Marco Arndt

Increased expression of extracellular signal-regulated kinase and angiotensin-converting enzyme in human atria during Atrial fibrillation

OBJECTIVES The purpose of this study was to determine whether atrial expression of the extracellular signal-regulated kinases Erk1/Erk2 and of the angiotensin-converting enzyme (ACE) is altered in patients with atrial fibrillation (AF). BACKGROUND Recent studies have demonstrated that atrial fibrosis can provide a pathophysiologic substrate for AF. However, the molecular mechanisms responsible for the development of atrial fibrosis are unclear. METHODS Atrial tissue samples of 43 patients undergoing open heart surgery were examined. Seventeen patients had chronic persistent AF (> or =6 months; CAF), 8 patients had paroxysmal AF (PAF) and 18 patients had no history of AF. Erk expression was analyzed at the mRNA (quantitative reverse transcription polymerase chain reaction), the protein (immunoblot techniques) and atrial tissue (immunohistochemistry) levels. Erk-activating kinases (MEK1/2) and ACE were analyzed by immunoblot techniques. RESULTS Increased amounts of Erk2-mRNA were found in patients with CAF (75 +/- 20 U vs. sinus rhythm: 31 +/- 25 U; p < 0.05). Activated Erk1/Erk2 and MEK1/2 were increased to more than 150% in patients with AF compared to patients with sinus rhythm. No differences between CAF and PAF were found. The expression of ACE was three-fold increased during CAF. Amounts of activated Erk1/Erk2 were reduced in patients treated with ACE inhibitors. Patients with AF showed an increased expression of Erk1/Erk2 in interstitial cells and marked atrial fibrosis. CONCLUSIONS An ACE-dependent increase in the amounts of activated Erk1/Erk2 in atrial interstitial cells may contribute as a molecular mechanism for the development of atrial fibrosis in patients with AF. These findings may have important impact on the treatment of AF.

Regulation of Angiotensin II Receptor Subtypes During Atrial Fibrillation in Humans

BACKGROUND Previous studies have suggested that atrial fibrillation (AF) is associated with the activation of the atrial angiotensin system. However, it is not known whether the expression of angiotensin II receptors changes during AF. The purpose of this study was to determine the atrial expression of angiotensin II type 1 and type 2 receptors (AT(1)-R and AT(2)-R) in patients with AF. METHODS AND RESULTS Atrial tissue samples from 30 patients undergoing open heart surgery were examined. Eleven patients had chronic persistent AF (> or =6 months; cAF), 8 patients had paroxysmal AF (pAF), and 11 patients were in sinus rhythm. AT(1)-R and AT(2)-R were localized in the atrial tissue by immunohistochemistry and quantified at the protein and mRNA level by Western blotting and quantitative polymerase chain reaction. Both types of AT-R were predominantly expressed in atrial myocytes in all groups. The amount of AT(1)-R was reduced to 34.9% during cAF (P<0.01) and to 51.7% during pAF (P<0.05) compared with patients in sinus rhythm. In contrast, AT(2)-R was increased during cAF (246%; P=NS) and pAF (505%; P<0.01). AT(1)-R/AT(2)-R mRNA content was similar in all groups. CONCLUSIONS AF is associated with the down-regulation of atrial AT(1)-R and the up-regulation of AT(2)-R proteins. These findings may help define the pathophysiological role of the angiotensin system in the structural remodeling of the fibrillating atria.

Increased expression of ADAM family members in human breast cancer and breast cancer cell lines

Purpose ADAMs (A Disintegrin and Metalloprotease) are multifunctional, membrane-bound cell surface glycoproteins, which have numerous functions in cell growth, differentiation, and motility. We wished to investigate the expression of ADAM 9, 10, 12, 15, and in human breast cancer.Methods Expression of ADAMs was determined in breast cancer specimens and the corresponding non-neoplastic breast tissue from 24 patients, and in the MCF-7 and MDA-MB 453 breast cancer cell lines via quantitative RT-PCR and immunohistochemistry. The effects of anti-ADAM antibodies on cell proliferation were assessed by measuring DNA-synthesis.Results Breast cancer tissue samples showed increased mRNA expression of ADAM 9, 12, and 17, whereas ADAM 10 and 15 were not differently expressed. Protein expression was studied by immunohistochemistry. All ADAMs were expressed in MCF-7 and MDA-MB453 cell lines, with the highest expression levels being observed for ADAM 9, 12, and 17. Application of anti-ADAM 15 and anti-ADAM 17 antibodies significantly inhibited the proliferation of both MCF-7 and MDA-MB453 breast cancer cell lines. In contrast, the growth of MCF-7 cells appeared to be stimulated by the administration of anti-ADAM 12 antibody.Conclusion The results of this study suggest that ADAMs are differentially expressed in human breast cancer and are capable of modulating tumour cell growth.

Matrix Metalloproteinases and Tace Play A Role in The Pathogenesis of Endometriosis

Endometriosis, a benign gynecologic disorder, occurs in about 10% of women in reproductive age and in up to 50% of women with infertility. The basic etiologic factors causing this disease are unknown as yet. Matrix metalloproteinases (MMP) are involved in degradation of the extracellular matrix (ECM). Their proteolytic activity is regulated by tissue inhibitors of metalloproteinases (TIMPs). Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-alpha to its mature soluble form. TNF-alpha induces the secretion of several MMPs. In order to study the expression of MMP-1, -2, -3 and -9, TIMP-1 and -2, TACE and TNF-alpha in endometrium and endometriotic tissue, we investigated formalin-fixed paraffin sections of endometriotic tissues and normal endometrium with immunohistochemical techniques and in situ hybridisation. Furthermore, quantitative PCR was used for quantification of TACE-mRNA in fresh tissue. We found in this study significant higher protein expression of MMP-1 and TACE and significant lower protein expression of TMP-1 and -2 in endometriotic tissue compared to endometrium. This data may suggest that high TACE expression causes the increased conversion of membrane-bound proTNF-alpha into its soluble form, which stimulates the increased secretion of MMP-1. The simultaneous deficiency of TIMP-1 and -2 in endometriotic tissue suppose an additional proteinase inhibitor imbalance in endometriosis.

Review: The Role of Membrane Peptidase in Immune Functions

Uwe Lendeckel, Thilo Kahne, Dagmar Riemann, Klaus Neubert, Marco Arndt and Dirk Reinhold Institute of Experimental Internal Medicine, Center of Internal Medicine, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany Institute of Medical Immunology,Martin-Luther-University Halle-Wittenberg, Strasse der Opfer des Faschismus 6, D-06097 Halle, Germany Institute of Biochemistry, Dept. Biochemistry & Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany

Extracellular cysteines define ectopeptidase (APN, CD13) expression and function.

Abstract Alanyl aminopeptidase (APN) is a surface-bound metallopeptidase that processes the N-terminals of biologically active peptides such as enkephalins, angiotensins, neurokinins, and cytokines. It exerts profound activity on vital processes such as immune response, cellular growth, and blood pressure control. Inhibition of either APN gene expression or its enzymatic activity severely affects leukocyte growth and function. We show here that oxidoreductase-mediated modulations of the cell surface thiol status affect the enzymatic activity of APN. Additional evidence for the pivotal role of extracellular cysteines in the APN molecule was obtained when substitution of any of these six cysteines caused complete loss of surface expression and enzymatic activity. In contrast, the transmembrane Cys24 appears to have no similar function. Enzymatically inactive cysteine mutants were retained in the endoplasmic reticulum as shown by high-resolution imaging and Endoglycosidase H digestion. In the absence of any crystal-structure data, the demonstration that individual extracellular cysteines contribute to APN expression and function appears to be of particular importance. The data are the first to show thiol-dependent modulation of the activity of a typical surface-bound peptidase at the cell surface, probably reflecting a general regulating mechanism. This may relate to various disease processes such as inflammation or malignant transformation.

Modulation of WNT-5At Eexpression by Actinonin: Linkage of APN to the WNT-Pathway?

Inhibition of alanyl-aminopeptidase gene expression or enzymatic activity compromises T cell proliferation and function. Molecular mechanisms mediating these effects are not known as yet. Applying the cDNA array technique we identified the proto-oncogen Wnt-5a strongly affected by APN-inhibition. Wnt-5a and other members of the Wnt family of secreted factors are implicated in cell growth and differentiation. Wnt-5a was moderately expressed in resting T cells, but strongly down-regulated in response to activation by OKT3/IL-4/IL-9. Actinonin increased Wnt-5a-mRNA contents as confirmed by RT-PCR. In addition, expression of GSK-3 beta, an inherent component of the Wnt-pathway, was found to be increased in response to activation, but suppressed by actinonin at both the mRNA and protein level. These findings may provide a rationale for the strong growth inhibitory effects resulting from an inhibition of alanyl aminopeptidase expression or activity.

Rapid mitogen-induced aminopeptidase N surface expression in human T cells is dominated by mechanisms independent of de novo protein biosynthesis

Abstract The membrane bound metalloprotease aminopeptidase N (APN, CD13, EC 3.4.11.2) is a well established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. Recently, the expression of the APN gene in T cell lines as well as the induction of APN gene and surface expression in human peripheral T cells by mitogenic activation have been demonstrated. Here, by means of cytofluorimetric analysis evidence is provided, that the induction of APN surface expression is partially resistent to the action of the inhibitors of protein biosynthesis, puromycin and cycloheximide, and is not prevented by tunicamycin, an inhibitor of glycosylation. These data suggest that the rapid mitogen-induced surface expression of APN, detectable 20 hours after stimulation is dominated by mechanisms not dependent on de novo protein biosynthesis or glycosylation. As shown by simultaneous analyses, the inhibitors used did also differently modify the induction of surface expression of other inducible glycosylated leukocyte surface antigens, namely CD25, CD69 and CD95.

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets.

Abstract Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanylaminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAPspecific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The nondiscriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.

Antisense-Mediated Inhibition of Aminopeptidase N (CD13) Markedly Decreases Growth Rates of Hematopoietic Tumour Cells

There are several reports describing neutral aminopeptidase activities expressed in immune cells1–7. One of the best studied members of these enzymes is the Zn-dependent aminopeptidase N (CD13, E.C.3.4.11.2, APN) Within the hematopoietic system the expression of this leukocyte surface antigen was thought to be restricted to myelomonocytic cells 1,8,9. During recent years, it has been proven that lymphoid cells contain APN-mRNA and express corresponding enzymatic activity, too 2–4,7,10,11. These cells contain very low amounts of CD13 only, and therefore, are predominantly CD13-negative1,9,12,13. Recently, different groups have been provided conclusive evidence that CD13 is strongly induced during processes such as T cell activation or inflammation, suggesting that this antigen may well be involved in the regulation of proliferation and differentiation processes3,12,14–15.