Adiponectin and p53 mRNA in epicardial and subcutaneous fat from heart failure patients.

OBJECTIVE: Heart failure (HF) is associated with a pro-inflammatory state in epicardial fat, but the involved mechanisms are not entirely clear. The aim of our study was to assess the relationship between p53 and adiponectin mRNA in epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT) in patients with heart failure and its sympathetic regulation. METHODS: Epicardial adipose tissue and SAT samples were obtained from 63 patients undergoing elective cardiac surgery. EAT and SAT explants culture from seven patients were stimulated with isoprenaline 0.1 or 1 uM for 6 h. p53 and adiponectin mRNA expression was measured in frozen biopsies or explants culture from both fat pads by real-time polymerase chain reaction (PCR). RESULTS: We observed that EAT expressed more p53 mRNA than SAT (1.73 ± 0.07 vs. 1.69 ± 0.04, P < 0.001) and its levels were higher in HF patients (1.75 ± 0.07 vs. 1.70 ± 0.04, P < 0.01 in EAT and 1.70 ± 0.04 vs. 1.67 ± 0.04, P < 0.05 in SAT). Moreover, p53 mRNA expression was negatively correlated with adiponectin in EAT. After analysing the p53 mRNA regulation by isoprenaline, we observed that only EAT p53 expression increased after adrenergic stimulation (1.63 ± 0.01 vs. 1.66 ± 0.02; P = 0.024). CONCLUSIONS: p53 mRNA expression levels, inversely correlated with adiponectin, increase in EAT of HF patients and can be regulated by sympathetic activation pathway. Our findings can help to explain the deleterious effect of sympathetic activation in HF.

Coronary artery disease is associated with higher epicardial retinol-binding protein 4 (RBP4) and lower glucose transporter (GLUT) 4 levels in epicardial and subcutaneous adipose tissue.

OBJECTIVE: Retinol-binding protein 4 (RBP4), produced by adipocytes and hepatocytes, contributes to an unfavourable lipid profile and insulin resistance, which can contribute to the development of coronary artery disease (CAD). Recently, several studies have shown that epicardial adipose tissue (EAT) differs from subcutaneous adipose tissue (SAT) and plays a role on the physiopathology of CAD because of its proximity to the coronary arteries. We aimed to study the expression and secretion levels of RBP4 in both fat tissues and explore its possible association with CAD. RESEARCH DESIGN AND METHODS: Fifty-eight patients undergoing heart surgery were included in the study. We analysed RBP4 mRNA expression by real-time PCR, protein expression by Western blot and immunohistochemistry, and secretion of EAT and SAT explants from CAD and non-CAD patients by Enzyme Immunoassay. RESULTS: Retinol-binding protein 4 is expressed at similar levels in EAT and SAT, mainly from adipocytes. Protein levels were higher in EAT from CAD than non-CAD patients (0·63 ± 0·09 arbitrary units (a.u).; n = 10) vs (0·41 ± 0·04 a.u.; n = 13, P = 0·039). In contrast, GLUT4 mRNA levels were lower in EAT from CAD than non-CAD patients (6·55 ± 0·16 a.u.; n = 13) vs (7·21 ± 0·18 a.u.; n = 14, P = 0·012). We also found differential expression in SAT between samples from CAD and non-CAD patients [(6·63 ± 0·16 a.u.; n = 14) vs (7·21 ± 0·14 a.u.; n = 14, P = 0·009)]. Besides, EAT releases higher RBP4 levels than SAT after 3, 6, 24 and 48 h of culture. These levels were independent of CAD but significantly higher in diabetic than nondiabetic patients. CONCLUSION: Retinol-binding protein 4 levels behave differently in EAT and SAT with respect to CAD. However, both adipose tissues have lower GLUT4 levels in patients with CAD. These findings suggest a differential regulation of RBP4 production in EAT and SAT that may be influenced by local factors.

Baseline epicardial adipose tissue adiponectin levels predict cardiovascular outcomes: a long-term follow-up study.

OBJECTIVE: Epicardial adipose tissue (EAT) produces a wide range of adipokines and has recently been linked to the physiopathology of cardiovascular (CV) and metabolic diseases. We aimed to study whether EAT and subcutaneous (SAT) adiponectin and leptin expression levels are associated with CV complications during long-term follow-up. METHODS AND RESULTS: EAT and SAT samples were obtained during surgery - mainly CABG (n=77) - from 137 patients (mean age 69.9 years, 31% women). Adiponectin and leptin mRNA levels were analyzed by RT-PCR. Plasma adiponectin levels were determined in a subsample of subjects (n=43). Thirty-four patients developed CV complications during 41 (SD 23) months of mean follow-up. Patients with CV events had lower EAT and SAT adiponectin levels at baseline (12.4 (3.0) vs. 15.7 (3.8) a.u., P=0.001; and 13.7 (2.6) vs. 15.7 (4.4) a.u., P=0.048, respectively). However, baseline EAT and SAT leptin levels and plasma adiponectin levels were not significantly different between patients with/without CV events during follow-up. Cox proportional hazards models adjusting for covariates in stages revealed that only baseline EAT adiponectin levels and heart failure could predict CV events. CONCLUSIONS: EAT adiponectin levels are strong predictors of CV prognosis in patients with CV diseases. EAT is likely to play a major role in the development of CV complications mainly through local effects.

Variations in platelet proteins associated with ST-elevation myocardial infarction: novel clues on pathways underlying platelet activation in acute coronary syndromes.

OBJECTIVE: Our aim in this study was to provide novel information on the molecular mechanisms playing a major role in the unwanted platelet activation associated with ST-elevation myocardial infarction (STEMI). METHODS AND RESULTS: We compared the platelet proteome of 11 STEMI patients to a matched control group of 15 stable chronic ischemic cardiopathy patients. In addition, we did a prospective study to follow the STEMI patients over time. Proteins were separated by high-resolution 2D gel electrophoresis, identified by mass spectrometry, and validated by Western blotting. Platelets from STEMI patients on admission displayed 56 protein spot differences (corresponding to 42 unique genes) compared with the control group. The number of differences decreased with time during the patients' follow-up. Interestingly, the adapter protein CrkL and the active form of Src (phosphorylated in Tyr418) were found to be upregulated in platelets from STEMI patients. Major signaling pathways related to the proteins identified include integrin, integrin-linked kinase, and glycoprotein VI (GPVI) signaling. Interestingly, a study on an independent cohort of patients showed a higher degree of activation of GPVI signaling in STEMI patients. CONCLUSIONS: CrkL, the active form of Src, and GPVI signaling are upregulated in platelets from STEMI patients.

Proteomic analysis of epicardial and subcutaneous adipose tissue reveals differences in proteins involved in oxidative stress.

Epicardial adipose tissue (EAT) is an endocrine organ adjacent to coronary arteries and myocardium without anatomy barriers. Locally produced adipokines may reflect or affect to cardiovascular physiology and pathology. Our aim was to study the protein expression profiles of EAT and subcutaneous adipose tissue (SAT) to identify local candidate molecules characterizing EAT in patients with cardiovascular disease. EAT and SAT samples were collected from 55 patients undergoing heart surgery. Proteins from these tissues were separated by two-dimensional (2D) gel electrophoresis, and differences between them were identified by MALDI-TOF/TOF spectra. Differences in protein levels were further investigated by real-time RT-PCR and Western blots, and production of reactive oxygen species (ROS) in EAT and SAT was evaluated by nitroblue tetrazolium chloride assays. ROS production was higher in EAT than SAT. We have found mRNA differences for catalase, glutathione S-transferase P, and protein disulfide isomerase, and 2D Western blots additionally showed post-translational differences for phosphoglycerate mutase 1; all four are related to oxidative stress pathways. EAT suffers greater oxidative stress than SAT in patients with cardiovascular diseases and exhibits associated proteomic differences that suggest the possibility of its association with myocardial stress in these patients.

Relationship between epicardial adipose tissue adipocyte size and MCP-1 expression.

Adipocyte size has been associated to increase in inflammatory cytokines expression that can be related to the cardiovascular risk of obesity. Epicardial adipose tissue (EAT) was discovered to play a key role in cardiovascular diseases by producing several inflammatory adipokines. We sought to study whether EAT and subcutaneous adipose tissue (SAT) mean adipocyte sizes are related to the expression of adipokines in patients with cardiovascular diseases. We collected EAT, SAT and blood samples from 22 patients aged 70.9 (s.d. 10.3) undergoing heart surgery. Monocyte chemoattractant protein (MCP)-1, interleukin (IL)-10 and tumor necrosis factor (TNF)-alpha were analyzed by real time RT-PCR, ELISA or immunohistochemistry. Hematoxylin-eosin staining was used for adipocyte area calculations. Adipocyte size is negatively correlated to MCP-1 expression (r=-0.475; p=0.034) in EAT and positively correlated in SAT (r=0.438; p=0.047). These trends persisted after stratification for sex and coronary artery disease (CAD), but only the relationship between EAT MCP-1 and adipocyte size reached statistical significance in the larger group of men with CAD. We have observed that SAT adipocyte size is correlated to BMI (r=0.601; p=0.003); whereas only a non-statistically significant trend was observed in EAT. IL-10 and TNF-alpha expression were not associated to adipocyte size in EAT nor SAT. Secondarily, we found that EAT IL-10 expression is higher in patients with CAD. These results suggest that adipocyte size is a negative determinant of MCP-1 expression in EAT and a positive determinant in SAT. These data might partly explain the different implications of EAT and SAT in cardiovascular diseases.

Why and when do patients with heart failure and normal left ventricular ejection fraction die? Analysis of >600 deaths in a community long-term study.

BACKGROUND: The aim of the study was to examine the causes of the death of patients with heart failure (HF) and evaluate the differences in this respect between patients with and without depression of left ventricular ejection fraction (LVEF). METHOD: All patients hospitalized with HF between 1995 and 2002 in the cardiology service of a tertiary hospital were assessed. LVEF was evaluated by echocardiography during hospitalization and was considered normal when it was > or =50%. After a mean follow-up time of 3.7 +/- 2.8 years, 615 cases had terminated in death. RESULTS: The most common cause was refractory HF, both in the whole group (39%) and in both the subgroups defined with respect to LVEF (normal and depressed). There was no statistically significant difference between the normal and depressed subgroups as regard the distribution of deaths, although the depressed group showed a somewhat greater incidence of sudden death (21% as against 16% in the normal group) and a somewhat smaller incidence of death due to refractory HF (37% as against 47%). However, in the depressed LVEF group, the cumulative risk of death due to acute myocardial infarction in the first 1.5 years first increased rapidly and then more slowly, whereas the reverse pattern was held in the normal left ventricular systolic function group, in which it was the cumulative risks of death from noncardiovascular or vascular noncardiac causes that initially increased more rapidly than later. CONCLUSIONS: The spectrum of causes of death among patients with HF who have been hospitalized is independent of LVEF in the long term. In the short term, there are differences between patients with normal LVEF and depressed LVEF as regard the dynamics of the risks of death from acute myocardial infarction, noncardiac vascular causes, and noncardiovascular causes. These results may help orient the short-term and long-term management of HF, especially for patients with normal LVEF, for whom there is still no well-established consensus strategy.

Extension of coronary artery disease is associated with increased IL-6 and decreased adiponectin gene expression in epicardial adipose tissue.

UNLABELLED: Epicardial adipose tissue (EAT) expresses lower levels of adiponectin in patients with CAD and higher levels of inflammatory mediators such as IL-6 and leptin than subcutaneous adipose tissue. This showed one important role of EAT in coronary artery disease. However, the relationship of EAT adiponectin and IL-6 levels to the extension of coronary artery disease has not hitherto been determined. We sought to determine whether the levels of adiponectin and interleukin-6 (IL-6) mRNA in epicardial adipose tissue are associated with the extension of coronary artery disease (CAD). METHODS: Angiographic and hormones expression were evaluated from epicardial and subcutaneous adipose tissue. 92 patients (58 CAD, 34 non-CAD) who underwent cardiac surgery. Adiponectin and IL-6 mRNA levels were measured by real time RT-PCR in epicardial and subcutaneous adipose tissue (SAT) following angiographic evaluation of their coronary arteries. RESULTS: We found that epicardial adipose tissue of CAD expressed lower levels of adiponectin mRNA and higher levels of IL-6 mRNA than that of non-CAD patients. As the number of injured arteries rose, adiponectin mRNA levels decreased (r=-0.402, p<0.001) and IL-6 mRNA increased (r=0.514, p<0.001) in epicardial adipose tissue. CONCLUSIONS: The extension of CAD is significantly associated with the expression of adiponectin and IL-6 mRNA in EAT. These findings suggest that low adiponectin and high IL-6 expression by EAT may contribute to CAD extension.

Influence of contact force on voltage mapping: A combined magnetic resonance imaging and electroanatomic mapping study in patients with tetralogy of Fallot.

BACKGROUND: Voltage criteria for ventricular mapping have been obtained from small series of patients and prioritizing high specificity. OBJECTIVE: The purpose of this study was to analyse the potential influence of contact force (CF) on voltage mapping and to define voltage cutoff values for right ventricular (RV) scar using the tetralogy of Fallot as a model of transmural RV scar and magnetic resonance imaging (MRI) as reference. METHODS: Fourteen patients (age 32.6 ± 14.3 years; 5 female) with repaired tetralogy of Fallot underwent high-resolution cardiac MRI (1.25 × 1.25 × 2.5 mm). Scar, defined as pixels with intensity >50% maximum, was mapped over the RV geometry and merged within the CARTO system to RV endocardial voltage maps acquired using a 3.5-mm ablation catheter with CF technology (SmartTouch, Biosense Webster). RESULTS: In total, 2446 points were analyzed, 915 within scars and 1531 in healthy tissue according to MRI. CF correlated to unipolar (ρ = 0.186; P <.001) and bipolar voltage in healthy tissue (ρ = 0.245; P <.001) and in scar tissue. Receiver operating characteristic curve analysis excluding points with very low CF (<5g) identified optimal voltage cutoffs of 5.19 mV for unipolar voltage and 1.76 mV for bipolar voltage, yielding sensitivity/specificity of 0.89/0.85 and 0.9/0.9, respectively. CONCLUSION: CF is an important factor to be taken into account for voltage mapping. If good CF is applied, unipolar and bipolar voltage cutoffs of 5.19 mV and 1.76 mV are optimal for identifying RV scar on endocardial mapping with the SmartTouch catheter. Data on the diagnostic accuracy of different voltage cutoff values are provided.

Pulmonary vein stenosis: Etiology, diagnosis and management.

Pulmonary vein stenosis (PVS) is rare condition characterized by a challenging diagnosis and unfavorable prognosis at advance stages. At present, injury from radiofrequency ablation for atrial fibrillation has become the main cause of the disease. PVS is characterized by a progressive lumen size reduction of one or more pulmonary veins that, when hemodynamically significant, may raise lobar capillary pressure leading to signs and symptoms such as shortness of breath, cough, and hemoptysis. Image techniques (transesophageal echocardiography, computed tomography, magnetic resonance and perfusion imaging) are essential to reach a final diagnosis and decide an appropriate therapy. In this regard, series from referral centers have shown that surgical and transcatheter interventions may improve prognosis. The purpose of this article is to review the etiology, assessment and management of PVS.

Differential behavior between S100A9 and adiponectin in coronary artery disease. Plasma or epicardial fat.

AIMS: S100A9 is a new inflammatory marker associated with obesity and cardiovascular disease. Because epicardial adipose tissue (EAT) is an inflammatory source in coronary artery disease (CAD), our aim was to evaluate the S100A9 levels in plasma and EAT and its association with CAD. MAIN METHODS: Blood, EAT and/or subcutaneous adipose tissue (SAT) biopsies were obtained from 89 patients undergoing elective cardiac surgery. Plasma S100A9 and adiponectin were analyzed by enzyme-linked immunosorbent assay (ELISA) and mRNA expression in both fat pads and were measured by real-time polymerase chain reaction (PCR). KEY FINDINGS: Our results have shown higher levels of plasma S100A9 in patients with CAD than those without (29 [10-50] vs. 17 [3-28] ng/mL; p=0.007). They were dependent on the number of injured-coronaries (p=0.002) with tendency toward negative association with plasma adiponectin (p=0.139). Although EAT expressed higher levels than SAT and their levels were higher in CAD patients, this last difference did not reach statistical significance. However, there was a positive correlation between neutrophils and EAT S100A9 expression (p=0.007) that may reveal an increase of neutrophil filtration on this fat pad. SIGNIFICANCE: Plasma S100A9 levels are increased in chronic CAD. The absence of differences regarding EAT S100A9 expression levels indicates a differential inflammatory process between fat tissues and blood in CAD process.

Changes in lipid transport-involved proteins of epicardial adipose tissue associated with coronary artery disease.

OBJECTIVE: Recent studies have focused on the potential role of epicardial adipose tissue (EAT) in the physiopathology of several metabolic and cardiovascular diseases, especially coronary artery disease (CAD). We aimed to study whether there are differences in the proteome and the secretome between epicardial and subcutaneous adipose tissue (SAT) from patients with and without CAD. METHODS: EAT and SAT samples were collected from 64 patients undergoing elective cardiac surgery either for coronary artery bypass grafting or valve surgery. One or two-dimensional electrophoresis were performed on tissue samples and media collected at 3, 6, 24 or 48 of tissue culture. Protein identification was performed with mass spectrometry, and the results were then validated with Western blot or enzyme immunoassay. mRNA expression levels were analysed by real time polymerase chain reaction. RESULTS: The release of several proteins was found to be higher in EAT that in SAT. Remarkably, there were higher levels of apolipoprotein A-I and glutation S-transferase P release, whereas mRNA expression of fatty acid binding protein 4 was lower in EAT. Although apolipoprotein A-I protein quantity in EAT was similar between CAD and non CAD patients, its released levels from this fat pad were lower in CAD. CONCLUSION: EAT and SAT show different profiles of protein release and a different pattern was also found in samples from patients with CAD. These findings might support the hypothesis that EAT plays an interesting role in the physiopathology of atherosclerosis and CAD.

High-resolution two-dimensional gel electrophoresis analysis of atrial tissue proteome reveals down-regulation of fibulin-1 in atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia found in clinical practice. We combined high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to compare the atrial proteome of subjects with AF versus controls with sinus rhythm (SR). Our aim was to identify novel differentially regulated proteins that could be related to the development of the arrhythmia. METHODS: Human atrial appendage tissue samples from patients undergoing heart surgery with AF or SR were analyzed by high-resolution 2-DE. Proteins of interest were identified by MS and validated by western blotting and inmunohistochemistry. RESULTS: Our analysis allowed the detection of over 2300 protein spots per gel. Following differential image analysis, we found 22 spot differences between the AF and SR groups in the 4-7 isoelectric point range, leading to the identification of 15 differentially regulated proteins. The main group of proteins identified was that of heat shock proteins (HSPs), including TRAP-1, HspB3, HspΒ6 and AHA1. Some of the differences detected between AF and SR for the above proteins were due to post-translational modifications. In addition, we identified the structural protein fibulin-1 as down-regulated in atrial tissue from AF patients. CONCLUSIONS: High-resolution 2-DE analysis of human atrial tissue revealed that AF is associated with changes in structural proteins and an important number of HSPs. The lower expression of the structural protein fibulin-1 in atrial tissue from AF patients might reflect the myocardial structural changes that take place in the arrhythmia.

Proteins involved in platelet signaling are differentially regulated in acute coronary syndrome: a proteomic study.

BACKGROUND: Platelets play a fundamental role in pathological events underlying acute coronary syndrome (ACS). Because platelets do not have a nucleus, proteomics constitutes an optimal approach to follow platelet molecular events associated with the onset of the acute episode. METHODOLOGY/PRINCIPAL FINDINGS: We performed the first high-resolution two-dimensional gel electrophoresis-based proteome analysis of circulating platelets from patients with non-ST segment elevation ACS (NSTE-ACS). Proteins were identified by mass spectrometry and validations were by western blotting. Forty protein features (corresponding to 22 unique genes) were found to be differentially regulated between NSTE-ACS patients and matched controls with chronic ischemic cardiopathy. The number of differences decreased at day 5 (28) and 6 months after the acute event (5). Interestingly, a systems biology approach demonstrated that 16 of the 22 differentially regulated proteins identified are interconnected as part of a common network related to cell assembly and organization and cell morphology, processes very related to platelet activation. Indeed, 14 of those proteins are either signaling or cytoskeletal, and nine of them are known to participate in platelet activation by αIIbß3 and/or GPVI receptors. Several of the proteins identified participate in platelet activation through post-translational modifications, as shown here for ILK, Src and Talin. Interestingly, the platelet-secreted glycoprotein SPARC was down-regulated in NSTE-ACS patients compared to stable controls, which is consistent with a secretion process from activated platelets. CONCLUSIONS/SIGNIFICANCE: The present study provides novel information on platelet proteome changes associated with platelet activation in NSTE-ACS, highlighting the presence of proteins involved in platelet signaling. This investigation paves the way for future studies in the search for novel platelet-related biomarkers and drug targets in ACS.

[Adipokines as novel cardiovascular disease markers. Pathological and clinical considerations]. / Adipocitocinas como nuevos marcadores de la enfermedad cardiovascular. Perspectivas fisiopatológicas y clínicas.

Adipose tissue exhibits a high level of metabolic activity that stems from the synthesis and secretion of hormones associated with energy metabolism. These hormones have a direct effect on the structure and function of the heart and on the cardiovascular system as a whole. Since the discovery of leptin, a group of adipokines produced mainly by adipose tissue, but also by other tissues, has been identified. Interestingly, these adipokines may also be synthesized and secreted by cardiomyocytes, and they have a direct influence on cardiomyocyte structure and metabolism. Epicardial fat is an important part of visceral fat and has a high level of metabolic activity. Compared with subcutaneous fat, this fat compartment expresses less adiponectin but more interleukins, and it is linked to the severity of coronary artery disease and the presence of hypertension. The adipokines form a family of hormones whose actions exert significant effects on the cardiovascular system. In particular, they are associated with atherothrombosis and could provide a new therapeutic target for reducing cardiovascular risk.