Biomedical subjects

Wilhelm K Aicher

Publications and source records attributed to Wilhelm K Aicher.

13 recordsLinked to original sources

PubMed2007

Nanostructured thermosensitive polymers with radical scavenging ability.

The thermosensitive [60]fullerene end-capped poly(N-isopropylacrylamide) was successfully synthesized by the reaction of C(60) with dithiobenzoate-terminated poly(N-isopropylacrylamide), which was prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization in the presence of azobisisobutyronitrile (AIBN). Its structure was determined by FTIR, UV/Vis, and carbon and proton NMR spectroscopy as well as by size exclusion chromatography (SEC). The novel fullerenated polymer retained the thermosensitivity of poly(N-isopropylacrylamide). Moreover, it is soluble in water and most of the common organic solvents. Interestingly, it was able to form nanoparticle clusters in methanol and exhibited significant radical scavenging ability in cell viability and metabolic activity tests with fibroblasts and NOR-3 radicals.

Acrylic Resins↗

PubMed2006

Attachment to laminin-111 facilitates transforming growth factor beta-induced expression of matrix metalloproteinase-3 in synovial fibroblasts.

BACKGROUND: In the synovial membrane of patients with rheumatoid arthritis (RA), a strong expression of laminins and matrix degrading proteases was reported. AIM: 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 beta (TGFbeta). METHODS: SFs were seeded in laminin-coated flasks and activated by addition of TGFbeta. 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. RESULTS: Attachment of SF to LM-111 did not activate the expression of MMPs, but addition of TGFbeta induced a fivefold higher expression of MMP-3. Incubation of SF on LM-111 in the presence of TGFbeta 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 TGFbeta 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. CONCLUSION: 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 TGFbeta-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)1beta or tumour necrosis factor (TNF)alpha.

Aged↗

PubMed2006

The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression.

BACKGROUND/AIMS: To elucidate the influence and mode of action of HMR1726 (the active metabolite of leflunomide) on TNF-alpha and IL-17 activated metalloproteinases expression in synoviocytes. METHODS: Synovial fibroblasts from RA and OA patients were stimulated with both cytokines and altered gene expression in the presence or absence of leflunomide was detected by microarray analyses and quantitative RT-PCR. Protein expression was detected by western blotting and commercial ELISAs. RESULTS: Microarray analyses revealed that the addition of HMR1726 (50 microM) to TNF-alpha and IL-17- stimulated synoviocytes induced gene expression of metallo-proteinases, especially MMP-1 and -3 in comparison to activated synoviocytes in the absence of leflunomide. To confirm these data, we examined the influence of different concentrations of HMR1726 in synoviocytes from further 5 OA and 7 RA patients by quantitative PCR. HMR1726 gradually induced MMP-1 and MMP-3 gene expression in a dose-dosedependent manner. Similar results were observed on protein levels. Examination of signal transduction pathways participating in the regulation of leflunomideinduced MMPs expression showed that the mechanism underlying activation of MMP-1 is in part p38- and activation of MMP-3 was MEK1/2- dependent. CONCLUSION: Leflunomide was not able to abolish expression of metallo-proteinases in synoviocytes activated with TNF-a and IL-17.

Adult↗

PubMed2005

Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. METHODS: Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. RESULTS: MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). CONCLUSION: The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

Animals↗

PubMed2004

Ribozymes that inhibit the production of matrix metalloproteinase 1 reduce the invasiveness of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To investigate whether retroviral gene transfer of ribozymes targeting matrix metalloproteinase 1 (MMP-1) inhibits the production of MMP-1 in rheumatoid arthritis synovial fibroblasts (RASFs) and reduces the invasiveness of these cells in vivo. METHODS: MMP-1-specific ribozymes (RzMMP-1) were designed and cloned into the pLNSX retroviral vector. Cleavage of MMP-1 was determined in vitro, and the most effective ribozyme was selected for further investigation. RASFs were transduced with replication-deficient viruses carrying RzMMP-1 or with empty viruses (mock). Quantitative polymerase chain reaction with cleavage site-spanning fluorescent probes was used to measure the levels of MMP-1, MMP-9, and MMP-13 messenger RNA. In addition, protein levels of MMP-1 in cell culture supernatants were determined by enzyme linked immunosorbent assay. The effects of stimulation with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFalpha) on the production of MMP-1 were assessed accordingly. The invasiveness of RzMMP-1-transduced, mock-transduced, and untransduced RASFs was analyzed in the SCID mouse in vivo model of RA. RESULTS: Transduction of RASFs with RzMMP-1 significantly decreased the production of MMP-1 in RASFs without affecting other MMPs, such as MMP-9 and MMP-13. RzMMP-1 not only reduced the spontaneous production of MMP-1, but also prevented the LPS- and TNFalpha-induced increase in MMP-1 production. Inhibition of MMP-1 was maintained for at least 2 months and was accompanied by a significant reduction of the invasiveness of RASFs in the SCID mouse model of RA. CONCLUSION: Intracellular expression of ribozymes constitutes a feasible tool for inhibiting the production of matrix-degrading enzymes. Inhibition of MMP-1 alone results in a significant reduction of cartilage invasion by RASFs.

Animals↗

PubMed2004

Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads.

OBJECTIVE: For autologous chondrocyte transplantation (ACT) chondrocytes are expanded in vitro. During expansion these cells may dedifferentiate. This change in phenotype is characterized by a raised expression of type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of hyaline cartilage, we investigated if the growth factor bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures and in three-dimensional culture systems. DESIGN: Chondrocytes from articular knee cartilage of 11 individuals (average age: 39.8 years) with no history of joint disease were isolated and seeded either in monolayer cultures or embedded in alginate beads in presence or absence of human recombinant BMP-2 (hr-BMP-2). Then, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA. RESULTS: Addition of BMP-2 to chondrocytes expanded in two-dimensional (2D) cultures during the first subculture (P1) had no effect on mRNA amounts encoding type II collagen and interleukin-1beta (IL-1beta). In contrast, seeding chondrocytes in three-dimensional (3D) alginate cultures raised type II collagen expression significantly and addition of BMP-2 enhanced this effect. CONCLUSIONS: We conclude that chondrocytes during expansion for ACT may benefit from BMP-2 activation only when seeded in an appropriate 3D culture system.

Adult↗

PubMed2004

Synovial fibroblasts from rheumatoid arthritis patients differ in their regulation of IL-16 gene activity in comparison to osteoarthritis fibroblasts.

BACKGROUND: In rheumatoid arthritis (RA), synovial fibroblast-like cells (SF) contribute significantly to articular inflammation. They express distinct patterns of genes associated with cell proliferation and differentiation and elevated levels of cytokines and chemoattractant factors, including IL-16. Here we investigated pathways regulating IL-16 expression in fibroblasts from RA patients in comparison to fibroblasts from osteoarthritis (OA) patients. METHODS: Fibroblasts were isolated from dermal and articular biopsies, expanded and pathways of IL-16 induction were investigated by real time quantitative RT/PCR, immuno blot and ELISA. RESULTS: Stimulation of cAMP dependent signal transduction by forskolin induced prominent IL-16 RT/PCR signals in OA-DF and OA-SF. In contrast, in RA-DF and RA-SF staurosporine significantly augmented IL-16 RT/PCR signals, but forskolin induced less IL-16 transcript amounts. Activation of protein kinase C by PMA induced a significant IL-16 response only in RA-SF. Addition of IL-1beta or TNF-alpha did not upregulate IL-16 mRNA but secretion of the mature IL-16 cytokine was activated in serum starved cells in presence of IL-1beta. CONCLUSION: Our results suggest that RA fibroblasts differ from OA fibroblasts with respect to their sensitivities to kinase/phospatase signal transduction pathways. The enhanced expression of IL-16 in the synovial membrane early in RA vs OA may be associated in part with these distinct signaling responses.

Arthritis, Rheumatoid↗

PubMed2003

Interleukin-18 is regulated by G protein pathways and protein kinase signals in human fibroblasts.

Interleukin-18 (IL-18) is a member of the IL-1 cytokine family and has proinflammatory activity. It has been detected in osteoarthritic (OA) and at higher levels in rheumatoid arthritic (RA) synovial tissue. Therefore we investigated major signal transduction pathways for their contribution to IL-18 expression. Here we report that cyclic adenosine monophosphate reduced and ionomycin increased IL-18 mRNA in RA synovial fibroblasts (SF) but not in OA SF. Moreover, activation of G-proteins by Mas-7 augmented IL-18 reverse transcriptase polymerase chain reaction signals in OA SF but not in RA SF. Specific protein kinase C activator phorbol myristate acetate reduced transcription and secretion of IL-18 in RA SF and OA SF. Staurosporine changed spontaneous IL-18 mRNA levels and increased the secretion of IL-18 protein. We conclude that G-protein activation and protein kinase C activation might partially be responsible for elevated IL-18 levels during RA.

Arthritis, Rheumatoid↗

PubMed2003

Osteoclast-independent bone resorption by fibroblast-like cells.

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.

Adult↗

PubMed2003

Transcription factor early growth response 1 activity up-regulates expression of tissue inhibitor of metalloproteinases 1 in human synovial fibroblasts.

OBJECTIVE: To investigate the regulatory potential of early growth response 1 (Egr-1) on tissue inhibitor of metalloproteinases 1 (TIMP-1) expression in synovial fibroblasts. METHODS: Egr-1 and TIMP-1 transcripts were detected by in situ hybridization in synovial tissue. Egr-1-regulated TIMP expression was studied in immortalized fibroblast lines using gel retardation assays, RNase protection analysis, reporter gene studies using the human TIMP-1 promoter, and by enzyme-linked immunosorbent assay. RESULTS: TIMP-1 and Egr-1 were coexpressed in synovial fibroblasts of inflamed joints, and Egr-1 activated the expression of TIMP-1. Egr-1 binding to a recognition sequence in the TIMP-1 promoter was demonstrated in gel retardation and reporter gene assays. Since the same DNA sequence was also recognized by the transcription factor Sp-1, our results suggest that the expression of TIMP-1 in synovial fibroblasts may be differentially regulated by Egr-1 and Sp-1. In addition, fibroblasts expressing Egr-1 at high levels were found to express increased levels of TIMP-2 and TIMP-3 messenger RNA. CONCLUSION: The enhanced expression of Egr-1 may regulate the activity of matrix metalloproteinases in synovial fibroblasts by enhancing the expression of the TIMP-1, -2, and -3 genes.

Arthritis, Rheumatoid↗

PubMed2003

Cartilage destruction mediated by synovial fibroblasts does not depend on proliferation in rheumatoid arthritis.

The aim of the study was to investigate the relationship between invasion and proliferation in rheumatoid arthritis synovial fibroblasts (RASFs). In vitro, RASFs, normal synovial fibroblasts (NSFs), and RASFs transformed with SV40 T-antigen (RASF(SV40)) were analyzed for the expression of cell surface markers (Thy1, VCAM-1, ICAM-1, CD40, CD44) and their proliferation by flow cytometry. Furthermore, colony-forming unit assays were performed and the expression of matrix metalloproteinases (MMP)-14 and cathepsin K mRNA were determined by real-time polymerase chain reaction. In vivo, in the severe combined immunodeficiency (SCID) mouse co-implantation model, RASFs, NSFs, and RASF(SV40) were tested for cartilage invasion, cellular density, and for their expression of the cell cycle-associated protein Ki67. In the SCID mouse co-implantation model, RASFs invaded significantly stronger into the cartilage than NSFs and RASF(SV40). Of note, RASF(SV40) cells formed tumor-like tissues, and the cellular density adjacent to the cartilage was significantly higher than in RASFs or NSFs. In turn, the proliferation marker Ki67 was strongly expressed in the SV40-transformed synoviocytes in SCID mice, but not in RASFs, and specifically not at sites of cartilage invasion. Using the colony-forming unit assay, RASFs and NSFs did not form colonies, whereas RASF(SV40) lost contact inhibition. In vitro, the proliferative rate of RASFs was low (4.3% S phase) in contrast to RASF(SV40) (24.4%). Expression of VCAM-1 was significantly higher, whereas of ICAM-1 was significantly lower, in RASFs than in RASF(SV40). CD40 was significantly stronger expressed in RASF(SV40), whereas CD44 and AS02 were present at the same degree in almost all synoviocytes. Expression of cathepsin K and matrix metalloproteinase-14 mRNA was significantly higher in RASFs than in the RASF(SV40). Our data demonstrate clearly that invasion of cartilage is mediated by activated RASFs characterized by increased expression of adhesion molecules, matrix-degrading enzymes, but does not depend on cellular proliferation, suggesting the dissociation of invasion and proliferation in RASFs.

Animals↗

PubMed2002

Transcription factor Egr-1 activates collagen expression in immortalized fibroblasts or fibrosarcoma cells.

Synovial fibroblasts from rheumatoid arthritis patients express elevated levels of the transcription factor Egr-1. The metabolic consequences of Egr-1 overexpression in fibroblasts are not known in detail. Therefore we searched for gene products that are differentially expressed in Egr-1(high) versus Egr-1(low) fibroblasts. Immortalized synovial fibroblasts were transfected with two different Egr-1 expression vectors. Expression of recombinant Egr-1 was confirmed by RT-PCR and immunoblots. Random arbitrarily primed PCR revealed that Egr-1 induces enhanced transcription levels of the alpha1 chain of type I collagen. Increased expression of the alpha2 (I) chain could also be observed. We found enhanced levels of type I collagen propeptide in supernatants and stronger signals of alpha2 (I) protein in extracts of the Egr-1(high) expressing clone versus controls. Additionally, Egr-1 was transiently expressed in fibrosarcoma cells. These cells showed a pronounced elevation of type I collagen (alpha1) transcripts as well. Moreover, we could demonstrate that Egr-1 induces transcription of other genes including type II collagen (alpha1) and plateled-derived growth factor beta1. These data suggest that upregulation of Egr-1 might contribute tofibrosis observed in rheumatoid arthritis synovium by activation of genes encoding the alpha1 and alpha2 chains of type I collagen.

Cloning, Molecular↗

PubMedDate not supplied

Supramolecular conjugates of carbon nanotubes and DNA by a solid-state reaction.

DNA-wrapped nanotubes of both multiwalled and single-walled carbon nanotubes were obtained by a solid-state mechanochemical reaction. Scanning electron microscopic images show that the nanotubes were cut into shorter lengths and were fully covered with DNA, which was further confirmed by fluorescence microscopy. This resulted in a high aqueous solubility of the products with a stability of >6 months. The results show that nanotubes were cut also with uniform distribution where >90% of the multiwalled products were 500 nm to 3 microm and 80% of the single-walled products were 250 nm to 1 microm in length, respectively. UV-vis spectra and gel electrophorosis show that the DNA in the product is intact. This facile technique for obtaining supramolecularly masked, water-soluble carbon nanotubes by a solid-state reaction has a great potential for both biological and nonbiological applications of nanotubes.

Antigens↗