Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats.

Objective: In spontaneously hypertensive rats (SHR) atrial remodeling has been shown to involve increase in endothelin (ET) signaling. Furthermore, inflammatory processes may further contribute to tissue remodeling. The aimed of this study was to investigate whether an endothelin receptor antagonist, macitentan, could reduce left atrial (LA) remodeling in arterial hypertension. Methods: Molecular characterization of atria was performed in SHR at the age of 8 months and their age-matched normotensive control rats (WKY). SHR were treated with macitentan and, for comparison with a blood pressure reducing drug, with doxazosin. After two months of treatment, molecules involved in endocardial inflammation and atrial calcium handling were assessed. The molecular changes provoked by rapid-pacing (RAP) were analyzed in atrial tissue slices. Results: Doxazosin reduced the systolic blood pressure compared with the untreated SHR (159 ± 26 vs. 176 ± 17; P < 0.05) or macitentan (vs. 189 ± 21; P < 0.05). Macitentan lowered the increased levels of atrial ET-1 and abrogated the pacing-induced upregulation of preproET-1-mRNA in atrial slices from SHR. Macitentan reduced the elevated levels of atrial 8-isoprostanes, the increased expression of pro-inflammatory ICAM-1 and IL-8, the phosphorylation of MAP kinases, ERK and p38, the phosphorylation of NF-κB and the expression of VCAM-mRNA. Major Ca2+-regulating proteins and markers of hypertrophy and fibrosis, however, were not affected. Doxazosin elicited similar changes, except for the alterations in ET-1 levels, NF-κB phosphorylation and VCAM-mRNA. Conclusion: Macitentan reversed pro-inflammatory remodeling in hypertensive atria in a blood pressure-independent manner, which might prevent endocardial dysfunction and thereby, thrombogenesis in arterial hypertension.

CREM-transgene mice: An animal model of atrial fibrillation and thrombogenesis.

BACKGROUND: The molecular pathomechanisms underlying atrial thrombogenesis are multifactorial and still require detailed investigations. Transgenic mice with cardiomyocyte-directed expression of the transcriptional repressor CREM-IbΔC-X (CREM-TG) represent an experimental model of atrial fibrillation (AF) that shows a gradual, age-dependent progression from atrial ectopy to persistent AF. Importantly, this model develops biatrial thrombi. The molecular characteristics related to the thrombogenesis in CREM-TG mice have not been studied, yet. METHODS: The inflammatory and prothrombotic state was evaluated at the transcriptional (qRT-PCR) and protein level in the left (LA) and right atria (RA) from CREM-TG mice at the age of 20weeks and compared to wild-type controls. Moreover, histological analyses of atrial thrombi were performed. RESULTS: The endocardial dysfunction was mirrored by diminished levels of eNOS-mRNA in both atria (RA: 0.79±0.04, LA: 0.72±0.06; each P<0.05). Moreover, the PAI-1/t-PA mRNA ratio was significantly increased in both atria (RA: 3.6±0.6; P<0.01, LA: 4.0±1.0; P<0.05) indicating a high risk of thrombus formation. However, the inflammatory phenotype was more pronounced in the RA and was reflected by a significant increase in the mRNA levels encoding adhesion molecules ICAM-1 (2.1±0.2; P<0.01), VCAM-1 (2.3±0.5; P<0.05), and selectin P (3.6±0.5: P<0.05). CONCLUSIONS: CREM-TG mice represent a valuable model for studying atrial thrombogenesis and assessing therapeutic approaches preventing embolic events in the systemic and pulmonary circulation.

Protective regulation of the ACE2/ACE gene expression by estrogen in human atrial tissue from elderly men.

Data from animal experiments and clinical investigations suggest that components of the renin-angiotensin system are markedly affected by sex hormones. However, whether estrogen affects human atrial myocardium has not been investigated yet. In this study, we determined the effects of estrogen on key components of atrial renin-angiotensin system: angiotensin-converting enzyme, responsible for generation of angiotensin II and angiotensin-converting enzyme 2, counteracting majority of AngII effects, and different renin-angiotensin system receptors, AT1R, AT2R, and MAS. First, the expression levels of estrogen receptors mRNA were determined in right atrial appendages obtained from patients undergoing heart surgery. The amounts of estrogen receptor α and estrogen receptor ß mRNA were similar between women ( n = 14) and men ( n = 10). Atrial tissue slices (350 µm) were prepared from male donors which were exposed to estrogen (1-100 nM; n = 21) or stimulated at 4 Hz for 24 h in the presence or absence of 100 nM estrogen ( n = 16), respectively. The administration of estrogen did not change mRNA levels of estrogen receptors, but activated MAP kinases, Erk1/2. Furthermore, estrogen increased the amounts of angiotensin-converting enzyme 2-mRNA (1.89 ± 0.23; P < 0.05) but reduced that of angiotensin-converting enzyme-mRNA (0.78 ± 0.07, P < 0.05). In addition, the transcript levels of AT2R and MAS were upregulated by estrogen. Pacing of tissue slices significantly increased the angiotensin-converting enzyme/angiotensin-converting enzyme 2 ratio at both the mRNA and protein level. During pacing, administration of estrogen substantially lowered the angiotensin-converting enzyme/angiotensin-converting enzyme 2 ratio at the transcript (0.92 ± 0.21 vs. 2.12 ± 0.27 at 4 Hz) and protein level (0.94 ± 0.20 vs. 2.14 ± 0.3 at 4 Hz). Moreover, estrogen elicited anti-inflammatory and anti-oxidative effects on renin-angiotensin system-associated downstream effectors such as pro-oxidative LOX-1 and pro-inflammatory ICAM-1. An antagonist of estrogen receptor α reversed these anti-inflammatory and anti-oxidative effects of estrogen significantly. Overall, our results demonstrated that estrogen modifies the local renin-angiotensin system homeostasis and achieves protective effects in atrial myocardium from elderly men. Impact statement The present study demonstrates that estrogen affects the human atrial myocardium and mediates protective actions through estrogen receptors-(ER) dependent signaling. Estrogen substantially modulates the local RAS via downregulation of ACE and simultaneous upregulation of ACE2, AT2R and MAS expression levels. This is indicative of a shift of the classical RAS/ACE axis to the alternative, protective RAS/ACE2 axis. In support of this view, estrogen attenuated the expression of RAS-associated downstream effectors, LOX-1, and ICAM-1. A specific antagonist of ERα reversed the anti-inflammatory and anti-oxidative effects of estrogen in paced and non-paced atrial tissue slices. In summary, our data demonstrate the existence of protective effects of estrogen in atrial tissue from elderly men which are at least in part, mediated by the regulation of local RAS homeostasis.

Atrial fibrillation and rapid acute pacing regulate adipocyte/adipositas-related gene expression in the atria.

PURPOSE: Atrial fibrillation (AF) has been associated with increased volumes of epicardial fat and atrial adipocyte accumulation. Underlying mechanisms are not well understood. This study aims to identify rapid atrial pacing (RAP)/AF-dependent changes in atrial adipocyte/adipositas-related gene expression (AARE). METHODS: Right atrial (RA) and adjacent epicardial adipose tissue (EAT) samples were obtained from 26 patients; 13 with AF, 13 in sinus rhythm (SR). Left atrial (LA) samples were obtained from 9 pigs (5 RAP, 4 sham-operated controls). AARE was analyzed using microarrays and RT-qPCR. The impact of diabetes/obesity on gene expression was additionally determined in RA samples (RAP ex vivo and controls) from 3 vs. 6 months old ZDF rats. RESULTS: RAP in vivo of pigs resulted in substantial changes of AARE, with 66 genes being up- and 53 down-regulated on the mRNA level. Differential expression during adipocyte differentiation was confirmed using 3T3-L1 cells. In patients with AF (compared to SR), a comparable change in RA mRNA levels concerned a fraction of genes only (RETN, IGF1, HK2, PYGM, LOX, and NR4A3). RA and EAT were affected by AF to a different extent. In patients, concomitant disease contributes to AARE changes. CONCLUSIONS: RAP, and to lesser extent AF, provoke significant changes in atrial AARE. In chronic AF, activation of this gene panel is very likely mediated by AF itself, AF risk factors and concomitant diseases. This may facilitate the development of an AF substrate by increasing atrial ectopic fat and fat infiltration of the atrial myocardium.

Dronedarone prevents microcirculatory abnormalities in the left ventricle during atrial tachypacing in pigs.

BACKGROUND AND PURPOSE: Atrial fibrillation induces ischaemic microcirculatory flow abnormalities in the ventricle, contributing to the risk for acute coronary syndromes. We evaluated the effect of dronedarone on ventricular perfusion during rapid atrial pacing (RAP). EXPERIMENTAL APPROACH: Coronary and fractional flow reserve (CFR/FFR) were measured in the left anterior descending artery in 29 pigs. Six received RAP, six received RAP with dronedarone (RAP/D), seven received dronedarone alone, four received RAP with amiodarone (RAP/A), and six received neither (sham). In ventricular tissue, oxidative stress/ischaemia-related gene and protein expression was evaluated by RT-PCR and Western blotting; Isoprostanes were measured by GC-MS procedures. KEY RESULTS: CFR was decreased in the RAP group, compared with other groups. FFR was not different between groups. Effective refractory period was reduced in RAP compared with RAP/D. RAP-activated PKC phosphorylation tended to be decreased by dronedarone (P= 0.055) RAP induced NOX-1 and NOX-2 protein and the mRNA for hypoxia-inducible factor-1α (HIF-1α). Dronedarone reduced the pacing-dependent increase in the expression of NOX-2 protein and of HIF-1α mRNA. The oxidative stress marker, F(2)-isoprostane, was increased by RAP and this increase was attenuated by dronedarone. Other oxidative stress/ischaemia-related genes were induced by RAP compared with sham and were decreased by dronedarone treatment. In HL1 cells, dronedarone significantly inhibited the increased phosphorylation of PKCα after oxidative stress, with an almost significant effect (P= 0.059) on that after RAP. CONCLUSIONS AND IMPLICATIONS: Dronedarone abolished RAP-induced ventricular microcirculatory abnormalities by decreasing oxidative stress/ischaemia-related gene and protein expression in the ventricle.

Plasma aldosterone levels and aldosterone-to-renin ratios are associated with endothelial dysfunction in young to middle-aged subjects.

OBJECTIVE: Small clinical studies suggested a role for aldosterone in the development of endothelial dysfunction. We investigated whether the plasma aldosterone concentration (PAC) or the aldosterone-to-renin ratio (ARR) were associated with decreased endothelial function as measured by flow-mediated dilation (FMD) of the brachial artery in the general population. METHODS: Our study population comprised 972 participants from the Study of Health in Pomerania, who were not treated with antihypertensive medication. We performed age-stratified (<50 and ≥ 50 years) ordinal logistic regression analyses. FMD was categorised as decreased (1st quintile), moderate (2nd-4th quintile), or increased (5th quintile). PAC and ARR were divided into low, moderate, and high values according to age- and sex-specific tertiles. All models were re-calculated for 871 subjects with PAC and ARR within the study-specific reference ranges. Odds ratios (OR) and 95% confidence intervals (CI) are presented. RESULTS: Subjects <50 years with high PAC (OR 1.60; 95% CI 1.07-2.38) or ARR (OR 1.81; 95% CI 1.21-2.73) had higher odds for decreased FMD than subjects with low PAC or ARR, respectively. Similar results were obtained in analyses restricted to subjects with PAC and ARR within the reference range. High-normal PAC (OR 1.62; 95% CI 1.07-2.47) or ARR (OR 1.62; 95% CI 1.05-2.50) was associated with higher odds for decreased FMD when compared with low-normal PAC or ARR, respectively. These associations were not observed in subjects ≥ 50 years. CONCLUSIONS: High and high-normal PAC or ARR contribute to an impaired FMD and subsequently the progression of subclinical atherosclerosis in young to middle-aged subjects.

Ectopeptidases in tumour biology: a review.

Cell membrane-bound proteolytic enzymes (ectopeptidases) are integral membrane proteins, orientated asymmetrically with the catalytic site exposed to the extracellular surface, which enables a versatile range of physiological and pathological functions. Ectopeptidases may regulate the release of many growth factors and their receptors into the circulation, as well as activating or inactivating circulating signalling molecules, thereby regulating the availability of ligands for the corresponding receptors. Additionally, many of these ectopeptidases have functions not limited to proteolysis, but are able in themselves to function as receptors, transducing intracellular signals. A versatile range of functions, such as the modulation of cell-signalling, matrix degradation, cell adhesion and migration, which are particularly important for tumour cell growth and dissemination, are attributed largely to the ectopeptidases. Even a minor disruption in the normal proteolytic equilibrium can influence tumor progression, and a range of ectopeptidases, including neutral endopeptidase 24.11, aminopeptidase N, dipeptidyl peptidase IV, angiotensin-converting enzyme, and the disintegrin-metalloproteinases, have been shown to be involved in tumour development and metastasis. The ability to degrade and inactivate peptide hormones and growth factors, with the resultant modulation of the tumour-host interface, may play an important role in the pathogenesis, development or progression of a range of cancers, and the extracellular orientation of the ectopeptidases makes them particularly accessible, and therefore interesting, with regard to therapeutical applications.

Activation of the calcineurin signaling pathway induces atrial hypertrophy during atrial fibrillation.

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.

Expression of carbonic anhydrase 9 at the invasion front of gastric cancers.

BACKGROUND: Carbonic anhydrase IX (MN/Ca9) catalyses the reversible metabolism of carbon dioxide to carbonic acid and has also been linked to malignant transformation and hypoxia in various cancers. AIMS: To assess the expression and biological role of Ca9 in gastric cancer. METHODS: Using gastric cancer cell lines and tissues, we studied expression of Ca9 by western blot analysis, immunohistochemistry, and polymerase chain reaction. Biological changes after Ca9 transfection and after treatment with 5'-azadeoxycytidine were also analysed in cancer cell lines. RESULTS: Non-cancerous tissues strongly expressed Ca9 with membranous localisation. In contrast, Ca9 expression was frequently lost in gastric cancers (p<0.001). However, gastric cancers that retained Ca9 expression in cancer cells exhibited a shorter postoperative survival (p = 0.028). In vitro analysis revealed that loss of Ca9 expression in gastric cancer cell lines was restored after treatment with 5'-azadeoxycytidine and was associated with increased invasion (p<0.01). Moreover, AGS cells transfected with Ca9 exhibited significantly increased cell proliferation (p<0.05). CONCLUSIONS: A subgroup of gastric cancers retain Ca9 expression in cancer cells at the invasion front. While loss of Ca9 expression is regulated in part by methylation, re-expression of Ca9 is associated with increased invasion, supporting the hypothesis that increased Ca9 expression may contribute to invasion and thus advanced disease and tumour progression in a subset of gastric cancers.

[Molecular biology of the heart atrium. New insights into the pathophysiology of atrial fibrillation as well as its clinical implications]. / Molekularbiologie der Herzvorhöfe. Neue Erkenntnisse zur Pathophysiologie des Vorhofflimmerns sowie klinische Implikationen.

Atrial fibrillation (AF) is the most common clinical arrhythmia and one of the most important factors for embolic stroke. In recent years, a tremendous amount has been learned about the pathophysiology and molecular biology of AF. Thus, pharmacologic interference with specific signal transduction pathways appears promising as a novel antiarrhythmic approach to maintain sinus rhythm and to prevent atrial clot formation. This review highlights the underlying molecular biology of atrial fibrillation, which may also be relevant for AF therapy.

Expression of carbonic anhydrase IX in human pancreatic cancer.

BACKGROUND: Carbonic anhydrase IX has been linked to cancer development and progression. AIM: To analyse carbonic anhydrase IX expression and anhydrase inhibition in pancreatic cancer and to correlate these findings with p53 expression and microvessel density. MATERIALS AND METHODS: Seventy-seven pancreatic cancers were examined (43 males, 34 females; mean age, 64 years). The anti-carbonic anhydrase IX M75 antibody was used for immunohistochemistry and Western blot analysis. Microvessels were visualized using the anti-CD34 antibody, and p53 expression in cancer cells was assessed with a specific anti-p53 antibody. Quantitative polymerase chain reaction was performed in order to assess carbonic anhydrase IX mRNA levels in the pancreas. Furthermore, pancreatic cancer cell lines were treated with acetazolamide, a carbonic anhydrase inhibitor. RESULTS: In the normal pancreas, carbonic anhydrase IX immunoreactivity was observed at the basolateral membrane of ductal cells in 24 cases (31%). Carbonic anhydrase IX expression was found at the membrane and in the cytoplasm of pancreatic cancer cells in 16 pancreatic cancers (21%). Carbonic anhydrase IX expression was independent of the localization, stage, size, metastases and differentiation of the tumour. p53 expression was significantly more frequent in poorly differentiated cancers (P=0.0323); however, p53 expression and microvessel density were independent of carbonic anhydrase IX expression. Overall, carbonic anhydrase IX expression was not altered in pancreatic cancers vs. adjacent normal pancreatic tissue as assessed by Western blot and quantitative polymerase chain reaction analysis. However, incubation of pancreatic cancer cell lines with acetazolamide led to a significant inhibition of cell proliferation in AsPC-1 and PANC-1 pancreatic cancer cells. CONCLUSION: Carbonic anhydrase IX expression is observed in both ductal epithelial and cancer cells of the pancreas. Although the expression of carbonic anhydrase IX in pancreatic cancer is not associated with angiogenesis or advanced disease, it may well be a target for carbo-anhydrase inhibitors in a subset of pancreatic cancers.

Expression and activity of ectopeptidases in fibrillating human atria.

Recent studies have demonstrated that atrial fibrillation (AF) occurs in the presence of degenerative changes of atrial tissue. In contrast, bradykinin (BK) appears to have cardioprotective effects diminishing myocardial hypertrophy and fibrosis. It is unknown, however, whether AF has direct effects on BK metabolism. Therefore, the purpose of this study was to determine the atrial expression of the membrane-bound peptidases, also referred to as ectopeptidases, carboxypeptidase M (CPM), dipeptidyl peptidase IV (DPIV), and alanyl-aminopeptidase (APN) in patients with and without AF. Atrial tissue samples of 35 patients undergoing open heart surgery were examined. Seventeen patients had chronic persistent AF (> or = 6 months; CAF), the remaining 18 patients (controls) had no history of AF. Peptidase expression was analyzed at the mRNA (quantitative RT-PCR) level and apparent changes were confirmed at the protein level. In case of unaltered mRNA levels, enzyme activity was determined. Reduced amounts of CPM-mRNA were found in patients with CAF (41.3+/-9.7 U nu controls: 86.1+/-17.5 U P<0.05). CPM protein was decreased to 47.5% in patients with CAF compared with controls (P<0.01). DPIV and APN mRNA amounts were similar in both groups. DPIV activity, however, was increased during CAF (219.6+/-30 pkat/mg protein v controls: 195.8+/-21.8 pkat/mg P<0.05). APN activity was unchanged. In conclusion, atrial bradykinin metabolizing activities are significantly altered during AF in humans. The observed alterations in ectopeptidase expression/activity may play a role in the structural remodeling of fibrillating atria.

Dipeptidyl peptidase IV (DP IV/CD26) mRNA expression in PWM-stimulated T-cells is suppressed by specific DP IV inhibition, an effect mediated by TGF-beta(1).

Stimulation of human T-cells by pokeweed mitogen (PWM) results in a significant increase of IL-2, IFN-gamma, and DP IV mRNA expression as analyzed by quantitative RT-PCR. Here we show for the first time that the changes observed in cytokine mRNA expression are dose-dependently suppressed by the specific dipeptidyl peptidase IV inhibitor Lys[Z(NO(2))]-thiazolidide. Most interestingly, the inhibition of DP IV activity leads to a decrease in mRNA expression of the enzyme itself. Furthermore, evidence is provided that this suppression is mediated by TGF-beta(1). The presented data fit into the hypothesis that inhibition of DP IV leads to the induction of TGF-beta(1), which in turn provokes an arrest of cell cycle in late G(1).

Modulation of WNT-5A expression 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.

Specific inhibitors of dipeptidyl peptidase IV suppress mRNA expression of DP IV/CD26 and cytokines.

The dipeptidyl peptidase IV is an activation marker on T, B and NK cells. Specific inhibitors of DP IV suppress DNA synthesis, as well as cytokine protein production. Here, we describe for the first time the quantitative changes of mRNA expression of IFN-gamma, IL-2, IL-12 and DP IV after inhibition of DP IV. Due to the stimulation of human peripheral blood mononuclear cells (PBMC) with pokeweed mitogen (PWM) both cytokine as well as DP IV mRNA expression is increased significantly. Treatment with DP IV inhibitor suppresses dose-dependently these changes. Importantly, mRNA expression of DP IV itself was inhibited. The presented data are fully compatible with our hypothesis that inhibition of DP IV leads to cell cycle arrest in late G1 due to enhanced TGF-beta 1 expression.

DNA synthesis in cultured human keratinocytes and HaCaT keratinocytes is reduced by specific inhibition of dipeptidyl peptidase IV (CD26) enzymatic activity.

The ectopeptidase dipeptidyl peptidase IV (DP IV, CD26, EC 3.4.14.5) is present on most mammalian cells. Using specific inhibitors of DP IV, it has been shown that this enzyme is involved in the regulation of DNA synthesis and in production of various cytokines in lymphocytes. The aim of the present work was to investigate the expression of DP IV/CD26 on human keratinocytes and to answer the question, whether the proliferation (DNA synthesis) of human keratinocytes is influenced by inhibition of the enzymatic activity of DP IV. Using flow cytometry, RT-PCR, and specific enzymatic activity assays, expression of DP IV-mRNA and CD26 antigen were shown on primary keratinocyte strains and on the HaCaT keratinocyte cell line. The synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and -pyrrolidide suppress the DNA-synthesis of these cells in a dose-dependent manner. These data demonstrate that CD26 is also involved in the regulation of DNA synthesis of keratinocytes and that the enzymatic activity is required for mediating these effects.

Expression of transforming growth factor beta-1 in gastric cancer and in the gastric mucosa of first-degree relatives of patients with gastric cancer.

Transforming growth factors beta (TGF-beta) constitute a family of polypeptide growth factors that control cell growth, cell differentiation and migration, as well as the formation of the extracellular matrix. Recent analyses revealed the overexpression of TGF-beta1 in human gastric cancers and demonstrated increased cell proliferation in the stomach of patients with gastric cancer and their first-degree relatives. Using human gastric tissues obtained from patients with gastric cancer (n = 19), biopsies from healthy first-degree relatives of gastric cancer patients (n = 18) and healthy individuals (n = 19), we analysed the expression of TGF-beta1 using the reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Fifteen of 19 patients with gastric cancer expressed TGF-beta1 in the tumour. In 11 of these 15 cases TGF-beta1 mRNA was also detectable in the non-tumourous stomach. Interestingly, all but two individuals with a first-degree relative diagnosed with gastric cancer exhibited TGF-beta1 expression in either the antrum or corpus biopsy or both. In contrast, only one of 19 individuals without a family history of gastric cancer expressed TGF-beta1 in the stomach (P< 0.0001). TGF-beta1 expression is detectable in a large proportion of gastric cancers and in the stomach of healthy first-degree relatives of gastric cancer patients. Since individuals without gastric cancers in their family express TGF-beta1 only in one of 19 cases, the induction of TGF-beta1 expression in first-degree relatives of patients with gastric cancer points to the presence of specific molecular alterations in a subgroup of individuals with an increased risk of developing gastric cancer that may precede the development of gastric cancers.

Inhibition of alanyl-aminopeptidase suppresses the activation-dependent induction of glycogen synthase kinase-3beta (GSK-3beta) in human T cells.

Inhibition of alanyl-aminopeptidase (APN, CD13) gene expression or enzymatic activity compromises T cell proliferation and function. Molecular mechanisms mediating these effects are not known as yet. Recently, we found the expression of the proto-oncogen Wnt-5a to be strongly affected by APN-inhibition. Wnt-5a and other members of the Wnt family of secreted factors are implicated in cell growth and differentiation. Here, we analyzed by quantitative RT-PCR and immunoblotting the expression in mitogen-activated T cells of a major constituent of the Wnt-5a pathway, glycogen synthase kinase-3beta (GSK-3beta). T cell activation by phytohaemagglutinin or pokeweed mitogen results in a strong increase of GSK-3beta mRNA amounts. At the protein level, we observed an up-regulation of both GSK-3beta and phosphorylated GSK-3beta. This induction-dependent increase of GSK-3beta is markedly reduced in response to inhibitors of alanyl-aminopeptidase, actinonin, leuhistin, and RB3014. These findings may provide a rational for the growth inhibition resulting from a diminished expression or activity of alanyl aminopeptidase.