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Biomedical subjects

Michael Täger

Publications and source records attributed to Michael Täger.

13 recordsLinked to original sources

Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Aminopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bowel disease.

The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-beta1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])(2)]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p<0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p<0.0001). TGF-beta mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals. In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.

Animals↗

Dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) as regulators of T cell function and targets of immunotherapy in CNS inflammation.

The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.

Animals↗

Effect of L-arginine on asymmetric dimethylarginine (ADMA) or homocysteine-accelerated endothelial cell aging.

We investigated here the effect of l-arginine on asymmetric dimethylarginine (ADMA) or homocysteine-accelerated endothelial aging. Endothelial cells were cultured in medium containing 70micromol/L arginine until fourteenth passage. ADMA, dl-homocysteine, and l-arginine were replaced every 48h starting at the fourth passage. ADMA or homocysteine inhibited significantly the population doublings (PD) and accelerated the process of aging. Co-incubation with l-arginine enhanced PD, inhibited senescence associated beta-galactosidase activity, and increased telomerase activity. This effect was associated with an increase in NO synthesis and NO synthase protein expression. Furthermore, l-arginine-induced NO formation was accompanied by a reduction in oxidative stress and an increase in protein expression and enzyme activity of heme oxygenase (HO)-1. The NO synthase inhibitor l-NAME completely abolished the effect of l-arginine on ADMA or homocysteine-accelerated aging. These findings demonstrate that l-arginine prevents the onset of endothelial aging in ADMA or homocysteine-treated cells by increasing NO formation and consequently the induction of HO-1. This might provide a new strategy to delay ADMA or homocysteine-accelerated aging.

Aging↗

Rapid pacing of embryoid bodies impairs mitochondrial ATP synthesis by a calcium-dependent mechanism--a model of in vitro differentiated cardiomyocytes to study molecular effects of tachycardia.

Tachycardia may cause substantial molecular and ultrastructural alterations in cardiac tissue. The underlying pathophysiology has not been fully explored. The purpose of this study was (I) to validate a three-dimensional in vitro pacing model, (II) to examine the effect of rapid pacing on mitochondrial function in intact cells, and (III) to evaluate the involvement of L-type-channel-mediated calcium influx in alterations of mitochondria in cardiomyocytes during rapid pacing. In vitro differentiated cardiomyocytes from P19 cells that formed embryoid bodies were paced for 24 h with 0.6 and 2.0 Hz. Pacing at 2.0 Hz increased mRNA expression and phosphorylation of ERK1/2 and caused cellular hypertrophy, indicated by increased protein/DNA ratio, and oxidative stress measured as loss of cellular thiols. Rapid pacing additionally provoked structural alterations of mitochondria. All these changes are known to occur in vivo during atrial fibrillation. The structural alterations of mitochondria were accompanied by limitation of ATP production as evidenced by decreased endogenous respiration in combination with decreased ATP levels in intact cells. Inhibition of calcium inward current with verapamil protected against hypertrophic response and oxidative stress. Verapamil ameliorated morphological changes and dysfunction of mitochondria. In conclusion, rapid pacing-dependent changes in calcium inward current via L-type channels mediate both oxidative stress and mitochondrial dysfunction. The in vitro pacing model presented here reflects changes occurring during tachycardia and, thus, allows functional analyses of the signaling pathways involved.

Adenosine Diphosphate↗

Assigning the phenotype of a natural regulatory T-cell to the human T-cell line, KARPAS-299.

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.

Cell Line, Tumor↗

Aspirin reduces endothelial cell senescence.

We report here the effect of aspirin on the onset of replicative senescence. Endothelial cells that were cultured until cumulative population doublings 40 showed clear signs of aging. Incubation with aspirin inhibited senescence-associated beta-galactosidase activity and increased telomerase activity. Along with the delayed onset of senescence, aspirin decreased reactive oxygen species and increased nitric oxide (NO) and cGMP levels. Furthermore, aspirin reduced the elaboration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, and up-regulated the activity of dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA. These effects were specific in that other nonsteroidal anti-inflammatory drugs, such as ibuprofen or acetaminophen, did not prevent the onset of endothelial senescence. The NO synthase inhibitor l-NAME, but not its inactive d-enantiomer, led to complete inhibition of aspirin-delayed senescence. These findings demonstrate that aspirin delays the onset of endothelial senescence by preventing a decrease in NO formation/generation. This might provide a therapeutic strategy aimed at blocking aging-induced NO inhibition.

Aspirin↗

Erythropoietin increases asymmetric dimethylarginine in endothelial cells: role of dimethylarginine dimethylaminohydrolase.

Recombinant human erythropoietin therapy frequently causes hypertension in humans and animals with chronic renal failure. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase, and its accumulation has been associated with reducing NO bioavailability and increasing superoxide generation. Whether epoetin beta (EPO) or darbepoetin alpha (NESP) can modify the levels of ADMA in endothelial cells was investigated. Endothelial cells from the third passage were incubated for 24 h in the presence of various concentrations of EPO or NESP (0, 0.1, 1, 10, 50, 100, and 200 U/ml). The levels of ADMA, allantoin, nitrate, and nitrite in conditioned media and the activity of dimethylarginine dimethylaminohydrolase (DDAH), the content of thiols and reactive oxygen species in endothelial cells, were determined. When endothelial cells were exposed to EPO or NESP, ADMA concentration in the cell culture medium increased significantly in a dose-dependent manner versus control. This effect was associated with a reduced activity of DDAH, the enzyme that degrades ADMA. Furthermore, EPO- or NESP-induced accumulation of ADMA was accompanied by a significant reduction of NO synthesis and an increase in oxidative stress. Both allantoin, a marker of oxygen free radical generation, and reactive oxygen species increased significantly after EPO or NESP treatment compared with control. The antioxidant pyrrolidine dithiocarbamate preserved DDAH activity and reduced ADMA accumulation in the same way as the co-incubation with anti-EPO neutralizing antibody. EPO and NESP posttranslationally impair DDAH activity via increased oxidative stress, causing ADMA as an important cardiovascular risk factor to accumulate and inhibit NO synthesis.

Amidohydrolases↗

Expression of opioid receptor subtypes and their ligands in fibrillating human atria.

The delta- and kappa-receptor subtypes are both abundantly expressed in the human heart and participate in age- and stress-related alterations of cardiac function. Opioid receptor agonists mediate cardioprotection in response to ischemic preconditioning via increased intracellular Ca(2) (+) levels, opening mitochondrial K(ATP) channels, and PKC activation. We studied the expression of opioid receptor subtypes kappa and delta, and of their ligand precursors, proopiomelanocortin (POMC) and preproenkephalin A (PENKA), in human atrial tissue of patients in sinus rhythm (SR), or persistent atrial fibrillation (AF). The mitochondrial size was also compared between the two groups. The atrial mRNA expression of opioid peptide precursors and receptors was assessed by competitive and real-time RT-PCR in 16 patients in AF and 16 patients in SR. Mitochondria were analyzed in the atrial tissue by electron microscopy in four patients in AF and four patients in SR. Both PENKA (SR: 100 +/- 33% vs AF: 33 +/- 21%; P < 0.05) and kappa-receptor mRNA amounts (AF: 78 +/- 20% vs SR: 100 +/- 11%; P < 0.05) were both decreased in AF in comparison to SR. In addition, POMC mRNA levels were decreased in AF (SR: 100 +/- 54% vs AF: 37 +/- 26%; P < 0.05), whereas the expression of the corresponding delta-opioid receptor was unchanged (AF: 102 +/- 34% vs 100 +/- 44%). Mitochondrial size was increased during persistent AF. Persistent AF is associated with the down-regulation of the opioid receptor/ligand expression. This suggests a loss of protective capacity in the fibrillating atrial tissue, resulting in an ultrastructural remodeling of atrial myocytes.

Aged↗

Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence.

OBJECTIVE: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS), and its accumulation has been associated with cardiovascular disease. We aimed to investigate the role of ADMA in endothelial cell senescence. METHODS AND RESULTS: Endothelial cells were cultured until the tenth passage. ADMA was replaced every 48 hours starting at the fourth passage. ADMA significantly accelerated senescence associated beta-galactosidase activity. Additionally, the shortening of telomere length was significantly accelerated and the telomerase activity was significantly reduced. This effect was associated with an increase of oxidative stress: allantoin, a marker of oxygen free radical generation, and intracellular reactive oxygen species (ROS) increased significantly after ADMA treatment compared with control, whereas cellular thiol status and NOx synthesis decreased. Furthermore, ADMA-increased oxidative stress was accompanied by a decrease in the activity of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA, which could be prevented by the antioxidant pyrrolidine dithiocarbamate. Exogenous ADMA also stimulated secretion of MCP-1 and interleukin-8. Coincubation with the methyltransferase inhibitor S-adenosylhomocysteine abolished the effects of ADMA. CONCLUSIONS: These data suggest that ADMA accelerates senescence, probably via increased oxygen radical formation by inhibiting nitric oxide elaboration. This study provides evidence that modest changes of intracellular ADMA levels are associated with significant effects on slowing endothelial senescence.

Allantoin↗

Thiol-inducing and immunoregulatory effects of flavonoids in peripheral blood mononuclear cells from patients with end-stage diabetic nephropathy.

The cellular thiol status and its relationship to T-cell activation and cytokine synthesis of mononuclear cells was investigated in patients with end-stage diabetic nephropathy (ESDN) undergoing dialysis treatment. The functional effects of thiol repair by in vitro and in vivo treatment with flavonoids were elucidated. The thiol status of peripheral blood lymphocytes from 30 ESDN patients on hemodialysis and healthy controls was determined by flow cytometry. T-cell activation in response to pokeweed mitogen was analyzed by CD69 expression; cytokines were determined in cell culture supernatants. In result, compared to age-matched healthy subjects, a significant thiol deficiency in ESDN patients was obvious. The lowered total intracellular thiol levels correlated directly to a significant diminished T-cell activation and an elevated synthesis of TNF-alpha in the patient group. The treatment with flavonoids led to a restoration of the thiol status within 72 h in vitro and in vivo. This effect showed a biphasic kinetic that first utilized cell surface thiols and secondly intracellular thiols. In parallel, the T-cell activation was improved substantially along with a significant decrease in TNF-alpha release. These data provide the rational for clinical trials using flavonoids in ESDN to normalize immunoregulatory defects via restoration of the cellular thiol status.

Aged↗

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

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.

Amidohydrolases↗