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Next-generation sequencing (NGS)-based typings of HLA-A, B, C, DQB1 and DRB1 loci were performed from 2018 to 2019 in 23 595 newly recruited or re-typed adult potential bone marrow donors registered in Poltransplant Registry to characterize allele and haplotype frequencies of HLA system for loci important for hematopoietic stem cell transplantation. The donors were recruited for registry and not for any other purpose including controls in a disease association study. The population sample was collected in various regions of Poland including all voivodships. The data regarding the degree of relatedness among individuals in the sample were not collected. Typings were supported by public funds as a part of the Polish National Program for Transplant Medicine Development. HLA frequency data are available in the Allele Frequencies Net Database.

The aim of the present study was to evaluate advanced glycation end products (AGEs) and soluble form of receptor RAGE (sRAGE) concentrations as well as the AGEs/sRAGE ratio in mild (MH) and resistant (RH) hypertensive patients in comparison with normotensive individuals. We also evaluated the association between AGEs, sRAGE as well as AGEs/sRAGE ratio and circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPCs). The MH group consisted of 30 patients, whereas 30 patients were classified for the RH group. The control group (C) included 25 normotensive volunteers. AGEs and sRAGE were measured using enzyme-linked-immunosorbent assay (ELISA). The multicolor flow cytometry was used for analysis of CECs and CEPCs. Significantly higher levels of AGEs in RH cohort were observed as compared to C cohort. Furthermore, significantly lower sRAGE levels as well as a higher AGEs/sRAGE ratio were observed between MH and RH cohorts. Significant correlations were found in the MH cohort for sRAGE and CECs, and CEPCs. The elevation of AGEs levels suggests that oxidative modification of proteins occurs in hypertension pathogenesis. The decrease in sRAGE levels and elevation of the AGEs/sRAGE ratio in MH and RH groups may suggest that hypertensive patients are less protected against the side effects of AGEs as a consequence of an insufficient competitive role of sRAGE against the AGEs-RAGE axis. Finally, it may be concluded that the level of AGEs may be an independent predictor of the condition and function of the endothelium. Furthermore, sRAGE may be classified as a potential biomarker of inflammation and endothelium dysfunction.

There is growing evidence for the contribution of the activated coagulation factor X (FXa) in the development of chronic inflammatory lung diseases. Therefore, we aimed to investigate effects of exogenous FXa on mitochondrial and metabolic function as well as the induction of inflammatory molecules in type II alveolar epithelial cells. Effects of FXa on epithelial cells were investigated in A549 cell line. Activation of extracellular signal-regulated kinase (ERK) and induction of inflammatory molecules were examined by immunoblot and gene expression analysis. Mitochondrial function was assessed by the measurement of oxygen consumption during maximal oxidative phosphorylation and quantitative determination of cardiolipin oxidation. Apoptosis was tested using a caspase 3 antibody. Metabolic activity and lactate dehydrogenase assay were applied for the detection of cellular viability. FXa activated ERK1/2 and induced an increase in the expression of pro-inflammatory cytokines, which was prevented by an inhibitor of FXa, edoxaban, or an inhibitor of protease-activated receptor 1, vorapaxar. Exposure to FXa caused mitochondrial alteration with restricted capacity for ATP generation, which was effectively prevented by edoxaban, vorapaxar and GB83 (inhibitor of protease-activated receptor 2). Of note, exposure to FXa did not initiate apoptosis in epithelial cells. FXa-dependent pro-inflammatory state and impairment of mitochondria did not reach the level of significance in lung epithelial cells. However, these effects might limit regenerative potency of lung epithelial cells, particular under clinical circumstances where lung injury causes exposure to clotting factors.

Background Hepatitis E virus (HEV) infection is an emerging problem in industrialized countries, including Europe. Little data exists on HEV seroprevalence in Poland. Objectives The aim of this study was to assess the prevalence of anti-HEV IgG antibodies in Polish patients infected with the human immunodeficiency virus (HIV) and blood donors. Material and methods Two hundred and ten individuals (n = 105 of HIV-infected patients and n = 105 of ageand sex-matched blood donors from the same area; 178 men and 32 women), aged 18-50 (median age: 38 years), were tested for the presence of anti-HEV IgG antibodies with the EUROIMMUN Anti-Hepatitis E Virus (HEV) ELISA (IgG) tests (Lubeck, Germany). Additionally, some simple clinical and laboratory data was collected. Results The overall anti-HEV IgG prevalence was 2.4% (5/210). One HIV-positive patient (0.95%) and 4 blood donors (3.8%) were seropositive (p = 0.1745). All the HEV-exposed individuals were men with a history of travel abroad and no icteric disease in the past. Conclusions Exposure to HEV infection among Polish HIV patients and blood donors seems to be uncommon. Data on this issue is scarce and conflicting for HIV-infected individuals. Further investigations applying different serological tests and concomitant HEV RNA testing are needed to reliably assess the risk and practical impact of HEV infection in Poland.

Background The aim of the present study was to evaluate endothelial status by measuring the concentration of novel markers of endothelial dysfunction (ED): a number of circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPCs) and their ratio (CEPCs/CECs) as well as a traditional parameter - soluble thrombomodulin (sTM) in patients with resistant (RH) and mild hypertension (MH). Materials and methods Thirty patients with MH and thirty subjects with RH were involved in the study. The control group included thirty-three age and sex-matched normotensive volunteers. We used multicolor flow cytometry for CECs and CEPCs analysis and the commercial human sTM ELISA kit to measure plasma sTM concentration. Results An elevated CECs number and a decreased CEPCs/CECs ratio was found in MH as well as in RH patients in comparison with normotensive volunteers. CECs correlated positively with an increased triglycerides in MH patients and an elevated LDL-cholesterol and hsCRP in RH group. Positive correlation between CEPCs and LDL-cholesterol level was observed in both types of hypertension. Conclusions The results of the present study suggest that an endothelial alteration accompanies hypertension. The number of CECs reflecting the extent of endothelial damage does not appear to be related to the severity of disease. The drastically decreased ratio between CEPCs and CECs observed in both groups of patients suggests an inadequate process of endothelial regeneration. Among analyzed factors inflammation and lipid abnormalities may have significant contribution in endothelial pathology in hypertension.

BACKGROUND Hepatitis E virus (HEV) infection is an emerging problem in developed countries. At least 2 zoonotic genotypes of the virus (HEV-3 and HEV-4) infect human beings. There are some data suggesting that forest rangers (FRs) can be at a higher risk of contact with HEV. OBJECTIVES The aim of this study was to assess the prevalence of HEV exposure markers in FRs from a single forest district in Greater Poland in relation to anti-HAV (hepatitis A virus) IgG, and anti-Borrelia spp. IgM and IgG antibodies. MATERIAL AND METHODS In total, 138 participants (48 FRs and 90 blood donors - BDs) were tested for anti-HEV IgM and IgG (EUROIMMUN Medizinische Labordiagnostika AG, Luebeck, Germany) and 96 individuals (48 FRs and 48 BDs) were tested for anti-HAV IgG (ARCHITECT immunoassays, Abbott Laboratories, Wiesbaden, Germany); anti-Borrelia IgM and IgG (EUROIMMUN kits) were assessed in FRs only. RESULTS Anti-HEV markers were detected in 3 participants (2.2%; IgM in 1 FR, IgG in 2 BDs), less frequently than anti-HAV (16 out of 96 individuals, about 17%; FRs 19% vs BDs 15%) or anti-Borrelia antibodies (18 out of 48 individuals, 37.5%) (p < 0.0001 for both). Older study participants (≥45 years of age) were more frequently HAV-seropositive (29% vs 4% of the younger individuals; p = 0.0012). CONCLUSIONS We failed to unequivocally prove HEV exposure in FRs. The HAV seroprevalence in this study paralleled the situation in the general population. Exposure to Borrelia spp. in FRs was common.

Background Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and is a potential cause of thromboembolic events. AF induces significant changes in the electrophysiological properties of atrial myocytes and causes alterations in the structure, metabolism, and function of the atrial tissue. The molecular basis for the development of structural atrial remodeling of fibrillating human atria is still not fully understood. However, increased production of reactive oxygen or nitrogen species (ROS/RNS) and the activation of specific redox-sensitive signaling pathways observed both in patients with and animal models of AF are supposed to contribute to development, progression and self-perpetuation of AF. Scope of Review The present review summarizes the sources and targets of ROS/RNS in the setting of AF and focuses on key redox-sensitive signaling pathways that are implicated in the pathogenesis of AF and function either to aggravate or protect from disease. Major Conclusions NADPH oxidases and various mitochondrial monooxygenases are major sources of ROS during AF. Besides direct oxidative modification of e.g. ion channels and ion handling proteins that are crucially involved in action potential generation and duration, AF leads to the reversible activation of redox-sensitive signaling pathways mediated by activation of redox-regulated proteins including Nrf2, NF-κB, and CaMKII. Both processes are recognized to contribute to the formation of a substrate for AF and, thus, to increase AF inducibility and duration. General Significance AF is a prevalent disease and due to the current demographic developments its socio-economic relevance will further increase. Improving our understanding of the role that ROS and redox-related (patho)-mechanisms play in the development and progression of AF may allow the development of a targeted therapy for AF that surpasses the efficacy of previous general anti-oxidative strategies. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

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. 6months 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.

Background Patients (pts) with severely decreased left ventricular ejection fraction (LV-EF ≤35%) are at high risk for sudden cardiac death (SCD). We sought to investigate, if pts with primary prevention ICD hold alterations in enzyme-activities of the dipeptidyl-aminopeptidase IV (DPIV) and the renin–angiotensin system (RAS) before VT/VF occurrence. Methods 57 Pts (53 male, mean age 64.9 [42–84] years, mean LV-EF 26±5%) with ischemic (n=49) or non-ischemic cardiomyopathy (n=8) who had received an ICD/CRT-D for primary prevention, were included. Pts were assessed for appropriate ICD intervention for VT/VF during a mean follow-up of 365±90days. Serum levels of dipeptidyl-aminopeptidase IV (DPIV), aminopeptidase N (APN), aminopeptidase B (APB), insulin-regulated aminopeptidase (IRAP), and angiotensin-converting enzyme 2 (ACE2) were determined. Results Pts with appropriate ICD intervention (n=16) had higher serum activities of IRAP (mean difference=12.681pkat/mL; p=0.007), and DPIV (mean difference=117.557pkat/mL; p=0.032) than pts without appropriate ICD intervention. Furthermore, ACE2 activity was significantly higher (median: 223.7RFU/smL vs. 169.10RFU/smL; p=0.037). A Cox regression analysis indicated DPIV activity >50th centile to have a hazard ratio (HR) of 5.955 (CI 95%: 1.670–21.241; p=0.006) for prediction of appropriate ICD intervention. In a multivariate Cox regression model, DPIV and IRAP >50th centile remained predictive for appropriate ICD intervention. Conclusion Our prospective study shows that pts with primary prevention ICD, who receive appropriate ICD intervention during follow-up, can be identified by elevated activities of DPIV and several RAS proteases. Hence, theses biomarkers seem to be of prognostic relevance in a primary prevention collective. Our data has to be proven in larger cohorts.

The vasopressin- and oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP) is expressed in various organs including the brain. However, knowledge about its presence in human hypothalamus is fragmentary. Functionally, for a number of reasons (genetic linkage, hydrolysis of oxytocin and vasopressin, its role as angiotensin IV receptor in learning and memory and others) IRAP might play a role in schizophrenia. We studied the regional and cellular localization of IRAP in normal human brain with special emphasis on the hypothalamus and determined numerical densities of IRAP-expressing cells in the paraventricular, supraoptic and suprachiasmatic nuclei in schizophrenia patients and controls. By using immunohistochemistry and Western blot analysis, IRAP was immunolocalized in postmortem human brains. Cell countings were performed to estimate numbers and numerical densities of IRAP immunoreactive hypothalamic neurons in schizophrenia patients and control cases. Shape, size and regional distribution of IRAP-expressing cells, as well the lack of co-localization with the glia marker glutamine synthetase, show that IRAP is expressed in neurons. IRAP immunoreactive cells were observed in the hippocampal formation, cerebral cortex, thalamus, amygdala and, abundantly, hypothalamus. Double labeling experiments (IRAP and oxytocin/neurophysin 1, IRAP with vasopressin/neurophysin 2) revealed that IRAP is present in oxytocinergic and in vasopressinergic neurons. In schizophrenia patients, the numerical density of IRAP-expressing neurons in the paraventricular and the suprachiasmatic nuclei is significantly reduced, which might be associated with the reduction in neurophysin-containing neurons in these nuclei in schizophrenia. The pathophysiological role of lowered hypothalamic IRAP expression in schizophrenia remains to be established.

Aims More precise characterization of risk factors for occurring ventricular arrhythmia in patients (pts) with primary prevention implantable cardioverter–defibrillator (ICD) therapy is critical. We sought to investigate whether biomarkers of nitric oxide metabolism can predict the occurrence of ventricular tachyarrhythmias and might be used as risk markers in these pts. Methods and results Plasma levels of l-arginine (Arg), asymmetric dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), monomethyl l-arginine, and nitrite/nitrate were examined in 106 consecutive pts (mean age 65 years, 97 male, mean LV-EF 24 ± 6%), with ischaemic ( n = 82) or non-ischaemic cardiomyopathy ( n = 24) who underwent ICD implantation for primary prevention of SCD. Appropriate ICD intervention was assessed during a mean follow-up of 344 days, and occurred in 18 of 106 (17%) pts. Asymmetric dimethylarginine plasma levels were significantly higher in pts with appropriate ICD intervention compared with those without any ICD intervention (0.564 ± 0.083 μmol/L vs. 0.513 ± 0.088 μmol; P = 0.027). The Arg/ADMA ratio was found lower in pts with appropriate ICD intervention than in those without ICD intervention (144.71 ± 32.50 vs. 175.29 ± 41.29; P = 0.002). Univariate Cox regression showed that ADMA ( P = 0.028) and the Arg/ADMA ratio ( P = 0.003) were associated with a higher incidence of appropriate ICD intervention. In a multivariable Cox regression analysis, an ADMA concentration above the 50th centile was independently associated with appropriate ICD intervention, revealing a hazard ratio (HR) of 4.21 (CI 95 %: 1.14–15.63; P = 0.028, Table [4][1] ). An Arg/ADMA ratio below the 25th centile had a HR of 3.83 (1.360–10.87; P = 0.011). Conclusion Asymmetric dimethylarginine and the Arg/ADMA ratio seem to be new biomarkers for the prediction of ventricular tachycardia/ventricular fibrillation episodes and of appropriate ICD intervention in pts with left ventricular ejection fraction dysfunction (LV-EF ≤35%), suggesting a value for risk stratification in these pts. [1]: #T4

Cardiovascular diseases (CVD) and, in particular, coronary artery disease (CAD) are the leading causes of death in developed countries, especially in the elderly population. Males exhibit a higher risk for cardiovascular events than women. The pericardial fluid (PF) is in direct contact with the epicardial sections of the coronary arteries and the perimyocardium. A systematic analysis of gender-specific or age-related differences in angiotensin-related pathways like bradykinin metabolism however, has not been performed in the PF so far. Therefore, the amounts of angiotensin-converting enzyme (ACE) and the rate of the degradation of bradykinin (BK) and the amounts/activity of major BK-degrading enzymes, aminopeptidase N (APN) and dipeptidyl-aminopeptidase IV (DPIV), were assessed in the pericardial fluid (PF) of 44 patients undergoing coronary artery bypass grafting. We found BK being degraded within the PF. Interestingly, there was an age-dependent decrease in the amounts of ACE protein in women. In elderly women, ACE/APN and ACE/DPIV ratios were substantially reduced to 41.4% or 29.4% respectively (p

Insulin-degrading enzyme (IDE) is a neutral thiol metalloprotease, which cleaves insulin with high specificity. Additionally, IDE hydrolyzes Abeta, glucagon, IGF I and II, and beta-endorphin. We studied the expression of IDE protein in postmortem brains of patients with schizophrenia and controls because: (1) the gene encoding IDE is located on chromosome 10q23-q25, a gene locus linked to schizophrenia; (2) insulin resistance with brain insulin receptor deficits/receptor dysfunction was reported in schizophrenia; (3) the enzyme cleaves IGF-I and IGF-II which are implicated in the pathophysiology of the disease; and (4) brain gamma-endorphin levels, liberated from beta-endorphin exclusively by IDE, have been reported to be altered in schizophrenia. We counted the number of IDE immunoreactive neurons in the dorsolateral prefrontal cortex, the hypothalamic paraventricular and supraoptic nuclei, and the basal nucleus of Meynert of 14 patients with schizophrenia and 14 matched control cases. Patients had long-term haloperidol treatment. In addition, relative concentrations of IDE protein in the dorsolateral prefrontal cortex were estimated by Western blot analysis. There was a significantly reduced number of IDE expressing neurons and IDE protein content in the left and right dorsolateral prefrontal cortex in schizophrenia compared with controls, but not in other brain areas investigated. Results of our studies on the influence of haloperidol on IDE mRNA expression in SHSY5Y neuroblastoma cells, as well as the effect of long-term treatment with haloperidol on the number of IDE immunoreactive neurons in rat brain, indicate that haloperidol per se, is not responsible for the decreased neuronal expression of the enzyme in schizophrenics. Haloperidol however, might exert some effect on IDE, through changes of the expression levels of its substrates IGF-I and II, insulin and beta-endorphin. Reduced cortical IDE expression might be part of the disturbed insulin signaling cascades found in schizophrenia. Furthermore, it might contribute to the altered metabolism of certain neuropeptides (IGF-I and IGF-II, beta-endorphin), in schizophrenia.

Recent studies associate cathepsin K with schizophrenia. The endogenous substrates of this protease, however, remain to be identified. We show here that cathepsin K is capable of liberating met-enkephalin from beta-endorphin (beta-EP) in vitro. To verify if this process might possibly contribute in the pathogenesis of schizophrenia post mortem brains of patients suffering from this disease were analysed immunohistochemically for the presence and co-localization of cathepsin K and beta-EP. In support of a functional role of the observed formation of met-enkephalin on the expense of beta-EP increased numbers of cathepsin K immunoreactive cells, but diminished numbers of both beta-EP-positive cells and double-positive (cathepsin K/beta-EP) cells were found in left and right arcuate nucleus of schizophrenics. In addition a reduced density of beta-EP-immunoreactive neuropil (fibres, nerve terminals) was estimated in the left and right paraventricular nucleus (PVN) of individuals with schizophrenia. Our results imply that cathepsin K, which becomes up-regulated in its cerebral expression by neuroleptic treatment, might significantly contribute to altered opioid levels in brains of schizophrenics, which have previously been reported by us and others, and might reinforce the interest in the putative roles of endorphin and enkephalins in neuropsychiatric disorders.

Accumulating evidence links calcium-overload and oxidative stress to atrial remodeling during atrial fibrillation (AF). Furthermore, atrial remodeling appears to increase atrial thrombogeneity, characterized by increased expression of adhesion molecules. The aim of this study was to assess mitochondrial dysfunction and oxidative stress-activated signal transduction (nuclear factor-kappaB [NF-kappa B], lectin-like oxidized low-density lipoprotein receptor [LOX-1], intercellular adhesion molecule-1 [ICAM-1], and hemeoxgenase-1 [HO-1]) in atrial tissue during AF. Ex vivo atrial tissue from patients with and without AF and, additionally, rapid pacing of human atrial tissue slices were used to study mitochondrial structure by electron microscopy and mitochondrial respiration. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunoblot analyses, gel-shift assays, and enzyme-linked immunosorbent assay (ELISA) were applied to measure nuclear amounts of NF-kappa B target gene expression. Using ex vivo atrial tissue samples from patients with AF we demonstrated oxidative stress and impaired mitochondrial structure and respiration, which was accompanied by nuclear accumulation of NF-kappa B and elevated expression levels of the adhesion molecule ICAM-1 and the oxidative stress-induced markers HO-1 and LOX-1. All these changes were reproduced by rapid pacing for 24 hours of human atrial tissue slices. Furthermore, the blockade of calcium inward current with verapamil effectively prevented both the mitochondrial changes and the activation of NF-kappa B signaling and target gene expression. The latter appeared also diminished by the antioxidants apocynin and resveratrol (an inhibitor of NF-kappa B), or the angiotensin II receptor type 1 antagonist, olmesartan. This study demonstrates that calcium inward current via L-type calcium channels contributes to oxidative stress and increased expression of oxidative stress markers and adhesion molecules during cardiac tachyarrhythmia.

Telmisartan, in addition to blocking angiotensin (Ang) II type 1 receptor (AT 1 R), activates peroxisome proliferator activated receptor γ (PPARγ) signaling that interferes with nitric oxide (NO) system. Because aging of endothelial cells (ECs) is hallmarked by a reduction in NO synthesis, we hypothesized that telmisartan increases NO formation by regulated asymmetrical dimethylarginine (ADMA)-dimethylarginine dimethylaminohydrolase (DDAH)-system through blocking AT 1 R and activating PPARγ signaling. To test this hypothesis, ECs were cultured with telmisartan, eprosartan, Ang II, and GW9662 (PPARγ antagonist) until the twelfth passage. During the process of aging, PPARγ protein expression decreased significantly, whereas the expression of AT 1 R increased. Telmisartan reversed these effects and dose-dependently decreased reactive oxygen species and 8-iso-prostaglandin (PG) F 2α formation. This effect was associated with an upregulated activity and protein expression of DDAH, accompanied by a decrease in ADMA concentration, an increase in NO metabolites, and delayed senescence. Blockade of PPARγ signaling by GW9662 or PPARγ small-interference RNA prevented the effect of telmisartan on ADMA-DDAH-NO system. Coincubation with Ang II did not affect the effect of telmisartan-delayed senescence, whereas Ang II itself accelerated endothelial aging. Moreover, AT 1 R blocker eprosartan that did not influence PPARγ protein expression had no effect on ADMA system and senescence. We have demonstrated that telmisartan mainly by activating PPARγ signaling can alter the catabolism and release of ADMA as an important cardiovascular risk factor. We therefore propose that telmisartan translationally and posttranslationally upregulated DDAH expression via activation of PPARγ signaling, causing ADMA to diminish and increase NO synthesis sufficient to delay senescence.

Background— Increased levels of inflammatory markers are predictors of thromboembolic events during atrial fibrillation (AF). Increased endocardial expression of adhesion molecules (ie, vascular cell adhesion molecule [VCAM] and intercellular adhesion molecule [ICAM]) could be an important link between initiation of inflammatory and prothrombogenic mechanisms responsible for thrombus development at the atrial endocardium (endocardial remodeling). Methods and Results— Tissue microarrays were used to screen right atrial tissue specimens obtained from 320 consecutive patients for differences in atrial expression of the prothrombogenic proteins VCAM-1, ICAM-1, thrombomodulin, plasminogen activator inhibitor-1, and von Willebrand factor. An in vitro organotypic human atrial tissue model and a pig model of rapid atrial pacing were used to determine the therapeutic impact of angiotensin II receptor blockade. Immunohistochemical analyses showed that all prothrombogenic proteins are expressed by endocardial cells. Using multivariable analysis, only the intensity of VCAM-1 expression was increased in patients with AF ( P =0.03). Increased atrial VCAM-1 expression was confirmed by Western blotting in patients with persistent and paroxysmal AF (persistent AF 207±42% versus sinus rhythm 100±16%, P =0.028; paroxysmal AF 193±42%, P =0.024 versus sinus rhythm). In vitro pacing of ex vivo human atrial tissue slices confirmed that rapid activation causes VCAM-1 upregulation (mRNA and protein levels). Pacing-induced VCAM-1 expression was abolished by olmesartan. To confirm this finding in vivo, VCAM-1 expression was determined in 14 pigs after rapid atrial pacing (600 bpm). Atrial tachycardia caused an upregulation of VCAM-1 expression, which was prevented by irbesartan, consistent with the observed increase in plasma levels of angiotensin II. Alterations in the in vivo VCAM-1 expression were more pronounced in the left atrium (>5-fold compared with sham) than in the right atrium (3.5-fold compared with sham). Conclusions— AF and rapid atrial pacing both increase endocardial VCAM-1 expression, which can be attenuated by angiotensin II receptor blockade. This provides evidence that angiotensin II plays a pathophysiological role in prothrombotic endocardial remodeling.

Cigarette smoking (CS) promotes endothelial dysfunction and atherosclerosis in the vascular bed. The impact of smoking on atrial myocardium is not defined in humans.To determine the effect of CS on the development of interstitial fibrosis in atrial myocardium.Case-control study.95 patients (46 smokers and 49 non-smokers) undergoing coronary artery bypass grafting (CABG).Amount of atrial fibrosis, collagen I, III and IV expression pattern, and quantitative reverse transcriptase-PCR. Occurrence of postoperative atrial fibrillation (AF).In the study population, patient age correlated significantly with the amount of atrial fibrosis (r = 0.18; p0.05). Nicotine misuse (pack years) was identified as the only factor related to atrial fibrosis in smokers (r = 0.311; p0.05). The amount of fibrosis was higher in patients with postoperative AF (22.9% (6.2%) vs. 27.0% (8.2%); p0.05). To show a causal relationship between CS and atrial fibrosis, atrial tissue slices from non-smokers (n = 8) were cultured in the presence of nicotine base (185 and 740 nmol/l). Nicotine base induced mRNA expression of collagen III (up to 10-fold) in a concentration-dependent manner resembling the immunohistological collagen expression pattern observed in CS.CS contributes to the development of atrial fibrosis via nicotine. Atrial fibrosis by itself has been shown to provide an arrhythmogenic substrate, which may increase the likelihood of the occurrence of atrial arrhythmias, including postoperative AF.

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.

Aminopeptidase inhibitors strongly affect proliferation, differentiation, and function of immune cells and show therapeutic potential in inflammatory disorders. In psoriatic lesions, keratinocytes display increased cellular turnover and disturbed differentiation, leading to epidermal hyperplasia accompanied by the loss of stratum granulosum. Here, we report in the HaCaT hyperproliferative keratinocyte cell line as well as in two primary keratinocyte strains in vitro a molecular and biochemical analysis of the expression of both membrane and cytosol alanyl aminopeptidase (cAAP) on the mRNA, protein, and enzymatic activity level. We found a clear dose-dependent suppression of DNA synthesis in vitro in the presence of the inhibitors actinonin, bestatin, and the cAAP-specific inhibitor PAC-22 correlating well with the simultaneous decrease in enzyme activity. In vivo, actinonin dose-dependently restored the stratum granulosum and ameliorated the impaired keratinocyte differentiation in the mouse tail model of psoriasis. Taken together, these data suggest that targeting alanyl aminopeptidases may be beneficial for psoriasis and other inflammatory skin disorders.

Using immunohistochemical techniques we studied the light microscopic localization of ADAMs (A Disintegrin And Metalloprotease) 10 and 15 in different neocortical areas of the human brain during normal aging, and also in patients with Alzheimer disease (AD) and Down syndrome (DS). ADAM 10, a putative alpha-secretase involved in Notch signaling, was found in neurons of the perinatal cortex. During aging there is an increase in intraneuronal staining intensity and in the number of cortical nerve cells that contain the enzyme. Furthermore, in AD and DS brains ADAM 10 immunoreactivity was associated with diffuse and neuritic plaques. ADAM 15 was detected in perinatal cortical pyramidal cells; during aging there was also an increase in intracellular staining and the number of stained cells per volume (cell density). In AD brains ADAM 15 was seen in a few diffuse plaques. Morphometric analysis revealed a significant reduction of ADAM 10 but not ADAM 15 immunoreactive neurons in AD brains in comparison to controls. Our findings support the idea that ADAM 10 is involved in the pathophysiology of AD and DS. ADAM 15 might be linked to AD via interaction with integrin and/or src protein tyrosine kinases.

In recent years a tremendous amount has been learned about the pathophysiology of atrial fibrillation (AF) which induces electrophysiological changes in the right and left atrium. Besides calcium-dependent tissue changes which are induced by activation of proteases and phosphatases, such as calpain and calcineurin, concomitant cardiac diseases activate the atrial angiotensin II system. Experiments have shown positive effects of statins in AF models. In contrast, clinical studies have provided heterogeneous results. Of note, several studies have shown that therapy with angiotensin II receptor blockers or statins does not influence the recurrence of AF.

Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, which is activated during atrial fibrillation. Increasing evidence suggests that FXa influences cell signalling in various cell types by activating protease-activated receptors (PARs). It is so far not known if molecular effects of FXa affect atrial signal transduction. To study the effects of FXa, human atrial tissue slices were cultivated with FXa up to 24h. Additionally, rapid pacing was applied at 4Hz to resemble atrial fibrillation. The inhibitory impact of FXa antagonist (Rivaroxaban), protease-activated receptor 1 antagonist (SCH79797), and protease-activated receptor 2 antagonist (GB83) were analysed under experimental conditions. The exposure of atrial tissue to FXa resulted in the 1.7 fold upregulation of PAR2-mRNA, activation of MAP kinases (ERK1/2) and NF-κB signalling. Furthermore FXa increased the expression of adhesion molecule ICAM-1 (1.82 ± 0.20), chemokine IL-8 (1.94 ± 0.20), as well as prothrombotic molecule PAI-1 (1.52 ± 0.17). The combination of rapid pacing and FXa caused significant upregulation of PAR1 (2.82 ± 0.22), PAR2 (2.66 ± 0.40), ICAM-1 (2.13 ± 0.25), IL-8 (2.22 ± 0.24), LOX-1 (2.59 ± 0.35), and PAI-1 (2.65 ± 0.52) at the mRNA level. Rivaroxaban and GB83 prevented upregulation of PARs, ICAM-1, LOX-1, IL-8, and activation of MAP kinases. The elevation in the expression of PAI-1 was hindered in the presence of SCH79797, or Rivaroxaban. The present study indicates that FXa mediates inflammatory signalling in atrial tissue. Importantly, FXa and tachyarrhythmia act synergistically to increase expression of protease-activated receptors and inflammatory mediators. Rivaroxaban prevented effectively FXa-induced molecular effects in human atrial tissue particularly during rapid pacing.

Calpain is an intracellular Ca(2+)-activated protease and an important mediator of the actions of calcium. Cleavage by calpain is critical in a variety of calcium-regulated cellular processes such as muscle contraction, neuronal excitability, secretion, signal transduction, cell proliferation, differentiation, cell cycle progression, and apoptosis. Deregulation of calpain caused by a disruption of calcium homeostasis during cardiac pathologies such as atrial fibrillation, heart failure, hypertrophy, or ischemia reperfusion, is critically involved in the myocardial damage. This review will summarize the physiologic and pathophysiologic basis of calpain. Atrial fibrillation is chosen as one example to explain the specific consequences of an increased calpain activity in cardiac muscle.

The endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a well-established risk factor for oxidative stress, vascular dysfunction, and congestive heart failure. The aim of the present study was to determine the impact of rapid atrial pacing (RAP) on ADMA levels and eNOS expression.ADMA levels were studied in 60 age- and gender-matched patients. Thirty five patients had persistent atrial fibrillation (AF)≥ 4months. In AF-patients, parameters were studied before and 24h after electrical cardioversion. Moreover, ADMA, eNOS expression, and calcium-handling proteins were studied in pigs subjected to RAP as well as in endothelial cell (EC) cultures. ADMA level was significantly higher in AF compared to sinus rhythm patients (p=0.024). ADMA was highest in AF-patients, who also showed elevated troponin T (TnT) levels. Moreover, ADMA showed a significant linear correlation to TnT (r=0.47; p0.01). After electrical cardioversion ADMA returned to normal within 24h. In pigs, RAP for 7h increased ADMA levels (p=0.018) and TnI (p0.05), and reduced mRNA expression of ventricular and aortic eNOS (-80%; p0.05) compared to sham-control. However, ADMA per se did not affect eNOS mRNA level in EC cultures.The current study shows that acute and persistent episodes of atrial tachyarrhythmia are associated with elevated ADMA levels accompanied by increased ischemic myocardial markers. Moreover, RAP increases ADMA and down-regulates eNOS expression in an ADMA-independent manner. We conclude that the combination of these two separate and potentially synergistic mechanisms may contribute to long-term vascular injury during atrial tachyarrhythmia.

Background and objective: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Recent studies suggest there is an angiotensin II-dependent increase in adhesion molecules and oxidative stress parameters during AF. These alterations appear to contribute to inflammatory and prothrombotic changes in the atrial endocardium (‘endocardial remodelling’), suggesting that patients with increased levels of these factors might be at risk of thromboembolic events. The purpose of the CREATIVE-AF (Impact of Irbesartan on Oxidative Stress and C-Reactive Protein Levels in Patients with Persistent Atrial Fibrillation) trial is to prove the principle concept that blockade of angiotensin II type 1 receptors by irbesartan reduces levels of circulating adhesion molecules and oxidative stress parameters in patients with persistent AF by 25% compared with placebo. Methods: This is a prospective, randomized, double-blind, placebo-controlled, crossover study in patients with persistent/permanent AF. A total of 60 patients are planned to be included in the study. Patients will receive placebo and irbesartan therapy for 9 weeks each. Levels of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inflammatory markers (high sensitivity C-reactive protein, monocyte chemoattractant protein-1, von Willebrand factor, tumour growth factor-β1, tumour necrosis factor-α, interleukin-6) and oxidative stress parameters (8-iso-prostaglandin F2α) will be determined after each treatment phase and compared with baseline levels. Conclusion: Angiotensin II type 1 receptor antagonists may help reduce levels of circulating adhesion molecules and oxidative stress parameters in patients with persistent AF. This article summarizes the rationale and design of the CREATIVE-AF trial, which has been designed to test this hypothesis. ClinicalTrials.gov identifier: NCT00613496.

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.

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.

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.