Effects of dexamethasone on human synovial fibroblast-like cells, from osteoarthritic joints, in culture.

The effect of Dexamethasone (DEX) on cell division and macromolecular synthesis was investigated in a line (McCoy cells, A 9) of synovial fibroblast-like cells derived from human osteoarthritic joints. DEX markedly reduced the proliferation of McCoy cells in a time and dose-dependent manner. The maximal inhibition (45%) was found at 500 nM DEX 24 h after incubation and was accompanied by the appearance of giant macrophage-like cells. After DEX treatment cells showed increased content of DNA, proteins and RNA together with the reduction of [3H]-thymidine incorporation into the TCA-precipitable fraction.     

The synovial proteome: analysis of fibroblast-like synoviocytes

The present studies were initiated to determine the protein expression patterns of fibroblast-like synovial (FLS) cells derived from the synovia of rheumatoid arthritis patients. The cellular proteins were separated by two-dimensional polyacrylamide gel electrophoresis and the in-gel digested proteins were analyzed by matrix-assisted laser desorption ionization mass spectrometry. A total of 368 spots were examined and 254 identifications were made. The studies identified a number of proteins that have been implicated in the normal or pathological FLS function (e.g. uridine diphosphoglucose dehydrogenase, galectin 1 and galectin 3) or that have been characterized as potential autoantigens in rheumatoid arthritis (e.g. BiP, colligin, HC gp-39). A novel uncharacterized protein product of chromosome 19 open reading frame 10 was also detected as an apparently major component of FLS cells. These results demonstrate the utility of high-content proteomic approaches in the analysis of FLS composition.     

Stimulatory role of lysophosphatidic acid in cyclooxygenase-2 induction by synovial fluid of patients with rheumatoid arthritis in fibroblast-like synovial cells

While inflammatory cytokines are well-recognized critical factors for the induction of cyclooxygenase-2 (COX-2) in activated fibroblast-like synovial cells, the roles of biologically active components other than inflammatory cytokines in synovial fluid remain unknown. Herein, we assessed the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction using synovial fluid of patients with rheumatoid arthritis (RA) in fibroblast-like RA synovial cells. Synovial fluid from RA patients stimulated COX-2 induction, which was associated with prostaglandin E(2) production, in RA synovial cells. The synovial fluid-induced actions were inhibited by G(i/o) protein inhibitor pertussis toxin and LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1alpha- or IL-1beta-induced enzyme expression in a manner sensitive to pertussis toxin and Ki16425. RA synovial cells abundantly expressed LPA(1) receptor compared with other LPA receptor subtypes. Moreover, synovial fluid contains a significant amount of LPA, an LPA-synthesizing enzyme autotaxin, and its substrate lysophosphatidylcholine. In conclusion, LPA existing in synovial fluid plays a critical role in COX-2 induction in collaboration with inflammatory cytokines in RA synovial cells. Ki16425-sensitive LPA receptors may be therapeutic targets for RA.     

Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells

Introduction  

Advanced glycation end products (AGEs) have been introduced to be involved in the pathogenesis of osteoarthritis (OA). The influence of AGEs on osteoarthritic fibroblast-like synovial cells (FLS) has been incompletely understood as yet. The present study investigates a potential influence of AGE-modified bovine serum albumin (AGE-BSA) on cell growth, and on the expression of proinflammatory and osteoclastogenic markers in cultured FLS.     

Vascular endothelial growth factor signaling in VE-cadherin expression and tube-like formation by rheumatoid arthritic synovial fibroblast-like cells

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.     

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.     

Osteoclast-independent bone resorption by fibroblast-like cells

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.     

Effects of paclitaxel on cultured synovial cells from patients with rheumatoid arthritis

BACKGROUND: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA. METHODS: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected. RESULTS: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity. CONCLUSIONS: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA.     

Scanning electron microscopy of the surface of Chinese hamster fibroblast-like cells following glycerin treatment

The effect of glycerol upon the superficial structure of Chinese hamster fibroblast-like cells was studied. The analysis of scanning electron microscopy data made it clear that glycerol-induced changes in the surface structures were of various character depending on the duration of the contact period. A long-term (4 hour long) exposure to glycerol provoked significant structural changes. Nevertheless, the effects were reversible, judging by the fact that the washing-out of cell suspensions led to the repair of cell structures.     

Effects of ascorbic acid and analogs on the activity of testicular hyaluronidase and hyaluronan lyase on hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.     

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.     

The sculpturing role of fibroblast-like cells in morphogenesis

Cells of multicellular organisms are semi-fluid creatures. Even when they form specific cell-cell adhesions, they cannot create a defined shape or a tissue-specific architecture. Cartilaginous organs, such as ears and noses, exemplify the fact that form is imprinted in the extracellular matrix (ECM), which leads to the conclusion that cells must have the ability to shape the ECM in which they reside. This seems to be true for most tissues. The role of the ECM as an integrator of cells into functional assemblies with defined architecture is unique to multicellular organisms. The evolution of multicellularity became possible as a consequence of cells acquiring two new properties: first, cell surface macromolecular complexes that function in cell-cell binding; and, second, an ECM that integrates cells into three-dimensional structures. These two new properties allowed the evolution of the two basic types of cells-epithelial and mesenchymal. The appearance of the latter, a fibroblast-like cell with abundant filopodia, enabled the sculpturing of the ECM and the formation of complex tissue-specific architectures.