Rosa M. Agra

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 αIIbβ3 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.

Impaired Adipogenesis and Insulin Resistance in Epicardial Fat-Mesenchymal Cells From Patients With Cardiovascular Disease

The thickness of epicardial adipose tissue (EAT), which is an inflammatory source for coronary artery disease (CAD), correlates with insulin resistance. One trigger factor is impaired adipogenesis. Here, our aim was to clarify the underlying mechanisms of insulin resistance on EAT‐mesenchymal cells (MC). EAT and subcutaneous adipose tissue (SAT) were collected from 19 patients who were undergoing heart surgery. Their dedifferentiated adipocytes (DAs) and/or MCs were cultured. After the induction of adipogenesis or stimulation with insulin, the expression of adipokines was analyzed using real‐time polymerase chain reaction (PCR). Colorimetric assays were performed to measure glucose levels and proliferation rate. Proteins modifications were detected via the proteomic approach and Western blot. Our results showed lower adipogenic ability in EAT‐MCs than in SAT‐MCs. Maximum adiponectin levels were reached within 28–35 days of exposure to adipogenic inducers. Moreover, the adipogenesis profile in EAT‐MCs was dependent on the patients’ clinical characteristics. The low adipogenic ability of EAT‐MCs might be associated with an insulin‐resistant state because chronic insulin treatment reduced the inflammatory cytokine expression levels, improved the glucose consumption, and increased the post‐translational modifications (PTMs) of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1). We found lower adipogenic ability in EAT‐MCs than in SAT‐MCs. This lower ability level was dependent on gender and the presence of diabetes, obesity, and CAD. Low adipogenesis ability and insulin resistance in EAT‐MCs might shed light on the association between EAT dysfunction and cardiovascular disease. J. Cell. Physiol. 229: 1722–1730, 2014.

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

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.

Effects of dapagliflozin on human epicardial adipose tissue: modulation of insulin resistance, inflammatory chemokine production, and differentiation ability

Aims In patients with cardiovascular disease, epicardial adipose tissue (EAT) is characterized by insulin resistance, high pro-inflammatory chemokines, and low differentiation ability. As dapagliflozin reduces body fat and cardiovascular events in diabetic patients, we would like to know its effect on EAT and subcutaneous adipose tissue (SAT). Methods and results Adipose samples were obtained from 52 patients undergoing heart surgery. Sodium-glucose cotransporter 2 (SGLT2) expression was determined by real-time polymerase chain reaction (n = 20), western blot, and immunohistochemistry. Fat explants (n = 21) were treated with dapagliflozin and/or insulin and glucose transporters expression measured. Glucose, free fatty acid, and adipokine levels (by array) were measured in the EAT secretomes, which were then tested on human coronary endothelial cells using wound healing assays. Glucose uptake was also measured using the fluorescent glucose analogue (6NBDG) in differentiated stromal vascular cells (SVCs) from the fat pads (n = 11). Finally, dapagliflozin-induced adipocyte differentiation was assessed from the levels of fat droplets (AdipoRed staining) and of perilipin. SGLT2 was expressed in EAT. Dapagliflozin increased glucose uptake (20.95 ± 4.4 mg/dL vs. 12.97 ± 4.1 mg/dL; P < 0.001) and glucose transporter type 4 (2.09 ± 0.3 fold change; P < 0.01) in EAT. Moreover, dapagliflozin reduced the secretion levels of chemokines and benefited wound healing in endothelial cells (0.21 ± 0.05 vs. 0.38 ± 0.08 open wound; P < 0.05). Finally, chronic treatment with dapagliflozin improved the differentiation of SVC, confirmed by AdipoRed staining [539 ± 142 arbitrary units (a.u.) vs. 473 ± 136 a.u.; P < 0.01] and perilipin expression levels (121 ± 10 vs. 84 ± 11 a.u.). Conclusions Dapagliflozin increased glucose uptake, reduced the secretion of pro-inflammatory chemokines (with a beneficial effect on the healing of human coronary artery endothelial cells), and improved the differentiation of EAT cells. These results suggest a new protective pathway for this drug on EAT from patients with cardiovascular disease.

Differential Association of S100A9, an Inflammatory Marker, and p53, a Cell Cycle Marker, Expression with Epicardial Adipocyte Size in Patients with Cardiovascular Disease

ABSTRACTS100A9 (calgranulin B) has inflammatory and oxidative stress properties and was found to be associated with atherosclerosis and obesity. One of the proteins that can regulate S100A9 transcription is p53, which is involved in cell cycle, apoptosis and adipogenesis. Thus, it triggers adipocyte enlargement and finally obesity. Because epicardial adipose tissue (EAT) volume and thickness is related to coronary artery disease (CAD), we studied the gene expression of this pathway in patients with cardiovascular disease and its association with obesity. Adipocytes and stromal cells from EAT and subcutaneous adipose tissue (SAT) from 48 patients who underwent coronary artery bypass graft and/or valve replacement were obtained after collagenase digestion and differential centrifugation. The expression levels of the involved genes on adipogenesis and cell cycle like fatty acid-binding protein (FABP) 4, retinol-binding protein (RBP)4, p53 and S100A9 were determined by real-time polymerase chain reaction (PCR). Adipocyte diameter was measured by optical microscopy. We found that epicardial adipocytes expressed significantly lower levels of adipogenic genes (FABP4 and RBP4) and cell cycle-related genes (S100A9 and p53) than subcutaneous adipocytes. However, in obese patients, upregulation of adipogenic and cell cycle-related genes in subcutaneous and epicardial adipocytes, respectively, was observed. The enlargement of adipocyte size was related to FABP4, S100A9 and p53 expression levels in stromal cells. But only the p53 association was maintained in epicardial stromal cells from obese patients (p = 0.003). The expression of p53, but not S100A9, in epicardial stromal cells is related to adipocyte enlargement in obese patients with cardiovascular disease. These findings suggest new mechanisms for understanding the relationship between epicardial fat thickness, obesity and cardiovascular disease.

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.

Omentin treatment of epicardial fat improves its anti-inflammatory activity and paracrine benefit on smooth muscle cells

Epicardial adipose tissue (EAT) in coronary artery disease is insulin resistant and has a proinflammatory profile. This study examined the regulation of EAT by exogenous omentin and its consequence on vascular cells.

Orosomucoid as prognosis factor associated with inflammation in acute or nutritional status in chronic heart failure

BACKGROUND Inflammation and nutritional state are involved in the pathogenesis of heart failure (HF). OBJECTIVE To study the contribution of alpha-1-acid-glycoprotein (AGP) to these factors and its prognostic value in acute (AHF) or chronic HF (CHF). METHODS The observational study has included 147 patients (mean age 70years, 62% men) admitted to a cardiology department for HF and followed-up for an average 326.6±140.8days. Blood AGP values were measured by Enzyme-Linked ImmunoSorbent Assay. Monocytes subsets were determined with CD14 and CD16 antibodies by flow cytometry and body composition was measured by dual-energy X-ray absorptiometry. The regulation of tumor necrosis factor (TNF-α) and leptin by AGP in epicardial adipose tissue (EAT) were analyzed by real time polymerase chain reaction. RESULTS High AGP, that was associated with CD14+CD16+ monocytes, and proBNP levels at the discharge were indicators of rehospitalization for HF in AHF patients. However, low AGP levels determined a worse nutritional state in CHF patients. The leptin levels were downregulated by high AGP concentration in epicardial fat. CONCLUSION AGP is a dual indicator in HF because high levels are predictors of adverse outcomes in AHF but low levels are related to the worse nutritional status in CHF. The regulation of leptin by AGP in epicardial fat might suggest a new pathway as protective mechanism in CHF.

Nutrients restriction upregulates adiponectin in epicardial or subcutaneous adipose tissue: impact in de novo heart failure patients

Background: Hyperadiponectinemia is an indicator of worse outcomes in advanced heart failure (HF), its role in de novo HF is less clear. Objective: Because this protein is a hormone with starvation properties, we wanted to know its association with nutritional state and its regulator factors in de novo HF. Methods: Adiponectin circulating levels were determined by ELISA at discharge in patients admitted for de novo HF (n=74). Nutritional status was determined by CONUT score. Univariate and multivariate Cox regression analyses were employed to calculate the estimated hazard ratio (HR) with 95% confidence interval (CI) for death or all-cause readmission. Stromal vascular cells (SVC) of EAT and subcutaneous adipose tissue (SAT) from patients (n=5) underwent heart surgery were induced to adipogenesis for 18 days. Then, cells were cultured with complete or starved medium for 8 hours. At the end, adiponectin expression levels were analysed by real time polymerase chain reaction. Results: Patients were grouped regarding nutritional status. There was a strong association between high adiponectin levels and failing nutritional status. Those patients with worse nutritional state had the highest adiponectin and proBNP levels at discharge (p<0.01). Both proteins were slightly correlated (p<0.05). However, only high adiponectin levels were independently associated with death or all-cause readmission. Nutrients starvation upregulated adiponectin expression levels in adipogenesis-induced SVC from EAT or SAT. Conclusions: Worse nutritional state in de novo HF patients is associated with higher adiponectin plasma levels. Their levels were upregulated in adipose cells after being nutrients-starved. These results may help us to understand the adiponectin paradox in HF.