Fibroblasts-like Synoviocytes Research Articles

Mitochondria transfer restores fibroblasts-like synoviocytes (FLS) plasticity in LPS-induced, in vitro synovitis model

BackgroundSynovitis (SI) is one of the most common and serious orthopedic diseases in horses of different age, breed and sex, which contributes to the development of osteoarthritis. The burden of SI includes economic loss and represents a real challenge for current veterinary health care. At the molecular level, fibroblasts-like synoviocytes (FLS) are recognized as major cell populations involved in SI pathogenesis. In the course of SI, FLSs are losing their protective and pro-regenerative cytological features, become highly proliferative and initiate various stress signaling pathways.MethodsFibroblast-like synoviocytes were treated with LPS in order to generate SI in vitro model. Mitochondria were isolated from peripheral blood derived mononuclear cells and co-cultured with FLS. After 24 h of culture, cells were subjected to RT-qPCR, western blot, cytometric and confocal microscopy analysis.ResultsMitochondrial transfer (MT) was observed in vitro studies using confocal microscopy. Further studies revealed, that MT to LPS-treated FLS reduced cell proliferation, modulated apoptosis and decreased inflammatory response. Overall, MT Resulted in the considerable recovery of recipient cells cytophysiological properties.ConclusionsPresented data provides evidence that mitochondria transfersignificantly modulate FLS proliferative and metabolic activity through improved mitochondrial biogenesis and dynamics in activated FLS. Obtained results for the first time demonstrate that horizontal MT might be considered as a therapeutic tool for synovitis treatment; however, further clinical studies are strongly required.FTuaju_fdFNqjVa5MjyEASVideo abstract

Paeoniflorin-6′-o-benzene sulfonate ameliorates the progression of adjuvant-induced arthritis by inhibiting the interaction between Ahr and GRK2 of fibroblast-like synoviocytes

Aryl hydrocarbon receptor (Ahr) is thought to be a crucial factor that regulates immune responses, which may be involved in the pathogenesis of autoimmune inflammation including rheumatoid arthritis (RA). The results of our group in recent years have shown that Paeoniflorin-6′-O-benzene sulfonate (code: CP-25), a novel ester derivative of paeoniflorin, has a good effect on improving RA animal models. However, whether the anti-arthritis effect of CP-25 is related to Ahr remains unclear. Here, we showed that CP-25 treatment ameliorated adjuvant-induced arthritis (AA), a rat model of RA, by inhibiting Ahr-related activities in fibroblasts like synoviocytes (FLS). AA rats were treated with CP-25 or paroxetine from days 17 to 33 after immunization. We showed that CP-25 alleviated arthritis symptoms and the pathological changes. Treatment with CP-25 decreased the expression of Ahr in the synovium of AA rats. CP-25 inhibited the expression of Ahr and the G protein-coupled receptor kinase 2 (GRK2) as well as the co-expression of GRK2 with Ahr in FLS of AA rats. Furthermore, CP-25 down-regulated the production of Kyn in FLS of AA rats. These results suggested that CP-25 may inhibit the expression and activation of Ahr. Besides, treatment with CP-25 reduced the proliferation and migration of MH7A caused by Ahr activation. In addition, we also demonstrated that CP-25 down-regulated the total and nuclear expression of Ahr and the expression of GRK2 in Kyn-treated MH7A. Moreover, the co-expression and co-localization of Ahr and GRK2in Kyn-treated MH7A were also repressed by CP-25. The data presented here demonstrated that CP-25 suppressed FLS dysfunction in rats with AA, which were associated with reduced Ahr activation and the interaction between Ahr and GRK2.

Unique Inflammatory Signature in Hemophilic Arthropathy: Epigenetic Changes Due to Interaction between Blood and Fibroblast-like Synoviocytes

Context: Hemophilic patients bleeding occur mainly into large joints such as knee, elbow or ankle. Severe recurrence of hemarthrosis leads to irreversible arthropathy called hemophilic arthropathy (HA) characterized by a progressive complete destruction of the joint with permanent pain, musculoskeletal disability and poor quality of life. Physiopathology of blood joint disease remains unclear. Some data suggest that HA and rheumatoid arthritis (RA) may share similar pathogenicity including elevation of inflammatory cytokines, chronic proliferative synovitis and cartilage destruction. However, in these two diseases the origin of inflammation differs and is linked to autoimmunity in RA and bleeding in HA. HA is the unique model of arthropathy in which resident cells like fibroblasts-like synoviocytes (FLS) are directly in contact with blood. Therefore, we hypothesized that FLS in HA could have a unique inflammatory signature as a consequence of contact with blood.Materials and methods: Human FLS were isolated from synovial tissues from five different HA patients and two non-HA patients at the time of knee or ankle joint arthroscopic synovectomy after informed consent were obtained from patients. Experiments with FLS cultures were performed between the third and the ninth passage. During that time, cultures were constituted of a homogeneous population of fibroblastic cells. THP-1 cells line has been used as a control.Results: FLS from hemophilic patients have a singular inflammatory cytokines profile- FLS (5x105 cells) were activated or not (medium) with LPS from Salmonella abortus equi (1 µg/ml) for 6 h and activated-cells supernatants were analyzed by ELISA. ELISPOT were performed with a panel of 105 different cytokines. After LPS stimulation, HA FLS expressed a unique profile of cytokines secretion, which differs from non-HA FLS and THP-1 cell line including IL-1α, IL-6 and IL-8 involved in innate immunity, IFN-γ, IL-5, IL-17A and IL-22 involved in adaptive immunity and MCP-1, M-CSF, MIF and MIP-3α involved in macrophages polarization.- Constitutive expression of IL-1 and IL-6 mRNA was detectable in all cells, but in contrast, as shown previously in normal FLS, no TNF-α mRNA was detectable. To investigate mechanisms responsible for the lack of IL-1 protein synthesis in LPS-activated HA FLS, cells were incubated with LPS for 3 h and then with 5 µg/ml actinomycin D. After 2h of actinomycin D treatment, IL-1 mRNA became lower in LPS-activated HA FLS versus LPS-activated non-HA FLS and THP-1 cells. These results indicate that de novo synthesized IL-1 mRNA has a short half-life in LPS-activated HA FLS.- Since cytokines mRNA seems to be unstable, we then investigated microRNA involvement in the regulation of cytokines. A miRNOME (Transcriptome Profiling miRNA, AFFYMETRIX) analysis showed that miRNAs expression was different between HA and nonHA FLS. Five have a very high difference in expression (hsa-miR664b-5p, hsa-miR4516, hsa-miR4289, hsa-miR6719-3p, hsa-miR675-5p), which potentially target mRNA involved in inflammatory pathways (NFKB, MAPK, Wnt/beta-catenin).In conclusion our study demonstrates for the first time that the presence of blood in contact with FLS may induce durable epigenetic changes that may participate to the pathophysiology of hemophilic arthropathy. DisclosuresMignot:Shire: Research Funding. Delarue:Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Frenzel:Shire: Research Funding.

Proteomic analysis of Moringa oleifera's anti-arthritic effects on human fibroblasts-like synoviocytes

Rheumatoid arthritis (RA) affects more than 1.3 million Americans, making it the most common auto-immune arthritic disorder in the U.S. Current treatments are largely based on non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. However, these drugs are either ineffective, costly or have a plethora of side effects, notably immune suppression and increased risks of heart attacks. Therefore, here, we sought to examine the anti-arthritic effects of a tropical medicinal plant, Moringa oleifera (MO), and its underlying mechanism using proteomics analysis and primary human fibroblast-like synoviocytes (HFLS) harvested from healthy people and a patient with arthritis (HFLS-RA). Initial experiments optimized MO's optimal dosage by examining MO's effect on cell viability using MTT assay. Cells were divided into the following groups: (1) Negative control groups [(a) HFLS and (b) HFLS-RA, 0.1 M phosphate-buffered saline (PBS) only] and (2) MO treatment group (HFLS and HFLS-RA), dose-dependent treatment (500, 750, 1000, 50, 75, 100, 150 and 200 mg/mL). For proteomics studies, cells were divided into the following groups: (1) Negative control group (HFLS-RA), vehicle only (as above) and (2) MO Treatment group (HFLS-RA), 75 mg/mL (optimal concentration). After 24 h of treatment, cells were harvested and analyzed for cell viability (MTT assay) or proteome-wide expression (Proteomics). Data generated from proteomics were verified by confocal immunofluorescence. The MTT assay data shows that below 75 mg/mL, MO is not cytotoxic and therefore does not affect cell viability. Proteomics data revealed that MO alters expression of all the 40 proteins that are aberrantly expressed in HFLS-RA by either mitigating their expression (35 proteins) or enhancing (5 proteins) them. Specifically, the 35 proteins were mostly associated with pathological processes, such as inflammation, aberrant proliferation and cell adhesion, whereas the 5 proteins (Heat shock 70 kDa protein 1A/1B, Kallistatin, programmed cell death 6-interacting protein, Hemoglobin subunit alpha, and Aldo-keto reductase family 1 member C1) were associated overall with normal protective processes, such as anti-inflammatory and apoptotic activities, and were downregulated in HFLS-RA. We conclude that MO is potentially a good candidate for developing alternative therapy for managing arthritis.

Geldanamycin induces apoptosis and inhibits inflammation in fibroblast-like synoviocytes isolated from rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblasts-like synoviocytes (FLS), major cells in the synovium, together with infiltrated leukocytes, contribute greatly to RA progression. In our study, we hypothesized that geldanamycin (GA), a cancer drug might be able to inhibit RA FLS growth. To test the idea, RA FLS were isolated and cultured for cancer drug test. The results showed that GA can specifically inhibit the growth of RA FLS compared with normal FLS. Essentially, GA was found to promote reactive oxygen species production in RA FLS and induce programmed cell death. The annexinV/propidium iodide and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining confirmed that GA can directly induce apoptosis and subsequently inhibit the growth of RA FLS, which was also confirmed by Western blot assay. In addition, our data demonstrated that inflammation was inhibited by suppressed nuclear factor κB signaling pathway. The therapeutic effect of GA was explored in collagen-induced arthritis mice. In short, GA was a promising drug for the treatment of RA by specifically inhibiting the proliferation and inflammation of RA FLS.

Quercetin, a Plant Polyphenol, Has Potential for the Prevention of Bone Destruction in Rheumatoid Arthritis.

We investigated the immune-regulatory function of quercetin, in interleukin (IL)-17-produced osteoclastogenesis in rheumatoid arthritis (RA). RA fibroblasts-like synoviocytes (RA-FLS) were stimulated with IL-17, and the mRNA expression and secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. CD14+ monocytes (osteoclast precursors) were stimulated with IL-17, RANKL, with/without quercetin, and tartrate-resistant acid phosphatase activity was evaluated to assess osteoclast differentiation. Osteoclast differentiation was investigated after coculturing IL-17-stimulated RA-FLS and Th17 cells with monocytes. CD4+ T cells were cocultured with quercetin under Th17-inducing conditions, and their differentiation to Th17 cells and Treg cells was determined by flow cytometry analysis. We found that IL-17 stimulated RA-FLS to produce RANKL and quercetin decreased the IL-17-induced RANKL protein levels. Quercetin decreased the IL-17-produced activation of mammalian target of rapamycin, extracellular signal-regulated kinase and inhibitor of kappa B-alpha. When monocytes were stimulated with IL-17, macrophage colony-stimulating factor or RANKL, mature osteoclasts were formed, and quercetin decreased this osteoclastogenesis. When monocytes were cultured with IL-17-prestimulated RA-FLS or Th17 cells, osteoclasts were produced, and quercetin decreased this osteoclast differentiation. In Th17-differentiation conditions, quercetin suppressed Th17 cell and the production of IL-17, but quercetin did not affect Treg cells. Quercetin inhibits IL-17-stimulated RANKL production in RA-FLS and IL-17-stimulated osteoclast formation. Quercetin reduces Th17 differentiation. Quercetin could be an additional therapeutic option for bone destructive processes in RA.

PhosphoLipid transfer protein (PLTP) exerts a direct pro-inflammatory effect on rheumatoid arthritis (RA) fibroblasts-like-synoviocytes (FLS) independently of its lipid transfer activity.

Rheumatoid arthritis (RA) is a chronic inflammatory rheumatic disease with modification of lipids profile and an increased risk of cardiovascular events related to inflammation. Plasma phospholipid transfer protein (PLTP) exerts a lipid transfer activity through its active form. PLTP can also bind to receptors such as ATP-binding cassette transporter A1 (ABCA1). In addition to its role in lipoprotein metabolism and atherosclerosis, the latest advances came in support of a complex role of PLTP in the regulation of the inflammatory response, both with pro-inflammatory or anti-inflammatory properties. The aim of the present study was to decipher the role of PLTP in joint inflammation and to assess its relevance in the context of RA. PLTP expression was examined by western-blot and by immunochemistry. ABCA1 expression was analyzed by flow cytometry. Lipid transfer activity of PLTP and pro-inflammatory cytokines were measured in sera and synovial fluid (SF) from RA patients and controls (healthy subjects or osteoarthritis patients [OA]). FLS were treated with both lipid-transfer active form and inactive form of recombinant human PLTP. IL-8, IL-6, VEGF and MMP3 produced by FLS were assessed by ELISA, and proliferation by measuring 3H-Thymidine incorporation. RA synovial tissues showed higher PLTP staining than OA and PLTP protein levels were also significantly higher in RA-FLS. In addition, RA, unlike OA patients, displayed elevated levels of PLTP activity in SF, which correlated with pro-inflammatory cytokines. Both lipid-transfer active and inactive forms of PLTP significantly increased the production of cytokines and proliferation of FLS. ABCA1 was expressed on RAFLS and PLTP activated STAT3 pathway. To conclude, PLTP is highly expressed in the joints of RA patients and may directly trigger inflammation and FLS proliferation, independently of its lipid transfer activity. These results suggest a pro-inflammatory role for PLTP in RA.

Effect of quercetin regulating the expression of B cell lymphoma/leukemia-2, B cell lymphoma/leukemia-2 associated X protein on apoptosis of fibroblast-like synoviocytes in human rheumatoid arthritis

Objective To observe the effect of Quercetin regulating the expression levels of B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) on the apoptosis of human rheumatoid arthritis fibroblasts-like synoviocytes (RAFLSs). Methods The synovial tissues were collected from patients with rheumatoid arthritis (RA). The standard of diagnosis of RA was the criteria revised by American College of Rheumatology (ACR). Fresh synovial tissues were chopped into fragments, washed in sterile phosphate-buffered saline (PBS), and digested with collagenase to obtain synoviocytes. Human RAFLSs morphology was observed under light microscope. Flow cytometry was used to measure the expression of cluster of differentiation 3 (CD3), CD14, CD19 and CD90 on the cell surface of RAFLSs. Human RAFLSs were treated with different concentrations of quercetin (0, 100, 200, and 300 μmol/L) for 24 h. Western blotting was used to detect the expression of anti-apoptotic protein bcl-2 and pro-apoptotic protein bax. The effect of quercetin through bcl-2/bax gray ratio on the apoptosis of fibroblast-like synoviocytes (FLSs) was analyzed. Results As compared with control group (the bcl-2/bax value was 8.26±0.08), the bcl-2/bax in the quercetin-treated groups (100, 200, and 300 μmol/L) (bcl-2/bax values were 3.23±0.11, 0.26±0.05 and 0.12±0.03 respectively) significantly decreased (P<0.01). The apoptosis rate of RAFLSs in the control group (6.5%±0.9%) was significantly lower than in 100 μmol/L quercetin-treated group (19.9%±3.8%) (P<0.05), and 200, 300 μmol/L quercetin-treated groups (34.3%±6.7% and 42.5%±4.8% respectively) (P<0.01). Conclusion Quercetin can regulate the expression of bax and bcl-2 to influence RAFLSs apoptosis, and the apoptosis rate of RAFLSs is inversely proportional to the bcl-2/bax value. Key words: Quercetin; Rheumatoid arthritis; Fibroblastlike synoviocytes; B cell lymphoma/leukemia 2 associated X protein; B cell lymphoma/leukemia-2

Identification of genes regulating TRAIL-induced apoptosis in rheumatoid arthritis fibroblasts-like synoviocytes.

We previously described that sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis varied in rheumatoid arthritis fibroblasts-like synoviocytes (RAFLS) from one patient to another and was correlated with disease severity. Therefore, we screened for genes differentially expressed in RAFLS sensitive and resistant to TRAIL-induced apoptosis. The sensitivity of RAFLS was defined based on the percentage of TRAIL-induced apoptosis: 0-10% for resistant cells and >25% for sensitive RAFLS. We performed transcriptomic comparison between RAFLS-S (n=6) and RAFLS-R (n=6) and then examined the implication of identified candidates in the regulation of apoptosis using small interference RNA (siRNA). Microarray analysis revealed 10 functional genes differentially expressed according to TRAIL sensitivity. These factors are implicated in different functions, such as the respiratory chain (ND3), the transport of lipids (OSBP2, PLTP), the regulation of signaling linked to extracellular factors (SULF2, GALNT1, SIAE) or the regulation of gene expression (TET2 and LARP6). We confirmed differential expression for GALNT1 and LARP6 by quantitative reverse transcriptase-PCR. Using siRNA extinction, we demonstrated the implication of GALNT1, SULF2 and LARP6 in the control of TRAIL-induced responses. These results are of particular interest as GALNT1 and LARP6 have been implicated in the regulation of cell death and may represent interesting targets to induce apoptosis of RAFLS.

Decreased expression of alpha-enolase inhibits the proliferation of hypoxia-induced rheumatoid arthritis fibroblasts-like synoviocytes

Objectives. To investigate the effect of decreased alpha-enolase (ENO1) expression on rheumatoid arthritis fibroblasts-like synoviocytes (RA-FLSs) proliferation in response to hypoxia, and elucidate the possible mechanisms involved.Methods. RA-FLSs and osteoarthritis fibroblasts-like synoviocytes (OA-FLSs) were cultured in tri-gas incubators with different oxygen concentrations (3% O2, 7% O2, and 21% O2). 3% O2 (hypoxia) and 7% O2 conditions simulated intra-articular oxygen concentrations as observed in RA and healthy individual, respectively. 21% O2 represented oxygen condition for normal cell culture. ENO1-knockdown FLSs were established using ENO1-siRNA. The expression level of ENO1 was detected using reverse transcription polymerase chain reaction or RT-PCR and Western blot. Proliferation and apoptosis of RA-FLSs and OA-FLSs were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium or MTS assay and flow cytometry, respectively. Western blot analysis was used to detect key proteins involved in apoptosis.Results. ENO1 gene expression was remarkably upregulated, as well as its translation into protein, in RA-FLSs and OA-FLSs that were cultured in 3% O2 concentration. RA-FLSs and OA-FLSs that were cultured under hypoxic conditions hyperproliferated compared with similar cells under normaxic conditions. Neither 7% O2 nor 21% O2 condition had any significant effect on ENO1 expression. ENO1-siRNA-transfected FLSs, but not control-siRNA FLSs, showed markedly decreased proliferation. Additionally, ENO1 expression was found to promote significantly higher expression levels of the anti-apoptotic proteins Bcl-2, surviving, and cyclinB1, but inhibited the expression of cleaved caspase3.Conclusion. ENO1 may be crucial in the regulation of the proliferation and survival of synovial fibroblasts.

SAT0109 Immortal fibroblast-like synoviocytes in rheumatoid arthritis & disease severity: How far is the antiapoptotic protein BCL-2 expression involved?

BackgroundImmortality, aggressiveness and tumor-like behavior of fibroblasts-like synoviocytes (FLS) are to be blamed for the joint damage in rheumatoid arthritis (RA). The lack of apoptosis is a hallmark of these...

THU0004 The Role of Histone Deacetylases in Rheumatoid Arthritis Fibroblasts-Like Synoviocytes

BackgroundRheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease that affects synovial joints. A key characteristic of RA is hyperplasia of the synovial lining layer, which includes fibroblast-like synoviocytes (FLS)....

Role of Immune Response to Type II Collagen in the Pathogenesis of Rheumatoid Arthritis

Type II collagen (CII), major component of hyaline cartilage, has been considered as an auto-antigen in rheumatoid arthritis (RA). However, the clinical and biological significances with regard to the CII autoimmunity need to be clarified in human RA. The presence of antibodies to CII has been identified in sera, synovial fluid, and cartilage of patients with RA. In our study, the increased titer of IgG anti-CII in sera was well correlated with C-reactive protein, suggesting that this antibody may reflect the inflammatory status of RA. The titer of anti-CII antibodies (anti-CII Abs) tended to be higher in early stages of diseases. In our extending study, among 997 patients with RA, 269 (27.0%) were positive for circulatory IgG antibody to CII, those levels were fluctuated over time. It is hard to assess the significant amount of T cell responses to CII and CII (255∼274) in RA. By using a sensitive method of antigen specific mixed lymphocyte culture, we can detect the presence of CII-reactive T cells in peripheral blood mononuclear cells of RA patients. Sixty seven (46.9%) of 143 patients showed positive CII reactive T cell responses to CII or CII (255∼274). The frequencies of CII reactive T cells were more prominent in inflamed synovial fluid (SF) than in peripheral blood. These T cells could be clonally expanded after consecutive stimulation of CII with feeding of autologous irradiated antigen presenting cells (APC). Moreover, the production of Th1-related cytokine, such as IFN-γ, was strongly up-regulated by CII reactive T cells. These data suggest that T cells responding to CII, which are probably presenting the IFN-γ producing cells, may play an important role in the perpetuation of inflammatory process in RA. To evaluate the effector function of CII reactive T cells, we investigated the effect of CII reactive T cells and fibroblasts-like synoviocytes (FLS) interaction on the production of pro-inflammatory cytokines. When the CII reactive T cells were co-cultured with FLS, the production of IL-15 and TNF-α from FLS were significantly increased (2 to 3 fold increase) and this increase was clearly presented in accord to the expansion of CII reactive T cells. In addition, the production of IFN-γ and IL-17, T cell derived cytokines, were also increased by the co-incubation of CII reactive T cells with FLS. We also examined the impact of CII reactive T cells on chemokines production. When FLS were co-cultured with CII stimulated T cells, the production of IL-8, MCP-1, and MIP-1α were significantly enhanced. The increased production of these chemokines was strongly correlated with increase the frequency of CII reactive T cells. Conclusively, immune response to CII was frequently found in RA. Activated T cells in response to CII contributed to increase the production 1 Immune Network ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 책임저자:김호연, 강남성모병원 류마티스내과, ꂕ 137-701, 서울시 서초구 반포동 505 Tel: 02-590-2702, Fax: 02-537-4673, E-mail: ho@catholic.ac.kr 본 논문은 보건복지부의 연구비 지원(HMP-00-CH-08-0007)으로 이루어졌음. 2 Young Ok Jung, et al.

Adenoviral gene transfer of tissue inhibitors of metalloproteinases (TIMPs) reduces the invasive behaviour of rheumatoid fibroblast-like synovicytes

In rheumatoid arthritis (RA), an excess of proteolytic enzymes secreted at the synovium-cartilage junction results in the invasion of the articular cartilage by synovial cells. The aims of the present study were to investigate the effects of overexpression of TIMP-1 and TIMP-3 on: 1) the invasive behaviour of rheumatoid fibroblasts-like synoviocytes and 2) cell proliferation and apoptosis. The day before the experiments, the synoviocytes were infected with adenoviral vectors encoding TIMP-1 or TIMP-3 or with a control vector (AdLacZ). A Transwell system was used to study invasion of the cells. After 3 days, the invaded cells were counted using a microscope. Proliferation was assessed by measuring 3H-thymidine incorporation and cell counting. Apoptosis was assessed at 1-4 days after transduction using an Annexin V-FITC kit. Both TIMP-1 and TIMP-3 overexpression resulted in a significant reduction, respectively 60% (P < 0.001) and 80% (P < 0.001), of invasiveness of the synoviocytes as compared to the AdLacZ-transduced cells. In all cases, TIMP-3 was superior to TIMP-1 (P = 0.02). Cell proliferation was significantly reduced by TIMP-3 overexpression (40%; P < 0.05) and to a lesser extend by TIMP-1 (20%; P < 0.05) as compared to AdLacZ. There were little differences in % of apoptotic cells between the non-transduced, AdTIMP-1, AdTIMP-3 or Ad LacZ transduced cells up to 4 days after transduction. A maximum of 15% of the AdTIMP-3 transduced cells were in apoptotis as compared to a maximum of 12% in the other conditions. These results show that the invasive behavior of RA-FLSs can be strongly inhibited by overexpression of TIMPs. Both MMP inhibition and a reduction of proliferation appear to contribute to this effect. The superior effect of TIMP-3 may be due to a stronger effect on proliferation or to differences in the inhibitory profile of TIMP-1 and TIMP-3. To limit joint destruction in rheumatoid arthritis, inhibition of cartilage invasion by the pannus tissue by TIMP overexpression may be a useful approach.