RA synovial tissue fibroblasts. In conclusion, the dominance of Ang-1 mRNA and protein expression over Ang-2 is in agreement with an active neovascularization in RA synovial tissue.