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26.  Platelet-derived serotonin links vascular disease and tissue fibrosis 
Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage.
Vascular damage and platelet activation are associated with tissue remodeling in diseases such as systemic sclerosis, but the molecular mechanisms underlying this association have not been identified. In this study, we show that serotonin (5-hydroxytryptamine [5-HT]) stored in platelets strongly induces extracellular matrix synthesis in interstitial fibroblasts via activation of 5-HT2B receptors (5-HT2B) in a transforming growth factor β (TGF-β)–dependent manner. Dermal fibrosis was reduced in 5-HT2B−/− mice using both inducible and genetic models of fibrosis. Pharmacologic inactivation of 5-HT2B also effectively prevented the onset of experimental fibrosis and ameliorated established fibrosis. Moreover, inhibition of platelet activation prevented fibrosis in different models of skin fibrosis. Consistently, mice deficient for TPH1, the rate-limiting enzyme for 5-HT production outside the central nervous system, showed reduced experimental skin fibrosis. These findings suggest that 5-HT/5-HT2B signaling links vascular damage and platelet activation to tissue remodeling and identify 5-HT2B as a novel therapeutic target to treat fibrotic diseases.
PMCID: PMC3092343  PMID: 21518801
27.  Altered Expression of MicroRNA-203 in Rheumatoid Arthritis Synovial Fibroblasts and Its Role in Fibroblast Activation 
Arthritis and rheumatism  2011;63(2):373-381.
MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR-155 and miR-146a in rheumatoid arthritis (RA) and showed a repressive effect of miR-155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR-203 in RASFs and analyze its role in fibroblast activation.
Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real-time polymerase chain reaction (PCR)–based screening of 260 individual miRNA. Transfection of miR-203 precursor was used to analyze the function of miR-203 in RASFs. Levels of interleukin-6 (IL-6) and MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay. RASFs were stimulated with IL-1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5-azacytidine (5-azaC). Activity of IκB kinase 2 was inhibited with SC-514.
Expression of miR-203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR-203 did not change upon stimulation with IL-1β, TNFα, or LPS; however, DNA demethylation with 5-azaC increased the expression of miR-203. Enforced expression of miR-203 led to significantly increased levels of MMP-1 and IL-6. Induction of IL-6 by miR-203 overexpression was inhibited by blocking of the NF-κB pathway. Basal expression levels of IL-6 correlated with basal expression levels of miR-203.
The current results demonstrate methylation-dependent regulation of miR-203 expression in RASFs. Importantly, they also show that elevated levels of miR-203 lead to increased secretion of MMP-1 and IL-6 via the NF-κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.
PMCID: PMC3116142  PMID: 21279994
29.  Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis 
Annals of the Rheumatic Diseases  2007;66(9):1151-1156.
Regulatory T cells (Tregs) exert their anti‐inflammatory activity predominantly by cell contact‐dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA).
44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ‐secreting cells was quantified in synovial tissue cell cultures, CD3‐depleted synovial tissue cell cultures, synovial tissue cultures co‐cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real‐time PCR for CD3ε, T‐bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein.
RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co‐culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T‐bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity.
This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.
PMCID: PMC1955165  PMID: 17392348
30.  Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts 
Rheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation.
RASF were passaged for up to 8 passages. RNA was isolated after each passage and cDNA arrays were performed to evaluate the RNA expression pattern during passaging. In addition, doubling time of the cells was also measured.
From passages 2-4, mRNA expression did not change significantly. Gene expression in RASF started to change in passages 5-6 with 7-10% differentially expressed genes. After passages 7-8, more than 10% of the genes were differentially expressed. The doubling rate was constant for up to 5 passages and decreased after passages 6-8. After freezing, gene expression of the second passage is comparable to gene expression prior to freezing.
The results of this study show, that experiments, which examine gene expression of RASF and shall reflect or imitate an in vivo situation, should be limited to early culture passages to avoid cell culture effects. It is not necessary to stop culturing SF after a few passages, but to keep the problems of cell culture in mind to avoid false positive results. Especially, when large-scale screening methods on mRNA level are used. Of note, freezing does not affect gene expression substantially.
PMCID: PMC2911867  PMID: 20462438
31.  Attachment to laminin‐111 facilitates transforming growth factor β‐induced expression of matrix metalloproteinase‐3 in synovial fibroblasts 
Annals of the Rheumatic Diseases  2006;66(4):446-451.
In the synovial membrane of patients with rheumatoid arthritis (RA), a strong expression of laminins and matrix degrading proteases was reported.
To investigate the regulation of matrix metalloproteinases (MMPs) in synovial fibroblasts (SFs) of patients with osteoarthritis (OA) and RA by attachment to laminin‐1 (LM‐111) and in the presence or absence of costimulatory signals provided by transforming growth factor β (TGFβ).
SFs were seeded in laminin‐coated flasks and activated by addition of TGFβ. The expression of genes was investigated by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR), immunocytochemistry and ELISA, and intracellular signalling pathways by immunoblotting, and by poisoning p38MAPK by SB203580, MEK‐ERK by PD98059 and SMAD2 by A‐83‐01.
Attachment of SF to LM‐111 did not activate the expression of MMPs, but addition of TGFβ induced a fivefold higher expression of MMP‐3. Incubation of SF on LM‐111 in the presence of TGFβ induced a significant 12‐fold higher expression of MMP‐3 mRNA, and secretion of MMP‐3 was elevated 20‐fold above controls. Functional blocking of LM‐111–integrin interaction reduced the laminin‐activated MMP‐3 expression significantly. Stimulation of SF by LM‐111 and TGFβ activated the p38MAPK, ERK and SMAD2 pathways, and inhibition of these pathways by using SB203580, PD98059 or A‐83‐01 confirmed the involvement of these pathways in the regulation of MMP‐3.
Attachment of SF to LM‐111 by itself has only minor effects on the expression of MMP‐1 or MMP‐3, but it facilitates the TGFβ‐induced expression of MMP‐3 significantly. This mode of MMP‐3 induction may therefore contribute to inflammatory joint destruction in RA independent of the proinflammatory cytokines interleukin (IL)1β or tumour necrosis factor (TNF)α.
PMCID: PMC1856036  PMID: 17124250
32.  Hypoxia. Hypoxia in the pathogenesis of systemic sclerosis 
Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.
PMCID: PMC2688169  PMID: 19473554
33.  Increased levels of circulating microparticles in primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity 
Arthritis Research & Therapy  2009;11(5):R156.
Cell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).
We measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA.
Patients with pSS showed increased plasma level of total MPs (mean ± SEM 8.49 ± 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 ± 1.05 n PS Eq, P = 0.004) and SLE (7.3 ± 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 ± 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum β2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P ≤ 0.006).
Plasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS.
PMCID: PMC2787287  PMID: 19832990
34.  Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo 
Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.
PMCID: PMC4496125  PMID: 18774958
imatinib; scleroderma; sclerosis; endothelial cells; angiogenesis; vasculopathy; fibrosis; c-ab; PDGF; TGFβ
35.  Effect of the oral application of a highly selective MMP-13 inhibitor in three different animal models of rheumatoid arthritis 
Annals of the Rheumatic Diseases  2009;69(5):898-902.
To evaluate the decrease of cartilage destruction by a novel orally active and specific matrix metalloproteinase 13 (MMP-13) inhibitor in three different animal models of rheumatoid arthritis (RA).
Materials and methods
The SCID mouse co-implantation model of RA, the collagen-induced arthritis (CIA) model in mice and the antigen-induced arthritis model (AIA) in rabbits were used.
In the SCID mouse co-implantation model, the MMP-13 inhibitor reduced cartilage destruction by 75%. In the CIA model of RA, the MMP-13 inhibitor resulted in a significant and dose-dependent decrease in clinical symptoms as well as of cartilage erosion by 38% (30 mg/kg), 28% (10 mg/kg) and 21% (3 mg/kg). No significant effects were seen in the AIA model. No toxic effects were seen in all three animal models.
Although several MMPs in concert with other proteinases have a role in the process of cartilage destruction, there is a need for highly selective MMP inhibitors to reduce severe side effects that occur with non-specific inhibitors. Significant inhibition of MMP-13 reduced cartilage erosions in two of three tested animal models of RA. These results strongly support the development of this class of drugs to reduce or halt joint destruction in patients with RA.
PMCID: PMC2925150  PMID: 19497915
36.  Caveolin-1 Expression and Hemodynamics in COPD Patients 
Caveolin-1 is a regulator of both intracellular calcium homeostasis and endothelial nitric oxide synthase and may play a pathogenetic role in pulmonary hypertension. In the present study, we aimed to investigate the correlations between pulmonary hemodynamics and vessel morphology including the expression of Caveolin-1 in pulmonary arterioles from patients with chronic obstructive pulmonary disease (COPD) who underwent lung-volume reduction surgery. Staining and subsequent analysis was performed on paraffin-embedded lung tissue from COPD patients (n = 12). Pulmonary arteries with an external diameter of 100-500µm were analysed. Immunhistochemistry with antibodies against caveolin-1 was performed and intensity was assessed. Morphometric data were obtained by using computer-assisted imaging software. The findings were quantified and correlated to hemodynamic data obtained by right-heart catheterization. In COPD patients with pulmonary hypertension (n = 5), the expression of caveolin-1 within the medial smooth muscle cell layer was found to be increased, whereas the intimal caveolin-1 was more prominently expressed in COPD patients with normal pulmonary pressures (n = 7). The ratio between these expression patterns was positively correlated to the mean pulmonary artery pressure. Similar findings were observed for the ratio between intimal and medial thickness as well as for the expression of smooth muscle actin (SMA).
Taken together, the expression of caveolin-1 within medial smooth muscle cells of pulmonary arteries in patients with COPD is associated with pulmonary hypertension. Our results thus emphasize a potential novel player in the pathogenesis of COPD-associated pulmonary hypertension.
PMCID: PMC2703474  PMID: 19572028
Caveolin-1; chronic obstructive pulmonary disease (COPD); morphometry; pulmonary hypertension.
37.  Epigenetic modifications in rheumatoid arthritis 
Over the last decades, genetic factors for rheumatoid diseases like the HLA haplotypes have been studied extensively. However, during the past years of research, it has become more and more evident that the influence of epigenetic processes on the development of rheumatic diseases is probably as strong as the genetic background of a patient. Epigenetic processes are heritable changes in gene expression without alteration of the nucleotide sequence. Such modifications include chromatin methylation and post-translational modification of histones or other chromatin-associated proteins. The latter comprise the addition of methyl, acetyl, and phosphoryl groups or even larger moieties such as binding of ubiquitin or small ubiquitin-like modifier. The combinatory nature of these processes forms a complex network of epigenetic modifications that regulate gene expression through activation or silencing of genes. This review provides insight into the role of epigenetic alterations in the pathogenesis of rheumatoid arthritis and points out how a better understanding of such mechanisms may lead to novel therapeutic strategies.
PMCID: PMC2592785  PMID: 18947370
38.  DREAM is reduced in synovial fibroblasts of patients with chronic arthritic pain: is it a suitable target for peripheral pain management? 
The endogenous pain-relieving system depends in part on the regulation of nociceptive signals through binding of opioids to the corresponding opioid receptor. Interfering with the trans-repression effect of downstream regulatory element antagonist modulator (DREAM) on the transcription of the opioid dynorphin-encoding prodynorphin (pdyn) gene might enhance pain relief in the periphery.
Expression levels were measured in osteoarthritis (OA) synovial fibroblast-like cells (SFLCs) (n = 8) and in peripheral blood mononuclear cells (PBMCs) from OA patients (n = 53) and healthy controls (n = 26) by real-time polymerase chain reaction. Lysed OA SFLCs were analyzed by immunoprecipitation. Translation of DREAM mRNA was inhibited by small interfering RNAs (siRNAs). Expressions of DREAM, pdyn, and c-fos mRNAs were measured at 24, 48, and 72 hours after transfection.
The expression of DREAM mRNA was shown in both healthy and OA SFLCs as well as PBMCs. Inhibiting transcription using siRNAs led to a marked reduction in DREAM expression after 24, 48, and 72 hours. However, no significant changes in c-fos and pdyn expression occurred. In addition, DREAM mRNA expression was significantly reduced in OA patients with chronic pain (pain intensity as measured by a visual analog scale scale of greater than 40), but no pdyn expression was detectable.
To our knowledge, this is the first report showing the expression of DREAM in SFLCs and PBMCs on the mRNA level. However, DREAM protein was not detectable. Since repression of pdyn transcription persists after inhibiting DREAM translation, DREAM appears to play no functional role in the kappa opioid receptor system in OA SFLCs. Therefore, our data suggest that DREAM appears not to qualify as a target in peripheral pain management.
PMCID: PMC2483451  PMID: 18507845
39.  Cells of the synovium in rheumatoid arthritis. Synovial fibroblasts 
For some time synovial fibroblasts have been regarded simply as innocent synovial cells, mainly responsible for synovial homeostasis. During the past decade, however, a body of evidence has accumulated illustrating that rheumatoid arthritis synovial fibroblasts (RASFs) are active drivers of joint destruction in rheumatoid arthritis. Details regarding the intracellular signalling cascades that result in long-term activation and synthesis of proinflammatory molecules and matrix-degrading enzymes by RASFs have been analyzed. Molecular, cellular and animal studies have identified various interactions with other synovial and inflammatory cells. This expanded knowledge of the distinct role played by RASFs in the pathophysiology of rheumatoid arthritis has moved these fascinating cells to the fore, and work to identify targeted therapies to inhibit their joint destructive potential is underway.
PMCID: PMC2246247  PMID: 18177509
40.  Proteinases in the joint: clinical relevance of proteinases in joint destruction 
Proteinases are involved in essential steps in cartilage and bone homeostasis. Consequently, efforts have been made to establish their potential role in the pathology of rheumatic conditions such as rheumatoid arthritis, osteoarthritis and spondyloarthritis. Matrix metalloproteinases (MMPs) are sensitive markers of disease severity and response to treatment, and therefore they have potential in the assessment of rheumatic diseases. Despite disappointing early results with synthetic inhibitors of MMPs, there is still much scope for developing effective and safe MMPs inhibitors, and consequently to deliver new options to inhibit joint destruction.
PMCID: PMC2212555  PMID: 18001502
43.  Safety concerns on the development of novel therapeutic drugs 
Along with recent innovative approaches resulting in the development of new therapies such as small molecular inhibitors, therapeutic antibodies, recombinant proteins and gene therapy, there is increasing need for improved understanding of the basic molecular mechanisms that are exploited by such treatments. Helpful tools in the analysis of drug effects include high-throughput screening techniques such as microarrays, which are used in transcriptomics and pharmacogenomics. Although we are far from using these extensive and costly tests in our daily clinical routine, their application in basic research nevertheless takes us closer to individualized therapeutic strategies, in which the optimal therapeutic regimen is identified for each individual patient.
PMCID: PMC1779448  PMID: 16968526
44.  Somatic mutations in mitochondria: the chicken or the egg? 
Arthritis Research & Therapy  2005;7(5):179-180.
Somatic mutations of mitochondrial DNA have been detected in various pathologies such as cancer, neurodegenerative diseases, cardiac disorders and aging in general. Now it has been found that patients with rheumatoid arthritis also have a higher incidence of mitochondrial mutations in synoviocytes and synovial tissue compared with patients with osteoarthritis. Furthermore, it has been shown that these mutations possibly result in changed peptides that are presented by major histocompatibility complex II and thus might be recognized as non-self by the immune system. Further studies will show whether these mutations are actually able to trigger autoimmune inflammation in rheumatoid arthritis or whether they must be considered epiphenomena of cellular damage in chronic inflammation.
PMCID: PMC1257449  PMID: 16207343
45.  Higher susceptibility to Fas ligand induced apoptosis and altered modulation of cell death by tumor necrosis factor-α in periarticular tenocytes from patients with knee joint osteoarthritis 
Arthritis Research & Therapy  2003;5(5):R253-R261.
The aim of the present study was to investigate the expression of Fas in periarticular tenocytes of patients with osteoarthritis (OA) and to study their susceptibility to Fas ligand-mediated apoptosis. Tendon samples were obtained from the quadriceps femoris muscle of patients with knee OA and used for histological evaluation, for immunohistochemical detection of Fas, and to establish tenocyte cultures. The expression of Fas mRNA was determined by quantitative PCR. Levels of soluble Fas and soluble tumour necrosis factor (TNF) receptor I were measured using ELISA. Apoptosis was induced with recombinant human Fas ligand and measured by a histone fragmentation assay and flow cytometry. The effects of TNF-α were studied by stimulation with TNF-α alone or 24 hours before the induction of apoptosis. Tendon samples from non-OA patients were used as controls. Histological evaluation revealed degenerative changes in the tendons of all OA patients but not in the controls. Fas was detected by immunohistochemistry in all specimens, but quantitative PCR revealed significantly higher levels of Fas mRNA in OA tenocytes. In contrast, lower levels of soluble Fas were found in OA tenocytes by ELISA. OA tenocytes were significantly more susceptible to Fas ligand induced apoptosis than were control cells. TNF-α reduced the Fas ligand induced apoptosis in OA tenocytes but had no effects on control tenocytes. These data suggest that knee OA is associated with higher susceptibility of periarticular tenocytes to Fas ligand induced apoptosis because of higher expression of Fas but lower levels of apoptosis-inhibiting soluble Fas. These changes may contribute to decreased cellularity in degenerative tendons and promote their rupturing. The antiapoptotic effects of TNF-α in OA tenocytes most likely reflect regenerative attempts and must be taken into account when anti-TNF strategies are considered for OA.
PMCID: PMC193726  PMID: 12932288
apoptosis; osteoarthritis; Fas ligand; tenocytes; tumour necrosis factor-α
46.  Osteoclast-independent bone resorption by fibroblast-like cells 
Arthritis Research & Therapy  2003;5(3):R163-R173.
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.
PMCID: PMC165048  PMID: 12723988
aseptic prosthesis loosening; bone resorption; dentin; fibroblasts; severe combined immunodeficient mouse
47.  Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers 
Arthritis Research  2002;4(6):R11.
To examine whether the lack of sufficient neoangiogenesis in systemic sclerosis (SSc) is caused by a decrease in angiogenic factors and/or an increase in angiostatic factors, the potent proangiogenic molecules vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, and the angiostatic factor endostatin were determined in patients with SSc and in healthy controls. Forty-three patients with established SSc and nine patients with pre-SSc were included in the study. Serum levels of VEGF, basic fibroblast growth factor and endostatin were measured by ELISA. Age-matched and sex-matched healthy volunteers were used as controls. Highly significant differences were found in serum levels of VEGF between SSc patients and healthy controls, whereas no differences could be detected for endostatin and basic fibroblast growth factor. Significantly higher levels of VEGF were detected in patients with Scl-70 autoantibodies and in patients with diffuse SSc. Patients with pre-SSc and short disease duration showed significant higher levels of VEGF than healthy controls, indicating that elevated serum levels of VEGF are a feature of the earliest disease stages. Patients without fingertip ulcers were found to have higher levels of VEGF than patients with fingertip ulcers. Levels of endostatin were associated with the presence of giant capillaries in nailfold capillaroscopy, but not with any other clinical parameter. The results show that the concentration of VEGF is already increased in the serum of SSc patients at the earliest stages of the disease. VEGF appears to be protective against ischemic manifestations when concentrations of VEGF exceed a certain threshold level.
PMCID: PMC153841  PMID: 12453314
basic fibroblast growth factor; endostatin; fingertip ulcers; systemic sclerosis; vascular endothelial growth factor
48.  Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation 
Arthritis Research  2002;4(5):R8.
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.
PMCID: PMC125298  PMID: 12223111
differential gene expression; molecular profile; proliferation; rheumatoid arthritis; synovial fibroblasts
49.  Ex vivo gene transfer in the years to come 
Arthritis Research  2001;4(1):10-12.
Synovial fibroblasts (SFs) have become a major target for ex vivo gene transfer in rheumatoid arthritis (RA), but efficient transduction of RA-SFs still is a major problem. The low proliferation rate and heterogeneity of RA-SFs, together with their lack of highly specific surface receptors, have hampered a more extensive application of this technique. Improving transduction protocols with conventional viral vectors, therefore, as well as developing novel strategies, such as alternative target cells, and novel delivery systems constitute a major challenge. Recent progress in this field will lead to the achievement of high transgene expression, and will facilitate the use of gene transfer in human trials.
PMCID: PMC128912  PMID: 11879532
ex vivo approach; gene therapy; rheumatoid arthritis; viral vector
50.  Fibroblast biology: Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis 
Arthritis Research  2000;2(5):361-367.
There is growing evidence that activated synovial fibroblasts, as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. In recent years, significant progress has been made in elucidating the specific features of these fibroblasts. It has been understood that although macrophage and lymphocyte secreted factors contribute to their activation, rheumatoid arthritis synovial fibroblasts (RA-SFs) do not merely respond to stimulation by pro-inflammatory cytokines, but show a complex pattern of molecular changes also maintained in the absence of external stimulation. This pattern of activation is characterized by alterations in the expression of regulatory genes and signaling cascades, as well as changes in pathways leading to apoptosis. These together result in the upregulation of adhesion molecules that mediate the attachment of RA-SFs to the extracellular matrix and in the overexpression of matrix degrading enzymes that mediate the progressive destruction of the joints. In addition, activated RA-SFs exert specific effects on other cell types such as macrophages and lymphocytes. While the initiating step in the activation of RA-SFs remains elusive, several key pathways of RA-SF activation have been identified. However, there is so far no single, specific marker for this phenotype of RA-SF. It appears that activated RA-SFs are characterized by a set of specific properties which together lead to their aggressive behavior.
PMCID: PMC130137  PMID: 11094449
fibroblasts; rheumatoid arthritis

Results 26-50 (56)