Andreas Drynda

Molecular discrimination of responders and nonresponders to anti-TNFalpha therapy in rheumatoid arthritis by etanercept

IntroductionAbout 30% of rheumatoid arthritis patients fail to respond adequately to TNFα-blocking therapy. There is a medical and socioeconomic need to identify molecular markers for an early prediction of responders and nonresponders.MethodsRNA was extracted from peripheral blood mononuclear cells of 19 rheumatoid arthritis patients before the first application of the TNFα blocker etanercept as well as after 72 hours. Clinical response was assessed over 3 months using the 28-joint-count Disease Activity Score and X-ray scans. Supervised learning methods were applied to Affymetrix Human Genome U133 microarray data analysis to determine highly selective discriminatory gene pairs or triplets with prognostic relevance for the clinical outcome evinced by a decline of the 28-joint-count Disease Activity Score by 1.2.ResultsEarly downregulation of expression levels secondary to TNFα neutralization was associated with good clinical responses, as shown by a decline in overall disease activity 3 months after the start of treatment. Informative gene sets include genes (for example, NFKBIA, CCL4, IL8, IL1B, TNFAIP3, PDE4B, PPP1R15A and ADM) involved in different pathways and cellular processes such as TNFα signalling via NFκB, NFκB-independent signalling via cAMP, and the regulation of cellular and oxidative stress response. Pairs and triplets within these genes were found to have a high prognostic value, reflected by prediction accuracies of over 89% for seven selected gene pairs and of 95% for 10 specific gene triplets.ConclusionOur data underline that early gene expression profiling is instrumental in identifying candidate biomarkers to predict therapeutic outcomes of anti-TNFα treatment regimes.

Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.

Gene Transfer of Tissue Inhibitor of Metalloproteinases-3 Reverses the Inhibitory Effects of TNF-α on Fas-Induced Apoptosis in Rheumatoid Arthritis Synovial Fibroblasts

Apart from counteracting matrix metalloproteinases, tissue inhibitor of metalloproteinases-3 (TIMP-3) has proapoptotic properties. These features have been attributed to the inhibition of metalloproteinases involved in the shedding of cell surface receptors such as the TNFR. However, little is known about effects of TIMP-3 in cells that are not susceptible to apoptosis by TNF-α. In this study, we report that gene transfer of TIMP-3 into human rheumatoid arthritis synovial fibroblasts and MRC-5 human fetal lung fibroblasts facilitates apoptosis and completely reverses the apoptosis-inhibiting effects of TNF-α. Although TNF-α inhibits Fas/CD95-induced apoptosis in untransfected and mock-transfected cells, fibroblasts ectopically expressing TIMP-3 are sensitized most strongly to Fas/CD95-mediated cell death by TNF-α. Neither synthetic MMP inhibitors nor glycosylated bioactive TIMP-3 are able to achieve these effects. Gene transfer of TIMP-3 inhibits the TNF-α-induced activation of NF-κB in rheumatoid arthritis synovial fibroblasts and reduces the up-regulation of soluble Fas/CD95 by TNF-α, but has no effects on the cell surface expression of Fas. Collectively, our data demonstrate that intracellularly produced TIMP-3 not only induces apoptosis, but also modulates the apoptosis-inhibiting effects of TNF-α in human rheumatoid arthritis synovial fibroblast-like cells. Thus, our findings may stimulate further studies on the therapeutic potential of gene transfer strategies with TIMP-3.

In vitro and in vivo corrosion properties of new iron–manganese alloys designed for cardiovascular applications

The principle of biodegradation for the production of temporary implant materials (e.g. stents) plays an important role in the treatment of congenital heart defects. In the last decade several attempts have been made with different alloy materials-mainly based on iron and magnesium. None of the currently available materials in this field have demonstrated satisfying results and have therefore not found entry into broad clinical practice. While magnesium or magnesium alloy systems corrode too fast, the corrosion rate of pure iron-stents is too slow for cardiovascular applications. In the last years FeMn alloy systems were developed with the idea that galvanic effects, caused by different electrochemical properties of Fe and Mn, would increase the corrosion rate. In vitro tests with alloys containing up to 30% Mn showed promising results in terms of biocompatibility. This study deals with the development of new FeMn alloy systems with lower Mn concentrations (FeMn 0.5 wt %, FeMn 2.7 wt %, FeMn 6.9 wt %) to avoid Mn toxicity. Our results show, that these alloys exhibit good mechanical features as well as suitable in vitro biocompatibility and corrosion properties. In contrast, the evaluation of these alloys in a mouse model led to unexpected results-even after 9 months no significant corrosion was detectable. Preliminary SEM investigations showed that passivation layers (FeMn phosphates) might be the reason for corrosion resistance. If this can be proved in further experiments, strategies to prevent or dissolve those layers need to be developed to expedite the in vivo corrosion of FeMn alloys.

Biocompatibility of fluoride‐coated magnesium‐calcium alloys with optimized degradation kinetics in a subcutaneous mouse model

The principle of biodegradation has been considered for many years in the development of cardiovascular stents, especially for patients with congenital heart defects. A variety of materials have been examined with regard to their suitability for cardiovascular devices. Iron- and magnesium-based stents were investigated intensively during the last years. It has been shown, that iron, or iron based alloys have slow degradation kinetics whereas magnesium-based systems exhibit rapid degradation rates. Recently we have developed fluoride coated binary magnesium-calcium alloys with reduced degradation kinetics. These alloys exhibit good biocompatibility and no major adverse effects toward smooth muscle and endothelial cells in in vitro experiments. In this study, these alloys were investigated in a subcutaneous mouse model. Fluoride coated (fc) magnesium, as well as MgCa0.4%, MgCa0.6%, MgCa0.8%, MgCa1.0%, and a commercially available WE43 alloy were implanted in form of (fc) cylindrical plates into the subcutaneous tissue of NMRI mice. After a 3 and 6 months follow-up, the (fc) alloy plates were examined by histomorphometric techniques to assess their degradation rate in vivo. Our data indicate that all (fc) alloys showed a significant corrosion. For both time points the (fc) MgCa alloys showed a higher corrosion rate in comparison to the (fc) WE43 reference alloy. Significant adverse effects were not observed. Fluoride coating of magnesium-based alloys can be a suitable way to reduce degradation rates. However, the (fc) MgCa alloys did not exhibit decreased degradation kinetics in comparison to the (fc) WE43 alloy in a subcutaneous mouse model.

Metallic wear debris may regulate CXCR4 expression in vitro and in vivo

CXCR4, the chemokine receptor for CXCL12, also known as SDF-1 (stromal cell derived factor-1), has been shown to play a pivotal role in bone metastasis, inflammatory, and autoimmune conditions but has not been investigated in periprosthetic osteolysis. We co-cultured osteoblast-like cells with increasing concentrations of metallic (Co-35Ni-20Cr-10Mo and Co-28Cr-6Mo) and Co-ions simulating wear debris. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to quantify gene and protein expression of CXCR4. The expression of tumor necrosis factor-alpha (TNF-α) and the effects of AMD3100 (bicyclam) on both CXCR4 and TNF-α expression among these cells was investigated. RT-PCR showed an increase in CXCR4 mRNA (7.5-fold for MG63 and 4.0-fold for SaOs-2 cells) among cells co-cultured with metal alloy particles. Western blotting showed a time-dependent increase in protein expression of CXCR4. The attempted blockade of CXCR4 by its known competitive receptor agonist AMD3100 led to a significant inhibition TNF-α mRNA expression. Immunohistochemistry showed CXCR4 positivity among patients with failed metal-on-metal hip replacements and radiographic evidence of osteolysis. Our data collectively suggest that the CXCR4 chemokine is upregulated in a dose- and time-dependent manner in the presence of metallic wear debris.

The induction of CXCR4 expression in human osteoblast-like cells (MG63) by CoCr particles is regulated by the PLC-DAG-PKC pathway.

BACKGROUND Osteolysis which leads to aseptic loosening of implants is a fundamental problem in joint replacement surgery (arthroplasty) and the leading cause for implant failure and revision surgery. Metal (CoCr) particles separated from implants by wear cause osteolysis and the failure of orthopedic implants, but the molecular mechanism is not clear. The chemokine receptor CXCR4 has been shown to play a pivotal role in periprosthetic osteolysis. The aim of this study was to determine which signal transduction pathway (PLC-DAG-PKC or MAPK/ERK) induces CXCR4 expression in osteoblast-like cells (MG63) cells. METHODS MG63 and Jurkat cells were stimulated with different amounts of particles (107 , 106 , and 105 ) for different time periods (30 min to 24 h), in the presence and absence of specific inhibitors (chelerythrine for the PLC-DAG-PKC pathway and PD98059 for the MAPK/ERK pathway). The expression of CXCR4-specific mRNA was determined by real-time polymerase chain reaction (PCR), and the PKC activity was measured by Western Blot using an antibody specific for PKC-related phosphorylation. RESULTS Real-time PCR data showed that CXCR4 mRNA expression in MG63 cells induced by CoCr particles was significantly diminished by the PKC-specific inhibitor chelerythrine. This effect was not observed with the MAPK/ERK inhibitor PD98059. The involvement of PKC was also confirmed by an intensified phosphorylation pattern after stimulation with CoCr particles. In Jurkat cells, none of the inhibitors exhibited any effect. CONCLUSION The induction of CXCR4-specific mRNA expression in MG63 cells after stimulation with CoCr particles is regulated by the PLC-DAG-PKC pathway and not by the MAPK/ERK pathway.

The effects of cobalt and chromium ions on transforming growth factor-beta patterns and mineralization in human osteoblast-like MG63 and SaOs-2 cells: THE EFFECTS OF COBALT AND CHROMIUM IONS

Bone homeostasis, the balance of bone formation and resorption is affected by numerous influences, such as, hormones, inflammation, mechanical load, and external stimuli. The transforming growth factor-beta (TGF-β), which exists in three isoforms in humans, is a major factor in the maintenance of this balance by regulating osteoblast and osteoclast maturation, development, and function. In artificial joint replacements, release of particles or ions from arthroplasties may exert local effects on the periprosthetic tissue and modulate the expression of bone specific genes and functions. Therefore, the influence of cobalt (II) and chromium (III) ions on the expression levels of the three TGF-β isoforms in human osteosarcoma cell lines MG63 and SaOs-2 was analyzed and the impact on mineralization was studied. The osteosarcoma cell lines expressed all three TGF-β isoforms, with TGF-β1 being the most abundant isoform. A dose dependent reduction of all TGF-β isoforms by Co2+ ions was observed, the strongest effect was found on TGF-β2. The effect was lesser pronounced in SaOs-2 cells. However, the Cr3+ ions had no significant effect on the expression of all TGF-β isoforms. In contrast, Co2+ ions in a concentration range of 50-250 µM did not impair the mineralization, but Cr3+ exerted a strong inhibitory effect on the mineralization in a dose dependent fashion. These data suggest that the influence of cobalt ions on bone homeostasis may in part result from the inhibitory effect on the transcription of the bone regulating cytokines TGF-β1-3 whereas the chromium ions affect the process of mineralization.

Differential Effect of Cobalt and Chromium Ions as Well as CoCr Particles on the Expression of Osteogenic Markers and Osteoblast Function

The balance of bone formation and resorption is the result of a regulated crosstalk between osteoblasts, osteoclasts, and osteocytes. Inflammation, mechanical load, and external stimuli modulate this system. Exposure of bone cells to metal ions or wear particles are thought to cause osteolysis via activation of osteoclasts and inhibition of osteoblast activity. Co2+ ions have been shown to impair osteoblast function and the expression of the three transforming growth factor (TGF)-β isoforms. The current study was performed to analyze how Co2+ and Cr3+ influence the expression, proliferation, and migration profile of osteoblast-like cells. The influence of Co2+, Cr3+, and CoCr particles on gene expression was analyzed using an osteogenesis PCR Array. The expression of different members of the TGF-β signaling cascade were down-regulated by Co2+, as well as several TGF-β regulated collagens, however, Cr3+ had no effect. CoCr particles partially affected similar genes as the Co2+treatment. Total collagen production of Co2+ treated osteoblasts was reduced, which can be explained by the reduced expression levels of various collagens. While proliferation of MG63 cells appears unaffected by Co2+, the migration capacity was impaired. Our data may improve the knowledge of changes in gene expression patterns, and the proliferation and migration effects caused by artificial materials.

Effects of TIMP-1 and TIMP-3 gene transfer on invasiveness, proliferation and apoptosis of rheumatoid arthritis (RA) synovial fibroblasts (RA-SF)

TIMPs play a key role in counter balancing the action of MMPs and have been associated with cell proliferation, inhibition of angiogenesis and induction of apoptosis. Here, we investigated the effects of TIMP-1 and TIMP-3 gene transfer on cartilage invasion, proliferation and apoptosis of RA-SF. RA-SF were transduced with an adenoviral vector expressing human TIMP-1 (AdTIMP-1) or TIMP-3 (AdTIMP-3). Transduction efficacy was assessed by LacZ staining of RA-SF that were transduced with an adenoviral β-galactosidase construct. Untransduced and mock transduced RA-SF were used as controls. TIMP-1 was measured by ELISA in the culture supernatants of AdTIMP-1 transduced and mock transduced cells every 10 days until 60 days after transduction. Proliferation was assessed by 3H-thymidine incorporation, and the rate of spontaneous apoptosis as well as FasL induced cell death was determined by a histon fragmentation assay. AdTIMP-1 and AdTIMP-3 transduced RA-SF and control RA-SF were co-implanted with human articular cartilage under the renal capsule of SCID mice for 60 days and their invasiveness was evaluated on paraffin sections using a semiquantitative score. Transduction efficacy was 67%, and TIMP-1 levels in the supernatants of AdTIMP-1 transduced cells were 51.5 ± 6.5 μg/ml as compared to 8.7 ± 3.4 μg/ml in the mock transduced cells. These levels of TIMP expression were maintained for at least 60 days. AdTIMP-1 and AdTIMP-3 gene transfer resulted in an inhibition of proliferation (35% and 40% vs. mock, respectively; P < 0.05). Transduction of RA-SF with AdTIMP-3 but not TIMP-1 increased spontaneous apoptosis (+24%; vs. mock, P < 0.05) as well as susceptibility to FasL-induced cell death (+23% vs. mock, P < 0.05). In the SCID mouse model, untransduced and mock transduced RA.SF deeply invaded the cartilage (scores: 2.5 ± 0.2 and 3.2 respectively). In the AdTIMP-1 and AdTIMP-3 transduced RA-SF, invasion was inhibited clearly (scores 0.9 ± 0.4 and 1.2 ± 0.2 respectively) Both AdTIMP-1 and AdTIMP-3 gene transfer inhibit proliferation of RA-SF and reduce cartilage invasion. In contrast to TIMP-1, adenoviral gene transfer with TIMP-3. has a strong pro-apoptotic effect on RA-SF and facilitates Fas mediated cell death. These results indicate that gene transfer of TIMPs may be a useful approach to inhibit joint destruction in RA.