Objective. Rheumatoid arthritis (RA) is a chronic inflammatory disorder of unknown origin. Histone deacetylase (HDA) activity is considered to play a major role in the transcriptional regulation of proinflammatory genes. We undertook this study to investigate the balance of histone acetylase and HDA activity in synovial tissue from RA patients compared with that from patients with osteoarthritis (OA) and normal controls.Methods. Activity of histone acetylases and HDAs was measured in nuclear extracts of total synovial tissue samples, which were obtained from RA and OA patients undergoing surgical joint replacement, and compared with the activity in synovial tissues from patients without arthritis. Tissue expression of HDAs 1 and 2 was quantified by Western blotting. In addition, immunohistochemistry was performed for HDA-2.Results. Mean ؎ SEM HDA activity in synovial tissue samples derived from patients with RA was measured as 1.5 ؎ 0.3 moles/g, whereas the activity levels in OA (3.2 ؎ 0.7 moles/g) and normal (7.1 ؎ 4.2 moles/g) synovial tissue samples were significantly higher. Histone acetylase activity reached similar levels in RA and OA tissues and in normal tissues. The ratio of HDA activity to histone acetylase activity in RA synovial tissue was significantly reduced (12 ؎ 2%) compared with that in OA synovial tissue (26 ؎ 3%). The activity ratio in normal control samples was arbitrarily set at 100 ؎ 40%. In addition, the tissue expression of HDA-1 and HDA-2 proteins was clearly lower in RA samples than in OA samples.Conclusion. The balance of histone acetylase/ HDA activities is strongly shifted toward histone hyperacetylation in patients with RA. These results offer novel molecular insights into the pathogenesis of the disease that might be relevant to the development of future therapeutic approaches.
Objective. Tissue fibrosis is a hallmark compromising feature of many disorders. In this study, we investigated the antifibrogenic effects of the histone deacetylase inhibitor trichostatin A (TSA) on cytokinedriven fibrotic responses in vitro and in vivo.Methods. Skin fibroblasts from patients with systemic sclerosis (SSc) and normal healthy control subjects were stimulated with profibrotic cytokines in combination with TSA. Human Col␣1(I) and fibronectin were measured using real-time polymerase chain reaction, and levels of soluble collagen were estimated using the SirCol collagen assay. Electromobilty shift assay and confocal fluorescence microscopy were used to investigate the intracellular distribution of Smad transcription factors. For in vivo analysis, skin fibrosis was quantified by histologic assessment of mouse skin tissue in a model of bleomycin-induced fibrosis.Results. Reductions in the cytokine-induced transcription of Col␣1(I) and fibronectin were observed in both normal and SSc skin fibroblasts following the addition of TSA. Similarly, the expression of total collagen protein in TSA-stimulated SSc skin fibroblasts was reduced to basal levels. The mechanism of action of TSA included inhibition of the nuclear translocation and DNA binding of profibrotic Smad transcription factors. Western blot analysis revealed an up-regulation of the cell cycle inhibitor p21 by TSA, leading to reduced proliferation of fibroblasts. In addition, in bleomycininduced fibrosis in mice, TSA prevented dermal accumulation of extracellular matrix in vivo.Conclusion. These findings provide novel insights into the epigenetic regulation of fibrosis. TSA and similar inhibitory compounds appear to represent early therapeutic strategies for achieving reversal of the cytokine-driven induction of matrix synthesis that leads to fibrosis.
Systemic sclerosis (SSc) is characterised by a progressive microangiopathy that contributes significantly to the morbidity of patients with SSc. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers of endothelial precursor cells (EPCs) might also contribute to the vascular pathogenesis of SSc. However, different protocols for isolation, enrichment, culture and quantification of EPCs are currently used, which complicate comparison and interpretation of the results from different studies. The aim of the European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group expert panel was to provide recommendations for standardisation of future research on EPCs. Consensus statements and recommendations were developed in a face to face meeting by an expert panel of the basic science working group of EUSTAR. The findings were: cardiovascular risk factors and medications such as statins should be described in detail. A detailed description of methods considering isolation, culture, enrichment and detection of EPCs should be given. For in vitro culture of EPCs, no protocol has been shown to be superior to another, but coating with laminin and type IV collagen would resemble most closely the situation in vivo. The endothelial phenotype should be confirmed in all in vitro cultures at the end of the culture period. We recommend using CD133, vascular endothelial growth factor type 2 receptor (VEGFR2) and CD34 in combination with a viability marker for quantification of EPCs in the blood. Finally, exact standard operating procedures for fluorescence-activated cell sorting (FACS) analysis are given that should be strictly followed. In summary, the EUSTAR recommendations will help to unify EPC research and allow better comparison between the results of different studies. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in ARD and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://ARD.bmjjournals.com/ifora/licence.pdf). AbstractObjectives: Systemic sclerosis (SSc) is characterized by a progressive microangiopathy that contributes significantly to the morbidity of SSc patients. Besides insufficient angiogenesis, defective vasculogenesis with altered numbers of endothelial precursor cells (EPCs) might also contribute to the vascular pathogenesis of SSc. However, different protocols for isolation, enrichment, culture and quantification of EPCs are currently used, which complicate comparison and interpretation of the results from different studies. Methods: The aim of the EUSTAR expert panel was therefore to provide recommendations for standardization of future research on EPCs. Consensus statements and recommendations were developed in a face-to face meeting by an expert panel o...
Microparticles are membrane-derived vesicles that are released from cells during activation or cell death. These particles can serve as mediators of intercellular crosstalk and induce a variety of cellular responses. Previous studies have shown that macrophages undergo apoptosis after phagocytosing microparticles. Here, we have addressed the hypothesis that microparticles trigger this process via lipid pathways. In these experiments, microparticles induced apoptosis in primary macrophage cells or cell lines (RAW 264.7 or U937) with up to a 5-fold increase. Preincubation of macrophages with phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)BP) reduced the microparticle-induced apoptosis in a dose-dependent manner. PtdIns(3,5)BP is a specific inhibitor of the acid sphingomyelinase and thus can block the generation of pro-apoptotic ceramides. Similarly, the preincubation of macrophages with PtdIns(3,5)BP prevented microparticle-induced upregulation of caspase 8, which is a major target molecule of ceramide action in the apoptosis pathway. PtdIns(3,5)BP, however, had no effect on the spontaneous rate of apoptosis. To evaluate further signaling pathways induced by microparticles, the extracellular signal regulated kinase (ERK-) 1 was investigated. This kinase plays a role in activating phospholipases A2 which cleaves membrane phospholipids into arachidonic acid; microparticles have been suggested to be a preferred substrate for phospholipases A2. As shown in our experiments, microparticles strongly increased the amount of phosphorylated ERK1/2 in RAW 264.7 macrophages in a time-dependent manner, peaking 15 min after co-incubation. Addition of PD98059, a specific inhibitor of ERK1, prevented the increase in apoptosis of RAW 264.7 macrophages. Together, these data suggest that microparticles perturb lipid homeostasis of macrophages and thereby induce apoptosis. These results emphasize the importance of biolipids in the cellular cross-talk of immune cells. Based on the fact that in clinical situations with excessive cell death such as malignancies, autoimmune diseases and following chemotherapies high levels of circulating microparticles might modulate phagocytosing cells, a suppression of the immune response might occur due to loss of macrophages.
The systemic CD4+ T cell compartment in patients with rheumatoid arthritis (RA) is characterized by TCR repertoire contraction, shortened telomere lengths, and decreased numbers of recent thymic emigrants, suggesting a disturbed CD4+ T cell homeostasis. In mice, homeostatic proliferation of peripheral CD4+ T cells is regulated by TCR interaction with self peptide-MHC complexes (pMHC) and can be reproduced in vitro. We have established an ex vivo model of homeostatic proliferation, in which self-replication of human CD4+ T cells is induced by cell-cell contact with autologous monocytes. In healthy individuals, blockade of TCR-pMHC class II contact resulted in decreased CD4+ T cell division. In contrast, homeostatic proliferation in RA patients was not inhibited by pMHC blockade, but increased during the initial culture period. The anti-TNF-α Ab cA2 inhibited homeostasis-driven ex vivo proliferation in healthy controls and in RA patients. In addition, treatment of RA patients with infliximab decreased the ex vivo rate of homeostatic proliferation of CD4+ T cells. Our results suggest a disturbed regulation of CD4+ T cell homeostasis leading to the repertoire aberrations reported in RA. Membrane-anchored TNF-α appears to be a cell-cell contact-dependent stimulus of homeostatic proliferation of CD4+ T cells, possibly favoring self-replication of autoreactive CD4+ T cells in patients with RA.
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