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Viral infection activated alloreactive peripheral memory T-cells present in the host are considered as one of the causes of transplant related complications during post-transplant period of unrelated donor and haploidentical graft transplantation procedures. Antibody and T-cell receptor coding genes homology and their analogous rearrangement process help to understand cellular and humoral alloreactivity, mediated by alloreactive memory T-cells and antibodies, respectively. Simple tests using single antigen presenting cells or human leukocyte antigen (HLA)-mismatched recipient peripheral blood mononuclear cells vs. virus specific donor T-cells and more sophisticated natural killer cells degranulation marker assays based on flow cytometry enable to rule out or confirm alloreactivity thus increasing the chance of appropriate post-transplant treatment path selection.

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.

Background The main aim of present study is to evaluate the potential role of circulating endothelial cells (CECs) and endothelial progenitor cells (CEPCs) – representing specific markers of endothelial damage, in the prediction of left ventricular hypertrophy (LVH) in hypertensive patients categorized into two groups; mild (MH) and resistant hypertension (RH). Materials and Methods Thirty patients with MH and 28 subjects with RH were involved in the study. In both groups, patients were divided into an LVH and non-LVH group. The control group included 33 age and sex-matched normotensive volunteers. Physical examination, laboratory tests and echocardiography were conducted. Results In both the MH and RH group, patients with as well as without LVH demonstrated a higher number of CECs and a lower ratio of CEPCs/CECs as compared to the healthy control. Multiple linear regression analysis showed a positive association of CEPCs with left ventricular mass (LVM) and left ventricular mass index (LVMI), independently of other confounders. Conclusion Our results suggest that endothelial injury observed as an elevated CECs number and its impaired regeneration, reflected by a lowered CEPCs/CECs ratio, precede LVH occurrence and may play a significant role in LVH development regardless of the clinical severity of hypertension. Moreover, independent correlation of CEPCs with echocardiographic (ECG) incidences of LVH suggests their potential use as a screening biomarker to stratify the risk of LVH development.

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.

Dronedarone improves microvascular flow during atrial fibrillation and reduces the infarct size in acute models of myocardial infarction. However, dronedarone might be harmful in patients with recent decompensated heart failure and increases mortality in patients with permanent atrial fibrillation. A pathophysiological explanation for these discrepant data is lacking. This study investigated the effects of dronedarone on gene and protein expression in the infarcted area and border zone in pigs subjected to anterior ischemia/reperfusion myocardial infarction. The ischemia/reperfusion myocardial infarction was induced in 16 pigs. Eight pigs were treated with dronedarone for 28 days after myocardial infarction, the remaining pigs served as control. Microarray-based transcriptome profiling and 2D-DIGE-based proteome analysis were used to assess the effects of dronedarone on left ventricular gene expression in healthy (LV), infarcted (MI), and border zone tissue. Selected targets were validated by RT-qPCR or immunoblot analyses, with special emphasize given to the transcriptome/proteome overlap. Combined “omics” analysis was performed to identify most significant disease and function charts affected by dronedarone and to establish an integrated network. The levels of 879 (BZ) or 7 (MI) transcripts and 51 (LV) or 15 (BZ) proteins were significantly altered by dronedarone, pointing to a substantial efficacy of dronedarone in the border zone. Transcriptome and proteome data indicate that dronedarone influences post-infarction remodeling processes and identify matricellular proteins as major targets of dronedarone in this setting. This finding is fully supported by the disease and function charts as well as by the integrated network established by combined “omics”. Dronedarone therapy alters myocardial gene expression after acute myocardial infarction with pronounced effects in the border zone. Dronedarone promotes infarct healing via regulation of periostin and might contribute to the limitation of its expansion as well as cardiac rupture. Thus, there are no experimental hints that dronedarone per se has direct harmful effects after MI in ventricular tissue. Impact statement Dronedarone reduced the infarct size in models of acute myocardial infarction (MI). Here, we show that dronedarone attenuates many of the substantial changes in gene expression that are provoked by acute myocardial infarction (AMI) in pigs. Dronedarone modifies the expression of gene panels related to post-infarction cardiac healing and remodeling processes and, most remarkably, this occurs predominantly in the infarction border-zone and much less so in the vital or infarcted myocardium. Combined “omics” identified matricellular proteins and ECM as major dronedarone-regulated targets and emphasizes their relevance for Disease Charts and Tox Function Charts associated with tissue remodeling and cellular movement. The results demonstrate dronedarone’s capability of regulating cardiac repair and remodeling processes specifically in the infarction border zone and identify underlying mechanisms and pathways that might be employed in future therapeutic strategies to improve long-term cardiac tissue function and stability.

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.

Aim of the study: This study aimed at integrating research discussing the role of perceived psychosocial barriers in cervical cancer screening (CCS) uptake. In particular, we analyzed the evidence for the associations between CCS uptake and perceived psychosocial barriers and frequency of psychosocial barriers identified by women. Material and methods: A systematic search of peer-reviewed papers published until 2011 in 8 databases yielded 48 original studies, analyzing data obtained from 155 954 women. The majority of studies (k = 43) applied correlational design, while 5 had experimental design. Results: Experimental research indicated a positive effect of 75% of psychosocial interventions targeting barriers. The interventions resulted in a significant increase of CCS uptake. Overall 100% of correlational studies indicated that perceiving lower levels of barriers significantly predicted higher CCS uptake. 53 psychosocial barriers were listed in at least 2 original correlational studies: 9.5% of barriers were related to CCS facilities/environment, 67.9% dealt with personal characteristics of the patient, and 22.6% addressed social factors. As many as 35.9% of perceived barriers referred to negative emotions related to CCS examination procedures and collecting CCS results, whereas 25.7% of barriers referred to prior contacts with health professionals. Conclusions: Leaflets or discussion on psychosocial barriers between patients and health professionals involved in CCS might increase CCS uptake and thus reduce cervical cancer mortality rates. Communication skills training for health professionals conducting CCS might focus on the most frequently reported barriers, referring to emotions related to CCS examination and collecting CCS results.

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.

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, F2-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.

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

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.

Plans Not Needed if You Have High and Stable Self-Efficacy: Planning Intervention and Snack Intake in the Context of Self-Efficacy Trajectories Forming action plans is expected to move people from intention to action. We hypothesized that the effects of planning interventions may depend on changes in self-efficacy beliefs. Participants (182 nurses and midwifes, 89% women, aged 19-50) were assigned to the control or the planning intervention (three planning sessions) groups and reported their self-efficacy, sweet and salty snack intake at the baseline and four months later. The results suggest that an increase of efficacy beliefs over time augmented the effects of the planning intervention and resulted in the lowest snack intake (the enhancing effect of self-efficacy). Planning intervention also prompted lower unhealthy snacking if efficacy beliefs were decreasing (the protective effect of planning). Those who have stable-high self-efficacy were able to achieve low snack intake regardless of the group assignment (the buffering effect of self-efficacy).

Aims Recent studies suggest that atrial fibrillation (AF) substantially influences microvascular flow in ventricular myocardium. This process may contribute to the occurrence of heart failure in AF. In general, development of heart failure and renal dysfunction go hand-in-hand causing systemic fluid overload and oedema. So far, it is unknown whether AF itself influences renal function. The aim of the present study was to determine the impact of AF on renal gene expression in a closed chest rapid atrial pacing model. Methods and results A total of 14 pigs were studied. In five pigs, rapid atrial pacing (AT) was performed for 7 h (600 bpm); in five additional animals, rapid atrial pacing was performed in the presence of irbesartan infusion (irbesartan group). Four pigs were instrumented without interventions (sham). After the pacing period, renal expression of collagen Ia1 and Ia3, transforming growth factor-b (TGF-b), neutral endopeptidase (NEP; the main enzyme involved in natriuretic protein metabolism), and atrial natriuretic peptide (ANP) were determined by RT–PCR and immunoblot analysis. Functional in vitro experiments were performed using HEK-293 kidney cells. Renal mRNA expression of NEP was substantially downregulated during AT (AT: 12.7+ 9.3% vs. sham: 100+ 43.4%; P , 0.01). Results at the mRNA level were confirmed at the protein level. Irbesartan therapy did not prevent down-regulation of NEP. In contrast, TGF-b1 mRNA expression was up-regulated (AT: 208.5+ 79.3% vs. sham: 100+ 34.6% P , 0.05). Collagen and angiotensin II type 1 receptor (AT1R) expression were not significantly altered by AT. HEK-293 cells were used to determine the potential humoral factors involved in down-regulation of NEP. Application of aldosterone, ANP, asymmetric dimethylarginine, and angiotensin peptides failed to cause down-regulation of renal NEP expression in vitro. Conclusion AT reduces NEP expression and stimulates TGF-b1 signalling in the kidneys. Thus, even brief episodes of AT affect renal gene expression, which may account for structural renal changes and alterations of renal function in the long term.

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.

Aims Hypertension is a major risk factor for atrial fibrillation. We hypothesized that arterial hypertension would alter atrial myocyte calcium (Ca2+) handling and that these alterations would serve to trigger atrial tachyarrhythmias. Methods and results Left atria or left atrial (LA) myocytes were isolated from spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) controls. Early after the onset of hypertension, at 3 months of age, there were no differences in Ca2+ transients (CaTs) or expression and phosphorylation of Ca2+ handling proteins between SHR and WKY. At 7 months of age, when left ventricular (LV) hypertrophy had progressed and markers of fibrosis were increased in left atrium, CaTs (at 1 Hz stimulation) were still unchanged. Subcellular alterations in Ca2+ handling were observed, however, in SHR atrial myocytes including (i) reduced expression of the alpha 1C subunit of and reduced Ca2+ influx through L-type Ca2+ channels, (ii) reduced expression of ryanodine receptors with increased phosphorylation at Ser2808, (iii) decreased activity of the Na+/Ca2+ exchanger (at unaltered intracellular Na+ concentration), and (iv) increased SR Ca2+ load with reduced fractional release. These changes were associated with an increased propensity of SHR atrial myocytes to develop frequency-dependent, arrhythmogenic Ca2+ alternans. Conclusions In SHR, hypertension induces early subcellular LA myocyte Ca2+ remodelling during compensated LV hypertrophy. In basal conditions, atrial myocyte CaTs are not changed. At increased stimulation frequency, however, SHR atrial myocytes become moreprone to arrhythmogenic Ca2+ alternans, suggesting a link between hypertension, atrial Ca2+ homeostasis, and development of atrial tachyarrhythmias.

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.

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.

Patients with atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non ST-segment elevation myocardial infarction; NSTEMI) during an acute episode of AF. However, in a large proportion of these patients significant coronary artery disease is excluded by coronary angiography, which suggests that AF itself influences myocardial blood flow. The present review summarizes the effect of AF on the occurrence of ventricular oxidative stress, redox-sensitive signalling pathways & gene expression, and microcirculatory flow abnormalities in the left ventricle.

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.

Aims Patients with paroxysmal atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non ST-segment elevation myocardial infarction) during an arrhythmic event. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography. Here we explored the potential underlying mechanism of these events. Methods and results A total of 14 pigs were studied using a closed chest, rapid atrial pacing (RAP) model. In five pigs RAP was performed for 7 h (600 b.p.m.; n = 5), in five animals RAP was performed in the presence of angiotensin-II type-1-receptor (AT1-receptor) inhibitor irbesartan (RAP+Irb), and four pigs were instrumented without intervention (Sham). One-factor analysis of variance was performed to assess differences between and within the three groups. Simultaneous measurements of fractional flow reserve (FFR) and coronary flow reserve (CFR) before, during, and after RAP demonstrated unchanged FFR ( P = 0.327), but decreased CFR during RAP (RAP: 67.7 ± 7.2%, sham: 97.2 ± 2.8%, RAP+Irb: 93.2 ± 3.3; P = 0.0013) indicating abnormal left ventricular (LV) microcirculation. Alterations in microcirculatory blood flow were accompanied by elevated ventricular expression of NADPH oxidase subunit Nox2 ( P = 0.039), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1, P = 0.004), and F2-isoprostane levels ( P = 0.008) suggesting RAP-related oxidative stress. Plasma concentrations of cardiac troponin-I (cTn-I) increased in RAP (RAP: 613.3 ± 125.8 pmol/L vs. sham: 82.5 ± 12.5 pmol/L; P = 0.013), whereas protein levels of eNOS and LV function remained unchanged. RAP+Irb prevented the increase of Nox2, LOX-1, and F2-isoprostanes, and abolished the impairment of microvascular blood flow. Conclusion Rapid atrial pacing induces AT1-receptor-mediated oxidative stress in LV myocardium that is accompanied by impaired microvascular blood flow and cTn-I release. These findings provide a plausible mechanism for the frequently observed cTn-I elevation accompanied with typical angina pectoris symptoms in patients with paroxysmal AF and normal (non-stenotic) coronary arteries.

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

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

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

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.

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

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