Dirk Wernicke

CHARACTERIZATION OF COLLAGENASE 3 (MATRIX METALLOPROTEINASE 13) MESSENGER RNA EXPRESSION IN THE SYNOVIAL MEMBRANE AND SYNOVIAL FIBROBLASTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

OBJECTIVE To study the localization and cell type-specific expression of collagenase 3 messenger RNA (mRNA) in the synovial membrane, its regulation in primary synovial fibroblasts, and the correlation with systemic markers of inflammation and radiographic damage in rheumatoid arthritis (RA). METHODS The expression of collagenase 3 mRNA was characterized by Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization. Immunohistochemical detection of cell type-specific antigens was used in combination with in situ hybridization of collagenase 3 mRNA to characterize the cellular origin of collagenase 3 mRNA expression. RESULTS Collagenase 3 mRNA was detected in synovial membrane specimens of 21 of 36 RA patients (58%) and correlated with an increase in erythrocyte sedimentation rate (P<0.05) and C-reactive protein levels (P<0.005). Collagenase 3 mRNA was localized in fibroblast-like cells of the lining and sublining layers, and at the synovial membrane-cartilage interface. Four of 10 primary synovial fibroblast cell cultures showed basal expression of collagenase 3 mRNA, which was stimulated 2-4-fold upon interleukin-1beta or tumor necrosis factor alpha treatment and, in contrast to interstitial collagenase mRNA, 5-10-fold by increasing the intracellular level of cAMP. The stimulation by cAMP analogs was completely abolished by protein kinase A inhibitors. CONCLUSION Some RA patients show collagenase 3 mRNA expression in the synovial membrane, which correlates with elevated levels of systemic markers of inflammation in these patients. In synovial fibroblasts, the expression of collagenase 3 and interstitial collagenase mRNA is differentially regulated by distinct protein kinase signal transduction pathways.

Colour duplex sonography of finger arteries in vasculitis and in systemic sclerosis

Case reports: Three patients—two with Wegener’s granulomatosis and one with an overlap syndrome of rheumatoid vasculitis, systemic lupus erythematosus, and antiphospholipid syndrome—are described. All patients experienced a sudden onset of Raynaud’s phenomenon or acrocyanosis when they had a flare of their disease. Discussion: Ultrasonography (US) showed dark (hypoechoic) arteries without colour signals, resembling the US pattern of embolism. In contrast, US in patients with systemic sclerosis is entirely different, delineating a smaller artery lumen, reduced pulsation, and thickened, slightly hyperechoic artery walls.

Characterisation of the cell type-specificity of collagenase 3 mRNA expression in comparison with membrane type 1 matrix metalloproteinase and gelatinase A in the synovial membrane in rheumatoid arthritis

Objective: To study the pattern and cell type-specificity of collagenase 3, membrane-type 1 matrix metalloproteinase (MT1-MMP), and gelatinase A mRNA expression in the synovial membrane in rheumatoid arthritis (RA). Methods: The mRNA expression of collagenase 3, MT1-MMP, and gelatinase A was characterised by northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridisation. In situ hybridisation was performed in combination with the immunohistochemical detection of cell type-specific antigens. Results: Synovial membrane specimens from 19 of 21 patients with RA expressing collagenase 3 mRNA were positive for MT1-MMP and gelatinase A mRNA. In control samples from patients without destructive inflammatory joint diseases collagenase 3 mRNA was not expressed and only in two of seven cases was a coexpression of MT1-MMP and gelatinase A mRNA detected. Fibroblast-like cells of the synovial membrane were found to be the predominant source of collagenase 3, MT1-MMP, and gelatinase A mRNA expression in lining and sublining layers as well as at the synovial membrane-cartilage interface. Additionally, the expression of MT1-MMP mRNA was detected in endothelial cells. Collagenase 3 mRNA expression was found in about 5% of CD68 positive macrophages. Conclusions: Collagenase 3 mRNA is expressed simultaneously with MT1-MMP and gelatinase A mRNA in fibroblast-like cells of the synovial membrane in RA. These results suggest (a) a broad extracellular proteolytic potential of fibroblast-like cells and (b) an important role of cell surface associated procollagenase 3 activation by MT1-MMP and gelatinase A for cartilage degradation by invading fibroblast-like cells.

The expression of collagenase 3 (MMP-13) mRNA in the synovial tissue is associated with histopathologic type II synovitis in rheumatoid arthritis

The histopathologic analysis of the synovial tissue is important to distinguish rheumatoid arthritis (RA) from other forms of synovitis and to provide information about prognosis and therapeutic strategies at early stages of the disease. In this context, the present study was performed to investigate the correlation between immunohistopathological and morphological features of synovitis and the expression of collagenase 3 (MMP-13) known to contribute significantly to cartilage degradation in RA. In the histopathologic scoring system used in this study, type I synovitis is characterized by B lymphocyte infiltration and an intact lining, and is only mild destructive to cartilage and bone. Type II shows marked diffuse infiltrations of macrophages and T lymphocytes, an ulcerated lining, fibrin exudation, and invasive growth into cartilage and bone tissue. Investigating 36 patients with RA, 21 patients (58%) were positive for the expression of collagenase 3 mRNA in the synovial tissue. Among these patients, 19 showed a histopathologic type II synovitis and only 2 patients had undifferentiated synovitis. In contrast, synovial tissue samples from patients without collagenase 3 mRNA expression were characterized in 6 cases by type I, in 5 cases by type II and in 4 cases by undifferentiated synovitis. The analysis of the clinical data revealed that RA patients with a histopathologic type II synovitis and synovial tissue collagenase 3 mRNA expression had elevated levels of systemic markers of inflammation and received stronger therapies. The data suggest, that collagenase 3 expression and the histopathologic type II synovitis are associated with a severe and destructive course of RA.

Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three‐dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice

OBJECTIVE To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP-13]) and collagenase 1 (MMP-1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3-dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice. METHODS Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell-cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts. RESULTS Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3-dimensional collagen gels was associated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2-4-fold increase upon treatment with interleukin-1beta or tumor necrosis factor a. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3-dimensional collagen or by proinflammatory cytokines. CONCLUSION The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3-dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell-matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.

Autonomic cardiac control in animal models of cardiovascular diseases. I. Methods of variability analysis

Abstract Analysis of heart rate variability (HRV) and blood pressure variability (BPV) and baroreceptor sensitivity (BRS) has become a proven tool in clinical cardiovascular diagnostics and risk stratification. In the present work, traditional and new methodological approaches for analysis of HRV, BPV, and BRS data are summarized. HRV, BPV, and BRS parameters were obtained from animal studies designed to study pathogenetic mechanisms of distinct cardiovascular diseases. Different non-linear approaches for HRV and BPV analysis are presented here, in particular measures of complexity based on symbolic dynamics. The dual sequence method (DSM) was employed for BRS analysis. In comparison to the classical measure of BRS using the average slope [ms/mm Hg], DSM offers additional information about the time-variant coupling between BPV and HRV. Since cardiovascular regulation shares common features among different species, data on HRV and BPV, as well as BRS, in animal models might be useful for understanding the pathogenetic mechanisms of cardiovascular diseases in humans and in the development of new diagnostic approaches.

Autonomic cardiac control in animal models of cardiovascular diseases II. Variability analysis in transgenic rats with alpha-tropomyosin mutations Asp175Asn and Glu180Gly.

Abstract Animal models of cardiovascular diseases allow to investigate relevant pathogenetic mechanisms in detail. In the present study, the mutations Asp175Asn and Glu180Gly in α-tropomyosin (TPM1), known cause familiar hypertrophic cardiomyopathy (FHC) were studied for changes in hemodynamic parameters and spontaneous baroreflex regulation in transgenic rats in comparison to transgenic and non-transgenic controls by telemetry. Heart rate variability (HRV) and blood pressure variability (BPV) were analyzed using time- and frequency domain, as well as non-linear measures. The dual sequence method was used for the estimation of the baroreflex regulation. In transgenic rats harboring mutated TPM1, changes in HRV were detected during exercise, but not at rest. Both mutations, Asp175Asn and Glu180Gly, caused increased low frequency power. In addition, in animals with mutation Asp175Asn a reduced total HRV was observed. BPV did not show any differences between all transgenic animal lines. During exercise, a strong increase in the number of bradycardic and tachycardic fluctuations accompanied with decreased baroreflex sensitivity (BRS) was detected in animals with either TPM1 mutation, Asp175Asn or Glu180Gly. These data suggest, that the analysis of cardiac autonomic control, particularly of baroreflex regulation, represents a powerful non-invasive approach to investigate the effects of subtle changes in sarcomeric architecture on cardiac physiology in vivo. In case of mutations Asp175Asn or Glu180Gly in TPM1, early detection of alterations in autonomic cardiac control could help to prevent sudden cardiac death in affected persons.

Generation of recombinant CHO(dhfr−) cell lines by single selection for dhfr+ transformants

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.