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Inhibitors of alanyl-aminopeptidase e.g. phebestin increase the expression of transforming growth factor (TGF)-Bl in mononuclear cells. We investigated whether phebestin also produced this effect in CD4 + CD25 + T-cells and whether phebestin-treated CD4 + CD25 + T-cells were capable of ameliorating acute colitis in mice. The suppressive activity of mouse CD4 + CD25 + T-cells was assessed in vitro by co-culture with splenocytes. mRNA expression associated with the suppressive phenotype was determined in vitro and in vivo. The in vivo role of phebestin-exposed CD4 + CD25 + T-cells was studied in sodium dextran sulfate-induced acute colitis in mice. The proliferation of activated effector T-cells or splenocytes in vitro was inversely correlated with the number of CD4 + CD25 + T-cells. Phebestin pre-treatment substantially enhanced the suppressive activity of these cells and increased expression levels of TGF-s1 and FoxP3. Furthermore, transfer of CD4 + CD25 + T-cells exposed to phebestin for a short time ex vivo significantly reduced the mouse colitis disease activity index. We conclude that aminopeptidase inhibitors support the suppressive activity as well as TGF-s1 and FoxP3 expression of natural regulatory T-cells.

Inhibitors of alanyl-aminopeptidase e.g. phebestin increase the expression of transforming growth factor (TGF)-beta1 in mononuclear cells. We investigated whether phebestin also produced this effect in CD4+CD25+ T-cells and whether phebestin-treated CD4+CD25+ T-cells were capable of ameliorating acute colitis in mice. The suppressive activity of mouse CD4+CD25+ T-cells was assessed in vitro by co-culture with splenocytes. mRNA expression associated with the suppressive phenotype was determined in vitro and in vivo. The in vivo role of phebestin-exposed CD4+CD25+ T-cells was studied in sodium dextran sulfate-induced acute colitis in mice. The proliferation of activated effector T-cells or splenocytes in vitro was inversely correlated with the number of CD4+CD25+ T-cells. Phebestin pre-treatment substantially enhanced the suppressive activity of these cells and increased expression levels of TGF-beta1 and FoxP3. Furthermore, transfer of CD4+CD25+ T-cells exposed to phebestin for a short time ex vivo significantly reduced the mouse colitis disease activity index. We conclude that aminopeptidase inhibitors support the suppressive activity as well as TGF-beta1 and FoxP3 expression of natural regulatory T-cells.

The regional distribution and cellular localization of insulin-degrading enzyme (IDE) was studied in adult human brain and pituitary by means of immunhistochemistry. We show that the enzyme is widely but unevenly distributed in human brain, with hypothalamic neurons showing the strongest immunoreaction. Strong to moderate immunostaining for the enzyme was observed in multiple cortical areas, hippocampus, cerebellum, and brain stem. Cellularly, IDE was mainly confined to neurons, but it was also present in oligodendrocytes, choroid plexus, and some blood vessel endothelial cells. A strong immunoreaction was seen in a subset of adenohypophysial cells. Some immunolabeling was also present in the neurohypophysis. The putative importance of the distribution of the enzyme in brain and pituitary is discussed in relation to its main known substrates, insulin, Aβ, and β-endorphin.

In the central nervous system (CNS), neuregulin-1 (NRG-1) proteins function in neuronal migration, differentiation, and survival of oligodendrocytes. The NRG-1 gene codes for at least 15 different isoforms, which may be classified on the basis of their molecular structure. At least two different haplotypes of the NRG-1 gene may be associated with schizophrenia. An abnormal expression pattern of NRG-1 mRNA was found in the prefrontal cortex of schizophrenic patients in comparison to controls. We here show that the NRG-1alpha isoform is significantly reduced in white matter of the prefrontal cortex in schizophrenia but not in affective disorder. In the prefrontal gray matter, the density of NRG-1alpha expressing neurons was reduced in individuals with schizophrenia and in unipolar patients. We studied brains of 22 schizophrenics, 12 patients with affective disorders (7 unipolar and 5 bipolar), and 22 matched controls. NRG-1alpha immunoreactive material was detected with a polyclonal antiserum against the synthetic peptide from alpha-type EGF-like domain of human NRG. The demonstrated decreased number of NRG-1 immunoreactive neurons in the brains of schizophrenics and patients with unipolar depression points to an important role of this NRG-1alpha splice variant in neuropsychiatric disorders. Reduced NRG-1alpha protein concentrations were found in brains of schizophrenics after Western blot analysis. The diminished expression of NRG-1alpha strongly supports an early neurodevelopmental component to schizophrenia.

Recent studies suggest the importance of prothrombotic and proinflammatory cascades in vascular thrombus formation. However, the impact of platelet CD40 and CD40 ligand (CD40L) expression and its relation to inflammatory markers in atrial clot formation have not yet been determined. Therefore, we studied a total of 40 patients. A total of 20 patients with persistent atrial fibrillation (AF) and 20 matched patients with sinus rhythm (SR) were included to quantify platelet surface expression of CD40/CD40L, serum levels of intercellular adhesion molecule-1 (ICAM), vascular adhesion molecule-1 (VCAM), high-sensitivity C-reactive protein (hsCRP), and monocyte chemoattractant protein-1 (MCP-1). Using fluorescence-activated cell sorting analysis, baseline CD40 expression (antibody binding capacity [ABC]) was increased during AF (AF: 7776 +/- 8.46 ABC vs. SR: 7753 +/- 7.32 ABC; P0.05), whereas CD40L expression was not different. In contrast to the effect of adenosine diphosphate, ex vivo stimulation with thrombin receptor activating peptide (TRAP) increased CD40 and CD40L expression in both groups. MCP-1, hsCRP, ICAM, and VCAM levels were significantly increased during AF, reaching highest levels in patients with atrial thrombi. Importantly, VCAM and MCP-1 were independent predictors for atrial thrombi (P0.05) using multivariate analysis. In contrast to declining levels of hsCRP, levels of ICAM, VCAM, MCP-1, and platelet CD40 expression remained elevated 5 weeks after successful electrical direct current cardioversion (CV). In conclusion, prothrombogenic markers are substantially elevated in patients with AF, reaching highest levels in patients with AF and atrial thrombi. Interestingly, amounts of adhesion molecules and platelet CD40 levels remain elevated even 5 weeks after successful CV, which may imply a persistently increased risk for atrial thrombus formation. In addition to hsCRP, MCP-1 and VCAM may serve as new biomarkers, which may help to identify patients with an increased risk for thromboembolic events.

Approximately 90% of patients with atrial fibrillation have concomitant cardiovascular disease, such as hypertension, heart failure or valve disease. These diseases have been found to substantially affect the structure of atrial tissue, and thereby the occurrence of atrial fibrillation. At the molecular level, angiotensin II, oxidative stress and pro-inflammatory mediators are of particular importance in the induction of pro-arrhthymic atrial dilation, myocardial hypertrophy and interstitial atrial fibrosis. Elucidating the signalling pathways responsible for the process of structural atrial remodeling has helped to define novel non-ion channel drug targets for atrial fibrillation.

The human CD30-positive anaplastic large (T-) cell lymphoma cell line, KARPAS-299 (DSM ACC31), was established from blast cells in the peripheral blood from a case of non-Hodgkin lymphoma in 1988. We describe the mRNA and surface expression in KARPAS-299 cells of a panel of markers highly restricted to human natural regulatory T-cells and associated with their suppressive activity, including FOXP3, CD25, IL-10, TGF-beta1, CD62L, and Lag-3. Results obtained from co-culturing human peripheral blood leukocytes with KARPAS-299 cells assigned a suppressive phenotype to the latter ones. In conclusion, KARPAS-299 cells show characteristics typical of natural regulatory T-cells and, thus, represent a valuable model for studying regulatory T-cell function, which may also facilitate drug development aimed at the modulation of regulatory T-cell activity for the pharmacological therapy of, for example, autoimmune diseases.

Atrial tachyarrhythmia (AF) alters intracellular calcium homeostasis and induces cellular hypertrophy of atrial myocytes. The impact of the calcium-dependent calcineurin pathway on the development of AF-induced atrial hypertrophy has not yet been analyzed. In this study, atrial tissue samples from patients with sinus rhythm and chronic persistent atrial fibrillation (CAF) were used to determine changes in expression and activity of calcineurin A (CnA), and its relation to CnA-regulated transcription factors NFATc1-4, and hypertrophic markers ANP, troponin I, and beta-MHC. CnA phosphatase activity and CnAbeta protein contents were significantly upregulated in patients with CAF. Calcineurin activation led to dephosphorylation, redistribution, and subsequent accumulation of NFATc3 in nuclei during CAF, and expression of hypertrophic genes was increased. CAF-dependent changes were reproduced by ex vivo pacing (2-4 Hz) of human atrial tissue slices. FK506 abolished the hypertrophic response induced by electrical-field stimulation. Atrial tachyarrhythmia causes atrial hypertrophy by activation of the CnA signal pathway, which thereby contributes to structural remodeling of human atria.

Inhibitors of the enzymatic activity of alanyl-aminopeptidases severely affect growth and typical functions of human peripheral T cells both in vitro and in vivo. The most prominent changes observed include the activation of cellular signal transduction pathways such as MAP kinases Erk1/2 or the Wnt-pathway, a decrease of production and release of “pro-inflammatory” cytokines (IL-2, IL-12) and, most importantly, an induction of expression and release of the immunosuppressive cytokine, TGF-β1. Similar effects on T cell proliferation and function have been observed in response to inhibition of DPIV, which is strongly suggestive of a functional synergism of APN and DPIV. In support of this hypothesis evidence is provided showing that the simultaneous application of inhibitors of DPIV and APN further enhances the anti-inflammatory and immunosuppressive effects provoked by the inhibition of APN or DPIV alone. Therefore, the simultaneous inhibition of these enzymes represents a promising strategy for the pharmacological therapy of T cell mediated diseases such as autoimmune disease, inflammation, allergy, and allograft rejection.

Recently, proteome analysis has been introduced to analyze differential protein expression and cellular protein composition in cardiovascular medicine. Proteins expressed by diseased hearts (myocardial proteomics) were first investigated over a decade ago using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). However, while 2D-PAGE is very successful for the abundant and moderately expressed proteins, it struggles to identify proteins expressed at low levels. However, the sensitivity of mass spectrometry has increased considerably during recent years, and technical progress widens the detection limits of mass-spectrometric analysis. Proteomics now allows us to examine global alterations in protein expression in the diseased hearts, and will provide new insights into the cellular mechanisms involved in cardiac dysfunction. This review will summarize the present knowledge about the use of proteome analysis in myocardial diseases.

ADAM12 is a member of the large family of multidomain metalloprotease-disintegrins which possess cell-binding and metalloprotease properties. Typically, ADAM12 is expressed in mesenchymal cells, developing and regenerating heart and skeletal muscle, bone as well as in certain tumours. This report shows by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry that the protease ADAM12 is detectable in human and rat brain tissue as well as in cultured cells derived from rat brain. With the exception of a very few immunopositive pyramidal neurons in the developing rat brain, the cellular localization of ADAM12 was exclusively confined to oligodendroglial cells. Thus, ADAM12 may be regarded a new suitable marker for this cell type.

Inhibition of the enzymatic activity of alanyl-aminopeptidase leads to strong immunosuppression both in vitro and in vivo. Mechanisms involved include growth arrest, induction of immunosuppressive cytokines (TGF-s1), reduced expression of inflammatory or T cell stimulating cytokines (IL-2, IL-12), and modulation of T cell signalling pathways. Thus, T cells appear to represent a major cellular target for the pharmacological treatment of T cell mediated diseases by virtue of aminopeptidase inhibitor administration. Membrane (APN) and cytosol alanyl-aminopeptidase (ApPS), both implicated in a variety of cellular functions, show similar substrate specifity and inhibitor sensitivity. Furthermore, both enzymes are expressed in practically all T cell subsets, including the population of natural regulatory T cells that was shown recently to control the immunological tolerance to self-antigens. While the involvement of APN and ApPS in the pathological immune response is evident, the precise molecular mechanisms remain to be identified. The development of inhibitors specific for APN and ApPS is an attractive field of study and would allow determination of the individual contribution of either enzyme in the immune response.

Inhibitors of the enzymatic activity of alanyl-aminopeptidases severely affect growth and typical functions of human peripheral T cells both in vitro and in vivo. The most prominent changes observed include the activation of cellular signal transduction pathways such as MAP kinases Erk1/2 or the Wnt-pathway, a decrease of production and release of "pro-inflammatory" cytokines (IL-2, IL-12) and, most importantly, an induction of expression and release of the immunosuppressive cytokine, TGF-beta1. Similar effects on T cell proliferation and function have been observed in response to inhibition of DPIV, which is strongly suggestive of a functional synergism of APN and DPIV. In support of this hypothesis evidence is provided showing that the simultaneous application of inhibitors of DPIV and APN further enhances the anti-inflammatory and immunosuppressive effects provoked by the inhibition of APN or DPIV alone. Therefore, the simultaneous inhibition of these enzymes represents a promising strategy for the pharmacological therapy of T cell mediated diseases such as autoimmune disease, inflammation, allergy, and allograft rejection.

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 alanyl-aminopeptidase (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 cAAP-specific 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 non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4 + and that of cAAP in Treg cells.

Purpose: Hypertension induces cardiac remodelling at the structural and functional level. Endothelin-1 (ET-1) may contribute to cardiac remodelling. It is not known, however, whether atrial ET-1 signalling is altered in hypertension. Here we tested the hypothesis that ET-1 signalling is augmented in the atria of spontaneously hypertensive rats (SHR) and that this may contribute to hypertension-induced atrial arrhythmias. Methods: SHR and control Wistar-Kyoto (WKY) rats were studied at 6-8 months of age. Hearts were removed for morphometric analysis. Atria were snap frozen and analyzed for mRNA and protein expression by qPCR and Western blotting. Isolated atrial myocytes were field stimulated in the presence of 50 nM ET-1, and Ca transients (CaTs) were characterized at the global or subcellular level by epifluorescence (Fura-2/AM) and line scan confocal microscopy (Fluo-4/AM). To mimic atrial tachycardia, atrial tissue slices were stimulated at 5 Hz for up to 20 h and used for Western blotting. Results: At 6-8 months of age, SHR exhibited compensated LV hypertrophy, but left atrial hypotrophy, despite increased atrial mRNA levels of BNP (2-fold) and β-MHC (3.6-fold), while α-MHC was unaffected. There were no differences in global CaTs between atrial myocytes from WKY and SHR. Exposure of atrial myocytes to 50 nM ET-1 caused a 35% increase in systolic Ca in SHR (n=14), but only a 16% increase in WKY (n=21; P

Purpose: Arterial hypertension and age are the most predominant factors causing atrial fibrillation (AF). The exact proarrhythmogenic pathways involved are still unknown. The present study was designed to examine changes in the composition of atrial members of the renin-angiotensin system (RAS) in response to tachyarrhythmia in the presence of arterial hypertension at different ages. The local RAS plays a crucial role in remodelling processes in the heart, primarily through the action of angiotensin II (AngII), which is generated by angiotensin converting enzyme (ACE). ACE2 counterbalances the action of ACE by cleaving of AngII into peptides that act protective. Methods: Atrial tissue from spontaneously hypertensive male rats (SHR; n=6) and their normotensive controls, Wistar-Kyoto (n=6), were analysed at the age of 12 weeks and 6-8 months. Atrial slices were rapidly stimulated at 5 Hz up to 20h to resemble AF (n=4 per group and age). The changes in the expression of RAS components (ACE, ACE2, angiotensin receptors) as well as RAS-associated downstream effectors were estimated at the mRNA and protein levels. Moreover, the activation of MAPK and NF-κB signalling were analysed by immunoblotting. Results: Our data revealed that left atrium of SHR expressed constantly high levels of ACE protein (3.8±0.7; p=0.03) and mRNA (1.7±0.7; p=0.04) compared to normotensive controls (1.0) in each age group. ACE2-mRNA levels did not show any changes at 12 weeks between normotensive and hypertensive rats. With age the mRNA levels of ACE2 diminished in SHR (0.79±0.04; p=0.02). These molecular changes were accompanied by activation of MAPK, NF-κB signalling and the presence of the active caspase-3 in SHR. In response to the stimulation in the electrical field, the atrial tissue from hypertensive rats showed 1.6-fold (p=0.01) and of normotensive rats 3.0-fold increase (p=0.01) in the expression of ACE. In normotensive young rats, ACE2 counterbalanced this action at the mRNA (1.5±0.12; p=0.01) and protein level (2.7±0.80; p=0.11). This protective action of ACE2 decreased with age in normotensive rats, and it disappeared completely in SHR (mRNA: 0.94±0.13; protein: 0.98±0.11). Conclusions: Our data show that hypertension in SHR is accompanied by enhanced atrial expression of angiotensin-converting enzyme. This fact strongly implies a sustained exposure of atrial tissue to elevated levels of local AngII. The observed imbalance of the ACE/ACE2 ratio in aged hypertensive rats might facilitate the susceptibility to arrhythmia especially in this clinical scenario.