Ex vivo gene transfer in the years to come

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

Molekulare Signalwege der aseptischen Endoprothesenlockerung

Die Behandlung von immobilisierenden degenerativen und entzündlichen Gelenkerkrankungen mit der Implantation von Endoprothesen ist ein großer Erfolg und Fortschritt in der Medizin und hat stark zur Verbesserung der Lebensqualität der betroffenen Patienten beigetragen. Jährlich werden weltweit ca. 1,3 Mio. Endoprothesen implantiert, davon allein 500.000 in den USA. Dennoch sind die einmal implantierten Prothesen nicht von lebenslanger Dauer und unterliegen multiplen Einflüssen. Trotz immer neuer Entwicklungen müssen innerhalb der ersten 15 Jahre bis zu 10% der Implantate aufgrund vorzeitiger Prothesenlockerung gewechselt werden. Bei vorzeitiger Lockerung ohne Infekt oder Trauma spricht man von aseptischer Lockerung. Es ist allgemein bekannt, dass durch Abrieb entstandene Kleinstpartikel und aktivierte Makrophagen die Hauptrolle im Prozess der aseptischen Lockerung spielen. Die Pathophysiologie ist jedoch noch nicht vollständig erklärt. Die vorliegende Arbeit gibt eine Übersicht über die anerkannten molekularen Mechanismen und die Signalwege, die zur aseptischen Prothesenlockerung führen. Außerdem werden neue Therapieoptionen zur Vermeidung der aseptischen Lockerung diskutier

DREAM is reduced in synovial fibroblasts of patients with chronic arthritic pain: is it a suitable target for peripheral pain management?

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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

Molecular profile of synovial fibroblasts in rheumatoid arthritis depends on the stage of proliferation

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

Regulation and function of SIRT1 in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and destruction of synovial joints. The function of sirtuin (SIRT)1 in RA is inconclusive. In human synovial cells, SIRT1 was shown to promote cytokine production and apoptosis resistance. However, deletion of SIRT1 aggravated inflammatory arthritis in mice and increased production of pro-inflammatory cytokines in murine macrophages. In the current study, we investigated the regulation, expression, and function of SIRT1 in RA, in particular its role in adhesion and proliferation of human RA synovial fibroblasts (RASF). We found that expression of SIRT1 was increased in vivo in synovial tissues of RA smokers and in vitro by stimulation of RASF with TNFα, but decreased upon treatment with cigarette smoke extract. Synovial tissues of RA smokers showed higher leukocytic infiltration that positively correlated with enhanced levels of SIRT1. Global transcriptome analysis revealed that SIRT1 modulates expression of genes involved in the regulation of inflammatory response and cell adhesion. In functional studies, silencing of SIRT1 reduced proliferation and leukocytic adhesion to RASF but showed inconsistent results in the regulation of adhesion to plastic. In conclusion, SIRT1 modulates the proliferative and potentially also adhesive properties of RASF and can therefore promote progression of RA. KEY MESSAGES: SIRT1 is upregulated by TNFα but decreased upon CSE treatment of RASF. Upregulation of SIRT1 in RA smokers correlates with increased leukocytic infiltration. SIRT1 modulates expression of genes regulating cell adhesion and inflammation. SIRT1 regulates proliferation of RASF

AntagomiR directed against miR-20a restores functional BMPR2 signalling and prevents vascular remodelling in hypoxia-induced pulmonary hypertension

Aims Dysregulation of the bone morphogenetic protein receptor type 2 (BMPR2) is a hallmark feature that has been described in several forms of pulmonary hypertension. We recently identified the microRNA miR-20a within a highly conserved pathway as a regulator of the expression of BMPR2. To address the pathophysiological relevance of this pathway in vivo, we employed antagomiR-20a and investigated whether specific inhibition of miR-20a could restore functional levels of BMPR2 and, in turn, might prevent pulmonary arterial vascular remodelling. Methods and results For specific inhibition of miR-20a, cholesterol-modified RNA oligonucleotides (antagomiR-20a) were synthesized. The experiments in mice were performed by using the hypoxia-induced mouse model for pulmonary hypertension and animal tissues were analysed for right ventricular hypertrophy and pulmonary arterial vascular remodelling. Treatment with antagomiR-20a enhanced the expression levels of BMPR2 in lung tissues; moreover, antagomiR-20a significantly reduced wall thickness and luminal occlusion of small pulmonary arteries and reduced right ventricular hypertrophy. To assess BMPR2 signalling and proliferation, we performed in vitro experiments with human pulmonary arterial smooth muscle cells (HPASMCs). Transfection of HPASMCs with antagomiR-20a resulted in activation of downstream targets of BMPR2 showing increased activation of Id-1 and Id-2. Proliferation of HPASMCs was found to be reduced upon transfection with antagomiR-20a. Conclusion This is the first report showing that miR-20a can be specifically targeted in an in vivo model for pulmonary hypertension. Our data emphasize that treatment with antagomiR-20a restores functional levels of BMPR2 in pulmonary arteries and prevents the development of vascular remodellin

Effect of the oral application of a highly selective MMP-13 inhibitor in three different animal models of rheumatoid arthritis

OBJECTIVE: In the present study we evaluated the decrease of cartilage destruction by a novel orally active and specific MMP-13 inhibitor in three different animal models of rheumatoid arthritis (RA). MATERIALS AND METHODS: The SCID mouse co-implantation model of RA, collagen-induced arthritis (CIA) model in mice and the antigen induced arthritis model (AIA) in rabbits were used. RESULTS: In the SCID mouse co-implantation model this inhibitor resulted in 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 observed in the AIA model. No toxic effects were observed in all three animal models. CONCLUSION: Although several MMPs in concert with other proteinases play 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 out 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

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

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