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Biomedical subjects

W K Aicher

Publications and source records attributed to W K Aicher.

At least 19 recordsLinked to original sources
PubMed2005

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Adult↗
PubMed2004

[Frequency of terminally differentiated fibroblasts in the synovial membrane of rheumatoid arthritis patients].

The metabolic activation of synovial fibroblasts (SF) and their expression of matrix degrading enzymes and inflammatory cytokines contributes to the pathology of rheumatoid arthritis (RA). It is remarkable that SF of RA patients do not proliferate at higher rates when compared to SF of other patients, but they are resistant to apotposis inducing signals. The chronic inflammation in RA causes fibrosis of the synovial tissue and fibrosis has been associated with terminal differentiation. Therefore we investigated if there are increased numbers of terminally differentiated fibroblasts in the RA synovium and if there is a correlation between terminal differentiation of SF and increased levels of expression of interleukins and matrix metalloproteinases. We analyzed specimen of four RA patients, two patients with osteoarthritis (OA) and two healthy donors suffering from joint injuries. By use of RT-PCR techniques we examined mRNA expression of two genes in SF which are associated with terminal differentiation, p16INK4a and p21-cip. In addition, we labelled differentiated fibroblasts using the SA-beta-galaktosidase assay and investigated differences in protein expression patterns of factor PIVa and the tropomyosin 1 and 2 molecules. We report that the number of terminally differentiated fibrolasts are not increased in the synovial membrane of RA patients. On the contrary we show that the synovia of the much younger patients has higher levels of terminally differentiated fibroblasts. Consequently, the fibrosis of synovial tissues in RA patients at later stages of disorder is not associated with proliferation and differentiation of the fibroblasts but rather a consequence of chronic inflammation.

Adult↗
PubMed2004

Metabolic activation stimulates acid secretion and expression of matrix degrading proteases in human osteoblasts.

BACKGROUND: Both cellular and matrix components of healthy bone are permanently renewed in a balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases. Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix proteins. OBJECTIVES: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to proton secretion expressed on osteoblasts. METHODS: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer. RESULTS: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is associated partially with proton pump ATPases. CONCLUSION: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone degradation.

Biotransformation↗
PubMed2003

p53 in rheumatoid arthritis synovial fibroblasts at sites of invasion.

OBJECTIVE: To analyse the functional response of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 modulates the destructive process of RASF. METHODS: RASF and controls grown on chamber slides were either directly examined with DO7 anti-p53 antibodies by immunofluorescence or irradiated with 10 Gy x rays and analysed time dependently for the expression of p53. The percentage of positive cells was evaluated by a quantitative scoring system. RASF and normal (N) SF cultured in vitro were co-implanted with human cartilage in SCID mice for 60 days. Consecutively, the invasion score was evaluated, and the number of p53 positive cells was determined at the sites of invasion by immunohistochemistry. In addition, synovial tissues from RA, osteoarthritis, and normal synovia were stained with DO7 antibodies. RESULTS: In vitro the rate of expression of p53 in RASF was low (<5%), but transiently inducible by ionising irradiation (50%). In vitro low p53 expressing RASF disclosed, when invading articular cartilage, a nuclear p53 signal in 20% of the cells, indicating the induction of p53 in a distinct population of RASF during the invasive process. CONCLUSIONS: These data suggest an inductive p53 response at sites of cartilage invasion during the destructive process driven by activated RASF.

Animals↗
PubMed2000

Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis.

OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA-expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/-SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF.

Animals↗
PubMed2000

Biocompatibility correlation of polymeric materials using human osteosarcoma cells.

Metal implants are the preferred materials to generate articular prostheses, plates, or bone pegs in orthopedic surgery. Although titanium and titanium alloys show a relatively good biocompatibility, clinical experience revealed that coating of the metallic implant surface may increase the biocompatibility. In a search for optimum bone implant surfaces, we determined polarity and contact angle parameters of a variety of polymers and substances and correlated the findings in a biocompatibility assay using an in vitro bone cell model. We report that an optimum adherence of SAOS-2 cells to such surfaces and a good vitality for polymers are characterized by water-based contact angles of 80 degrees and 20 degrees for advancing and receding probes, respectively.

Biocompatible Materials↗
PubMed2000

Metabolic activation stimulates acid production in synovial fibroblasts.

OBJECTIVE: In rheumatoid arthritis (RA), synovial fibroblasts express proteases such as collagenases or cathepsins and inflammatory cytokines at elevated levels and so contribute to the inflammatory degradation process. Extracellular matrix degradation and cathepsin activity is dependent upon the presence of an acidic milieu. We examined whether activated synovial fibroblasts secrete acidic components. METHODS: Synovial fibroblasts were isolated and immortalized to study the mechanisms of metabolic activation. Naïve and immortalized fibroblasts were activated with different cytokines. The responses were investigated by immunoblot to detect Egr-1 and by a cytosensor microphysiometer analysis to evaluate acid secretion. Basic gene expression patterns were investigated in naïve and immortalized cells by RT-PCR analysis. RESULTS: We found RA synovial fibroblasts respond to different cytokines associated with the pathomechanisms of RA including interleukin 1, basic fibroblast growth factor, platelet derived growth factor, and tumor necrosis factor-alpha, with metabolic activation and enhanced secretion of acidic components. In addition, naive and SV40 TAg immortalized fibroblasts rapidly release acidic components after stimulation with phorbol ester or ionomycin as well. CONCLUSION: Activated synovial fibroblasts not only express inflammatory cytokines and matrix degrading proteases that are associated with the pathomechanisms of RA, but upon stimulation may release acidic components that lower pH and consequently enhance cathepsin activity and collagen solubilization.

Arthritis, Rheumatoid↗
PubMed1999

Efficient generation of transgenic BALB/c mice using BALB/c embryonic stem cells.

BALB/c is one of the most widely used and best characterized mouse strains in immunology. For various applications, it is necessary to generate BALB/c transgenic mice. However, using the conventional microinjection technique it is extremely inefficient to produce transgenic BALB/c mice since the one-cell stage BALB/c embryos are highly vulnerable to pronuclear DNA microinjection. To overcome this problem, we have investigated the generation of Egr-1 (early growth response gene) transgenic mice via the transfection of BALB/c embryonic stem cells. Transfectants carrying Egr-1 constructs comprising either the immunoglobulin heavy chain or the MHC class II promoter/enhancer system were injected into C57BL/6 host blastocysts resulting in chimeric mice. For both type of expression vectors, transgenic offspring of the germline chimeras expressed recombinant Egr-1 in lymphoid tissues containing B cells. This demonstrates the successful generation of Egr-1 transgenic BALB/c mice using transfected ES cell.

Animals↗
PubMed1999

Analysis of functional elements in the human Egr-1 gene promoter.

The early growth response (Egr)-1 gene encoding a zinc-finger transcription factor is transiently induced in many different cell types upon various differentiation signals. However, in synovial fibroblasts of rheumatoid arthritis patients, Egr-1 is constitutively expressed at high levels, and several genes with Egr-1 binding sites in their promoter regions have been associated with disease progression of RA. We analyzed the control of Egr-1 transcription by characterizing those regulatory elements in the Egr-1 promoter that induce Egr-1 expression in fibroblasts. Using reporter gene assays and deletion mutants of the Egr-1 promoter we could demonstrate that Egr-1 transcription is mainly activated by a single serum response element, whereas other transcription factor binding sites, including binding sites for AP-1 or Egr-1, were found to play a minor role. Furthermore, we identified a novel regulatory element in the human Egr-1 promoter similar to a NF kappa-B binding site. Deletion of this element enhanced Egr-1 promoter activity in 3T3 but not in L929 fibroblasts. Stimulation by phorbolester induced only transient Egr-1 expression in 3T3 fibroblasts but a extended expression of Egr-1 in L929 cells. These data suggest that in fibroblasts the most proximal serum response element in the Egr-1 promoter represents the major activation site, whereas binding of the NFkB-like factor may serve as negative regulatory signal for Egr-1 transcription in fibroblasts.

Arthritis, Rheumatoid↗
PubMed1999

Serum response elements activate and cAMP responsive elements inhibit expression of transcription factor Egr-1 in synovial fibroblasts of rheumatoid arthritis patients.

Analyzing the induction kinetics and promoter elements regulating the expression of the transcription factor Egr-1, we found elevated levels of Egr-1-encoding mRNA in synovial fibroblasts of rheumatoid arthritis (RA) patients when compared to controls. By contrast, synovial lymphocytes and macrophages do not show an elevated Egr-1 transcription. Therefore, the overexpression of Egr-1 may serve as a diagnostic marker to characterize synovial fibroblasts of RA patients. To study the regulatory mechanisms controlling Egr-1 expression we analyzed the function of transcription factor binding sites located in the Egr-1 promoter. Individual transcription factor binding sites within the Egr-1 promoter were specifically mutated and Egr-1 promoter activity was tested using reporter gene constructs. Our experiments demonstrate that serum response elements are the main positive regulators and binding to a cAMP responsive element represents the major negative regulator for Egr-1 expression in synovial fibroblasts. In addition, we functionally defined a new element, which was not yet described in the human Egr-1 promoter and which serves as a second negative regulatory element for Egr-1 expression. Therefore increased serum response factor activity or failure of Egr-1 repressing signals may account for Egr-1 overexpression in RA synovial fibroblasts.

Arthritis, Rheumatoid↗
PubMed1998

Interleukin-16, produced by synovial fibroblasts, mediates chemoattraction for CD4+ T lymphocytes in rheumatoid arthritis.

The massive infiltration of synovium with CD4+ T cells during the course of rheumatoid arthritis (RA) implies the expression of chemoattractant factors by resident synovial cells. Therefore, we analyzed the expression of IL-16, a potent chemoattractant for CD4+ T cells, to account for the accumulation of CD4+ T cells in RA. Indeed, IL-16 was found to be significantly elevated in synovial fluid (SF) from patients with RA as compared to non-RA arthritis (p < 0.001), osteoarthritis (p < 0.001) and controls (p < 0.001). Chemotaxis studies showed IL-16 to contribute to the strong chemotactic activities of RA-SF. In situ hybridization (ISH) revealed IL-16 mRNA-expressing cells located within the lining layer of rheumatoid synovial tissue. In the sublining area, only scattered IL-16 transcript-positive cells could be detected, mainly adjacent to blood vessels. By a double-labeling technique, combining ISH for IL-16 mRNA and immunohistochemistry for CD68, synovial fibroblast-like, CD68-negative cells were identified as a major source of IL-16 mRNA within RA synovium. This study demonstrates that synovial fibroblasts produce IL-16 in RA and thus mediate chemoattraction of CD4+ cells into synovial tissue.

Adult↗
PubMed1998

TNF-alpha induces the transcription factor Egr-1, pro-inflammatory cytokines and cell proliferation in human skin fibroblasts and synovial lining cells.

In rheumatoid arthritis (RA) cell proliferation and altered metalloproteinase expression of synovial lining cells are associated with increased levels of TNF-alpha in the rheumatoid joint. We previously showed that synoviocytes of RA patients express high levels of the transcription factor Egr-1. Here we report that TNF-alpha is capable of inducing high Egr-1 mRNA levels in human skin fibroblasts and in synoviocytes from both, RA and reactive arthritis patients. Moreover, we observed in vitro a marked increase in fibroblast proliferation, a loss of growth inhibition by cell-to-cell contact with pannus-like cell growth and an altered cytokine expression pattern when synoviocytes were cultured in presence of TNF-alpha.

Cell Division↗
PubMed1998

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids.

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.

Arthritis, Rheumatoid↗
PubMed1998

Cysteine proteinase cathepsin K mRNA is expressed in synovium of patients with rheumatoid arthritis and is detected at sites of synovial bone destruction.

OBJECTIVE: Cysteine proteinases B and L have been shown to be involved in matrix degradation of joints in patients with rheumatoid arthritis (RA). Since the cysteine proteinase cathepsin K is assumed to play a pivotal role in osteoclast mediated bone resorption, we investigated the expression of cathepsin K in RA joints. METHODS: We studied 10 RA and 4 normal synovial specimens and 5 articular heads with RA lesions by in situ hybridization, applying specific riboprobes for cathepsin K, human collagen type I, and cathepsin B. Antibodies against monocyte/macrophage associated CD68 antigen were applied in immunohistochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay (RPA) were performed on 4 RA, 1 normal, and 1 immortalized normal fibroblast cultures. RESULTS: Cathepsin K mRNA expression was upregulated in RA synovium compared to normal synovium. Cathepsin K mRNA was expressed mainly by synovial fibroblasts. These data were confirmed by RT-PCR and RPA. In RA articular heads, cathepsin K mRNA was detected at sites where synovium attached and invaded underlying bone. The cells at these sites represented collagen type I and cathepsin B mRNA expressing fibroblasts as well as CD68+ macrophages and giant cells. In addition, a distinct expression of cathepsin K mRNA was also detected around lymphocytic infiltrates in RA synovium. CONCLUSION: The data indicate that cathepsin K is not only expressed by osteoclasts but also by synovial fibroblasts, and suggest that cathepsin K contributes to bone destruction mediated by RA synovial cells. The expression of cathepsin K around lymphocytic infiltrates suggests further to facilitate the movement of mononuclear cells through the perivascular interstitial matrix and thereby contribute to interstitial matrix turnover.

Adult↗
PubMed1997

Transcription factor Egr-1 activity down-regulates Fas and CD23 expression in B cells.

Activation of mature B cells via Ag receptor cross-linking induces transient expression of the transcription factor Egr-1. Although the activating signals leading to Egr-1 induction have been studied extensively, little is known about the genes that are placed further downstream within this activation cascade and that are transcriptionally regulated by Egr-1. To identify such target genes, we established Egr-1-overexpressing transfectants from the murine B cell line K46 and from human Ramos B cells. All clones derived from K46 B cells showed increased expression of CD44. Most interestingly, expression of CD95 (Fas/Apo-1) and of CD23 was down-regulated in all K46 transfectants. As a consequence, they became resistant to apoptosis induced by anti-CD95 Ab treatment. Similarly, the Egr-1-expressing Ramos cells showed reduced levels of CD95 expression. Thus, Egr-1 seems to control the expression of downstream target genes not only as a transcriptional activator, but also as a repressor molecule. In B cells, Egr-1 therefore plays a critical role in integrating the short-lived signal delivered by triggering of the Ag receptor into phenotypic changes, including repression of CD95 and CD23 transcription.

Apoptosis↗
PubMed1997

Characterization of SV40T antigen immortalized human synovial fibroblasts: maintained expression patterns of EGR-1, HLA-DR and some surface receptors.

In rheumatoid arthritis (RA) synovial fibroblasts are activated by growth factors and cytokines to proliferate and to express matrix-degrading proteases and pro-inflammatory cytokines. This contributes to cartilage degradation and joint destruction. To analyse the parameters that lead to activation of synovial fibroblasts, we established a stable human synoviocyte line (K4IM) from a healthy donor by immortalization with SV40 T antigen (TAg). Characterizing the phenotype of the immortalized K4IM cells, we found that they maintained CD44, CD54 (intercellular adhesion molecule; ICAM-1) and CD95 (Fas) expression, but lost the expression of CD106 (vascular cell adhesion molecule 1; VCAM-1) and the receptors for interleukin 1 (IL-1) and platelet-derived growth factor (PDGF). We also monitored normal expression kinetics of transcription factor Egr-1 upon activation with tumor necrosis factor alpha (TNF-alpha) or synovial fluid from RA patients. In addition, we showed that HLA-DR expression could still be upregulated by recombinant interferon gamma (rINF-gamma). The immortalized K4IM cell line therefore represents a valuable and unique tool to study mechanisms that induce or maintain synoviocyte activation.

Antigens, Viral, Tumor↗
PubMed1997

Measurement of transcription factor c-fos and EGR-1 mRNA transcription levels in synovial tissue by quantitative RT-PCR.

The transcription factors Fos and EGR-1 are known to be involved in the regulation of the transcription of metalloproteinases and their specific inhibitors. Since the overexpression of metalloproteinases is responsible for the matrix degradation in rheumatoid arthritis (RA), exact analysis of transcription levels of c-fos and EGR-1 ex vivo may serve to monitor progression or remission of the disease activity in RA. Here we report on a method based on a quantitative reverse transcription polymerase chain reaction (RT-PCR) for rapid estimation of the transcription levels of the immediate early genes c-fos and EGR-1. Coamplification with suitable internal standards, easily generated by the use of hybrid primers, allows us to semiquantitatively measure c-fos and EGR-1 induction levels in low numbers of cultured cells or very small tissue samples obtained by synovial biopsy. The sensitivity of the method was 3.5 pg/ml for c-fos- and 10 pg/ml for EGR-1-specific cDNAs.

Arthritis, Rheumatoid↗