Expression profile of adrenomedullin and its specific receptors in liver tissues from patients with hepatocellular carcinoma and in tumorigenic cell line-secreted extracellular vesicles.

The transcriptional profile of adrenomedullin (AM), a new metastasis-related factor involved in hepatocellular carcinoma (HCC), and its specific receptors (CLR, RAMP1, RAMP3) were evaluated in liver tissues of HCV-positive HCC subjects undergoing liver transplantation (LR) and in donors (LD). AM and its specific receptor expression were also assessed in extracellular vesicles (EVs) secreted by tumorigenic (HepG2) and non-tumorigenic (WRL68) cells by Real-Time PCR. AM expression resulted significantly elevated in LR concerning LD (p = 0.0038) and, for the first time, significantly higher levels in HCC patients as a function of clinical severity (MELD score), were observed. RAMP3 and CLR expression increased in LR as a function of clinical severity while RAMP1 decreased. Positive correlations were found among AM, its receptors, and apoptotic markers. No AM mRNA expression difference was observed between HepG2 and WRL68 EVs. RAMP1 and RAMP3 resulted lower in HepG2 concerning WRL68 while significantly higher levels were observed for CLR. While results at tissue level characterize AM as a regulator of carcinogenesis-tumor progression, those obtained in EVs do not indicate AM as a target candidate, neither as a pathological biomarker nor as a marker involved in cancer therapy.

Discrimination capability of pretest probability of stable coronary artery disease: a systematic review and meta-analysis suggesting how to improve validation procedures.

OBJECTIVE: Externally validated pretest probability models for risk stratification of subjects with chest pain and suspected stable coronary artery disease (CAD), determined through invasive coronary angiography or coronary CT angiography, are analysed to characterise the best validation procedures in terms of discriminatory ability, predictive variables and method completeness. DESIGN: Systematic review and meta-analysis. DATA SOURCES: Global Health (Ovid), Healthstar (Ovid) and MEDLINE (Ovid) searched on 22 April 2020. ELIGIBILITY CRITERIA: We included studies validating pretest models for the first-line assessment of patients with chest pain and suspected stable CAD. Reasons for exclusion: acute coronary syndrome, unstable chest pain, a history of myocardial infarction or previous revascularisation; models referring to diagnostic procedures different from the usual practices of the first-line assessment; univariable models; lack of quantitative discrimination capability. METHODS: Eligibility screening and review were performed independently by all the authors. Disagreements were resolved by consensus among all the authors. The quality assessment of studies conforms to the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). A random effects meta-analysis of area under the receiver operating characteristic curve (AUC) values for each validated model was performed. RESULTS: 27 studies were included for a total of 15 models. Besides age, sex and symptom typicality, other risk factors are smoking, hypertension, diabetes mellitus and dyslipidaemia. Only one model considers genetic profile. AUC values range from 0.51 to 0.81. Significant heterogeneity (p<0.003) was found in all but two cases (p>0.12). Values of I2 >90% for most analyses and not significant meta-regression results undermined relevant interpretations. A detailed discussion of individual results was then carried out. CONCLUSIONS: We recommend a clearer statement of endpoints, their consistent measurement both in the derivation and validation phases, more comprehensive validation analyses and the enhancement of threshold validations to assess the effects of pretest models on clinical management. PROSPERO REGISTRATION NUMBER: CRD42019139388.

A possible role for ST2 as prognostic biomarker for COVID-19.

COVID-19 is a pandemic illness caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2). It has been estimated that 80% of subject infected are asymptomatic or have mild to moderate symptoms. Differently, in severe cases of COVID-19, cytokine storm, acute respiratory distress syndrome (ARDS), severe systemic inflammatory response and cardiovascular diseases were observed Even if all molecular mechanisms leading to cardiovascular dysfunction in COVID-19 patients remain to be clarified, the evaluation of biomarkers of cardiac injury, stress and inflammation proved to be an excellent tool to identify the COVID-19 patients with worse outcome. However, the number of biomarkers used to manage COVID-19 patients is expected to increase with the increasing knowledge of the pathophysiology of the disease. It is our view that soluble suppressor of tumorigenicity 2 (sST2) can be used as biomarker in COVID-19. sST2 is routinely used as prognostic biomarker in patients with HF. Moreover, high circulating levels of sST2 have also been found in subjects with ARDS, pulmonary fibrosis and sepsis. Keeping in mind these considerations, in this review the possible mechanisms through which the SARS-CoV2 infection could damage the cardiovascular system were summarized and the possible role of sST2 in COVID-19 patients with CVD was discussed.

PCSK9 and atherosclerosis: Looking beyond LDL regulation.

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) is involved in cholesterol homeostasis. After binding to the complex low-density lipoprotein (LDL)-receptor, PCSK9 induces its intracellular degradation, thus reducing serum LDL clearance. In addition to the well-known activity on the hepatic LDL receptor-mediated pathway, PCSK9 has been, however, associated with vascular inflammation in atherogenesis. Indeed, PCSK9 is expressed by various cell types that are involved in atherosclerosis (e.g. endothelial cells, smooth muscle cells and macrophages) and is detected inside human atherosclerotic plaques. We here analyse the biology of PCSK9 and its possible involvement in molecular processes involved in atherosclerosis, beyond the regulation of circulating LDL cholesterol levels.

Variations of circulating miRNA in paediatric patients with Heart Failure supported with Ventricular Assist Device: a pilot study.

Circulating miRNAs (c-miRNAs) are promising biomarkers for HF diagnosis and prognosis. There are no studies on HF pediatric patients undergoing VAD-implantation. Aims of this study were: to examine the c-miRNAs profile in HF children; to evaluate the effects of VAD on c-miRNAs levels; to in vitro validate putative c-miRNA targets. c-miRNA profile was determined in serum of HF children by NGS before and one month after VAD-implant. The c-miRNA differentially expressed were analyzed by real time-PCR, before and at 4 hrs,1,3,7,14,30 days after VAD-implant. A miRNA mimic transfection study in HepG2 cells was performed to validate putative miRNA targets selected through miRWalk database. Thirteen c-miRNAs were modified at 30 days after VAD-implant compared to pre-VAD at NSG, and, among them, six c-miRNAs were confirmed by Real-TimePCR. Putative targets of the validated c-miRNAs are involved in the hemostatic process. The in vitro study confirmed a down-regulatory effect of hsa-miR-409-3p towards coagulation factor 7 (F7) and F2. Of note, all patients had thrombotic events requiring pump change. In conclusion, in HF children, the level of six c-miRNAs involved in the regulation of hemostatic events changed after 30 days of VAD-treatment. In particular, the lowering of c-miR-409-3p regulating both F7 and F2 could reflect a pro-thrombotic state after VAD-implant.

Effects of cerium oxide nanoparticles on hemostasis: Coagulation, platelets, and vascular endothelial cells.

Cerium oxide nanoparticles (nanoceria [NC]) have attracted much attention in biomedicine due to their surface composition that confers interesting redox activities and regenerative properties. Studies have demonstrated that the application of NPs in biomedicine can influence components of hemostatic system, inducing blood clotting, alterations of blood cells, and endothelial cell functions. NC were tested in vitro to assess their hemocompatibility and anticoagulant, anti-inflammatory, and anti-senescence activity in human endothelial cells. Hemocompatibility has been evaluated in vitro looking at the impact of NC on coagulation times, fibrinogen, and platelet aggregation. The effect of NC on vascular endothelial cells were assayed by testing cell viability, antioxidant activity, anticoagulant (tissue factor [TF]-mRNA expression) and anti-inflammatory properties (VCAM-1 exposure, cytokine release), and senescence (telomere shortening). NC did not show significant effects on coagulation process, hemolysis, or platelet aggregation. In endothelial cells, NC did not affect cell viability, reduced oxidative stress, inhibited mRNA-TF expression, VCAM-1 expression, and cytokine release. Moreover, NC reduce telomere shortening, possibly counteracting premature senescence. The hemocompatibility combined with anticoagulant and anti-inflammatory phenotype and the ability of counteract the premature senescence in vascular cells make NC a promising therapeutic tool in oxidative stress-related conditions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019.

Osteopontin in hepatocellular carcinoma: A possible biomarker for diagnosis and follow-up.

Recently osteopontin (OPN), a protein of the extracellular matrix, has generated in hepatocellular carcinoma (HCC) a significant interest as a prognostic factor. Aim of this study was to confirm, in liver tissues of subjects with HCV-positive HCC undergoing liver transplantation (RL, n=10) and of donors (DL, n=14), the increase of OPN plasma and tissue concentration, the OPN splicing isoforms expression profiling together with those of thrombin, and to evaluate a possible association between OPN measurements. Their association with Notch-1, IV-Collagen-7s domain, IL-6 and TNF-α were also evaluated. Real-Time PCR experiments and immunometric assay were performed. mRNA expression resulted higher in RL than in DL for all analyzed genes and several correlations were found between them. The more relevant association were between OPN-a and OPN-b (p<0.0001), between thrombin and OPN-a (p=0.007), between 7s-collagen and OPN isoforms (p<0.05) and between Notch-1 with OPN-c (p=0.004). Both OPN plasma and liver tissue extract concentrations were assessed confirming the trend observed at the mRNA level. An important association was found between OPN plasma and protein (p<0.0001, r=0.96) even splitting patients in DL (p<0.0001, r=0.93) and RL (p<0.0001, r=0.96). A reduction of OPN plasma levels was found at 6months after transplantation. Considering MELD score as liver disease severity, the mRNA expression of our markers as well as of OPN plasma and tissue concentrations resulted increased as a function of clinical severity. Our results might be considered a useful starting point to validate OPN as a prognostic and diagnostic marker of HCC.

Effects of obesity on IL-33/ST2 system in heart, adipose tissue and liver: study in the experimental model of Zucker rats.

Suppression of tumorigenicity 2 (ST2) mediates the effect of Interleukin-33 (IL-33). Few data are reported on the relationship between IL-33/ST2 and obesity. We aimed to investigate effects of obesity on IL-33/ST2 system in heart, adipose tissue and liver in a rodent model of obesity. The relationship of cardiac expression of IL-33/ST2 system with natriuretic peptides (NPs) system and inflammatory mediators was also studied. mRNA expression of IL-33/ST2 system was evaluated in cardiac, adipose and hepatic biopsies from obese Zucker rats (O) and controls (CO). Expression levels of sST2 was significantly lower in O rats compared with CO (p<0.05) in all tissues. Besides, the mRNA levels of IL-33 decreased significant in fat of O respect to CO, while, expression levels of ST2L was significantly higher in liver of CO than in O. A strong relationship of IL-33/ST2 with NPs and classical inflammatory mediators was observed in cardiac tissue. Expression of sST2 in cardiac, adipose and liver tissue decreased in O compared with controls, suggesting an involvement for IL-33/ST2 system in molecular mechanisms of obesity. The strong relationships with NP systems and inflammatory mediators could suggest an involvement for IL-33/ST2 in molecular pathways leading to cardiac dysfunction and inflammation associated with obesity.

HDL cholesterol, leptin and interleukin-6 predict high risk coronary anatomy assessed by CT angiography in patients with stable chest pain.

OBJECTIVES: Coronary computed tomography angiography (CTA) describes several features of coronary plaques, i.e. location, severity, and composition. Integrated CTA scores are able to identify individual patterns of higher risk. We sought to test whether circulating biomarkers related with metabolism and inflammation could predict high risk coronary anatomy at CTA in patients with stable chest pain. METHODS: We evaluated a panel of 17 biomarkers in 429 patients (60.3 ± 0.4 years, 268 males) with stable chest pain who underwent coronary CTA having been enrolled in the Evaluation of Integrated Cardiac Imaging (EVINCI) study. The individual CTA risk score was calculated combining plaque extent, severity, composition, and location. The presence and distribution of non-calcified, mixed and calcified plaques were analyzed in each patient. RESULTS: After adjustment for age, sex and medical treatment, high-density lipoprotein (HDL) cholesterol, leptin, and interleukin-6 (IL-6) were independent predictors of CTA risk score at multivariate analysis (P = 0.050, 0.002, and 0.007, respectively). Integrating these biomarkers with common clinical variables, a model was developed which showed a better discriminating ability than the Framingham Risk Score and the Euro-SCORE in identifying the patients with higher CTA risk score (area under the receiver-operating characteristics curve = 0.81, 0.63 and 0.71, respectively, P < 0.001). These three biomarkers were significantly altered in patients with mixed or non-calcified plaques. CONCLUSIONS: In patients with stable chest pain, low HDL cholesterol, low leptin and high IL-6 are independent predictors of high risk coronary anatomy as defined by an integrated CTA risk score.

Adenosine receptor transcriptomic profile in cardiac tissue of a Zucker rat model.

To evaluate the possible variations of adenosine receptor (AR) profile together with TNF-α and IL-6 mRNA in cardiac tissue of obese Zucker rats (OZR) during fasting conditions (fc) and during the induction of acute hyperglycemia (AH). OZR (O, n=21) and age-matched lean control rats (CO, n=18) were studied during fc (COfc, n=8; Ofc, n=13) and during the induction of AH (COAH, n=10; OAH, n=8). The histopathologic analysis performed on O and CO heart samples did not show abnormalities of myocardial structure. The AR transcriptomic profile was analyzed in O and CO by real-time polymerase chain reaction (PCR) and a significantly lower mRNA expression was observed for A2AR in O with respect to CO (p=0.047), while a significant upregulation was observed for A3R in O with respect to CO (p=0.002). No significant differences between O and CO were observed for TNF-α or IL-6. Correlations were found between glycemia and A1R (p=0.03) and A2BR (p=0.002); total cholesterol and A2BR (p=0.02) and A3R (p=0.0002), as well as between IL-6 and A1R (p=0.05) and TNF-α and A2AR (p<0.0001). The modulation of ARs in these settings could represent a promising approach to pharmacological treatment, which must be supported by diet restrictions and physical exercise.

Myocardial interleukin-6 in the setting of left ventricular mechanical assistance: relation with outcome and C-reactive protein.

BACKGROUND: In left ventricular assist device (LVAD) recipients, plasma levels of interleukin (IL)-6 are associated with Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles, reflecting post-operative risk. However, it is not clear how the cardiac level of IL-6, detectable on the tissue samples at the time of implantation, can contribute to predict the post-operative outcome. METHODS: In 40 LVAD recipients, blood and myocardial samples from LV-apex were collected at the time of implantation to assess plasma and cardiac IL-6 levels. Serum C-reactive protein (CRP) levels were considered as inflammatory variable routinely used in LVAD-based therapy. RESULTS: Cardiac IL-6 levels did not correlate with either plasma IL-6 levels (R=0.296, p=0.063) and tissue IL-6 mRNA expression (R=-0.013, p=0.954). Contrary to what happened for the plasma IL-6 and CRP, no differences were observed in cardiac IL-6 levels with respect to INTERMACS profiles (p=0.090). Furthermore, cardiac IL-6 concentrations, unlike IL-6 and CRP circulating levels, were not correlated with the length of intensive care unit stay and hospitalization. CONCLUSIONS: Cardiac IL-6 levels do not contribute to improve risk profile of LVAD recipients in relation to clinical inpatient post-implantation. Instead, plasma IL-6 and serum CRP concentrations are more effective in predicting the severity of the clinical course in the early phase of LVAD therapy.

Transcriptional alterations of ET-1 axis and DNA damage in lung tissue of a rat obesity model.

Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (CO(fc)-O(fc)) or acute hyperglycemia (CO(AH)-O(AH)). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in O(AH) rats were significantly higher (p=0.019) than in the corresponding CO and CO(AH) rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection.

Uncovering the cathepsin system in heart failure patients submitted to Left Ventricular Assist Device (LVAD) implantation.

BACKGROUND: In end-stage heart failure (HF), the implantation of a left ventricular assist device (LVAD) is able to induce reverse remodeling. Cellular proteases, such as cathepsins, are involved in the progression of HF. The aim of this study was to evaluate the role of cathepsin system in HF patients supported by LVAD, in order to determine their involvement in cardiac remodeling. METHODS: The expression of cysteine (CatB, CatK, CatL, CatS) and serine cathepsin (CatG), and relative inhibitors (Cystatin B, C and SerpinA3, respectively) was determined in cardiac biopsies of 22 patients submitted to LVAD (pre-LVAD) and compared with: 1) control stable chronic HF patients on medical therapy at the moment of heart transplantation without prior LVAD (HT, n = 7); 2) patients supported by LVAD at the moment of transplantation (post-LVAD, n = 6). RESULTS: The expression of cathepsins and their inhibitors was significantly higher in pre-LVAD compared to the HT group and LVAD induced a further increase in the cathepsin system. Significant positive correlations were observed between cardiac expression of cathepsins and their inhibitors as well as inflammatory cytokines. In the pre-LVAD group, a relationship of cathepsins with dilatative etiology and length of hospitalization was found. CONCLUSIONS: A parallel activation of cathepsins and their inhibitors was observed after LVAD support. The possible clinical importance of these modifications is confirmed by their relation with patients' outcome. A better discovery of these pathways could add more insights into the cardiac remodeling during HF.

Physical activity support or weight loss counseling for nonalcoholic fatty liver disease?

AIM: To determine the clinical effectiveness of intense psychological support to physical activity (PA) in nonalcoholic fatty liver disease (NAFLD), compared with cognitive-behavioral treatment (CBT). METHODS: Twenty-two NAFLD cases received support to exercise, tailored to their motivational needs (PA group). The effects on body weight, physical fitness [6-min walk test, VO2max and the PA-rating (PA-R) questionnaire] and body fat (fatty liver indices and visceral adiposity index) were compared with data obtained in 44 NAFLD subjects enrolled in a CBT program for weight loss, after adjustment for propensity score, calculated on baseline data. Measurements were performed at baseline, at 4-mo and one-year follow-up. Changes in anthropometric, biochemical and PA parameters were tested by repeated measurement ANOVA. Outcome results were tested by logistic regression analysis. RESULTS: At the end of the intensive program, BMI was less significantly reduced in the PA group (-1.09 ± 1.68 kg/m(2) vs -2.04 ± 1.42 kg/m(2) in the CBT group, P = 0.019) and the difference was maintained at 1-year follow-up (-0.73 ± 1.63 vs -1.95 ± 1.88, P = 0.012) (ANOVA, P = 0.005). PA-R was similar at baseline, when only 14% of cases in PA and 36% in CBT (P = 0.120) recorded values ≥ 3. At 4 mo, a PA-R ≥ 3 was registered in 91% of PA and 46% of CBT, respectively (P < 0.001) and PA-R ≥ 5 (up to 3 h/wk of moderate-to-heavy intensity physical activity) was registered in 41% of PA and only 9% of CBT group (P < 0.007). The 6-min walk test increased by 139 ± 26 m in PA and by only 43 ± 38 m in CBT (P < 0.001) and VO2max by 8.2 ± 3.8 mL/kg per minute and 3.3 ± 2.7 mL/kg per minute, respectively (P < 0.002). After adjustment for propensity, weight loss > 7% was significantly associated with CBT group at one year (OR = 6.21; 95%CI: 1.23-31.30), whereas PA-R > 3 was associated with PA group (10.31; 2.02-52.63). Liver enzymes decreased to values within normal limits in 36% of PA cases and 61% of CBT (P < 0.070). Estimated liver fat (Kotronen index) fell below the fatty liver threshold in 36% of PA and 34% and CBT cases at one-year (not different). Also the fatty liver index and the visceral adiposity index improved to a similar extent. CONCLUSION: Intensive psychological counseling for PA produces hepatic effects not different from standard CBT, improving physical fitness and liver fat independent of weight loss. Strategies promoting exercise are worth and effective in motivated patients, particularly in lean NAFLD patients where large weight loss cannot be systematically pursued.

Adrenomedullin and intermedin gene transcription is increased in leukocytes of patients with chronic heart failure at different stages of the disease.

Adrenomedullin (ADM) is a vasodilatory peptide expressed in many tissues. Its levels are elevated in various diseases including chronic heart failure (CHF) and it has been suggested that the up-regulation of ADM in cardiac disease represents a protective mechanism. Similarly, intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is considered a potential endogenous protector of the heart. Previous studies demonstrated that in CHF patients, elevated plasma concentrations of ADM and IMD reflect the patient's disease severity and prognosis, while the behavior of mRNA expression is not known. The aim of this study was to evaluate ADM/IDM transcriptomic profiling in human leukocytes of CHF patients as a function of clinical severity, assessing possible changes with respect to healthy subjects (C). mRNA expression was evaluated by Real-Time PCR and total RNA was extracted from leukocytes of C (n=8) and from CHF patients (NYHA I-II n=10; NYHA III-IV n=14) with PAXgene Blood RNA Kit. Significantly higher levels of ADM and IMD mRNA were found in CHF as a function of clinical severity (ADM: C=0.03 ± 0.013, NYHA I-II=0.11 ± 0.084, NYHA III-IV=11.46 ± 4.72, p=0.037 C vs NYHA III-IV, p=0.028 NYHA I-II vs NYHA III-IV; IMD: C=0.158 ± 0.041, NYHA I-II=0.93 ± 0.40, NYHA III-IV=2.6 ± 0.67, p=0.014 C vs NYHA III-IV, p=0.014 NYHA I-II vs NYHA III-IV). This study highlights, for the first time, the possibility of evaluating ADM and IMD mRNA expression in human whole blood samples by Real-Time PCR study providing further relevant information and providing a more complete interpretation of the pathophysiology of the disease.

Adenosine receptor expression in an experimental animal model of myocardial infarction with preserved left ventricular ejection fraction.

Adenosine, a purine nucleoside and a "retaliatory metabolite" in ischemia, is ubiquitous in the body and increases 100-fold during ischemia. Its biological actions are mediated by four adenosine receptors (ARs): A(1), A(2A), A(2B) and A(3). The aim of this study was to determine possible myocardial alterations in AR expression in an experimental animal model of myocardial infarction (MI) with a preserved left ventricular (LV) ejection fraction. LV tissue was collected from sexually mature male farm pigs with MI (n = 6) induced by permanent surgical ligation of the left anterior descending coronary artery and from five healthy pigs (C). mRNA expression of A(1)R, A(2A)R, A(2B)R, A(3)R and TNF-α was determined by real-time PCR in tissue collected from border (BZ) and remote zones (RZ) of the infarcted area and from LV of C. BZ, RZ and samples of C were stained immunohistochemically to investigate A(3)R immunoreaction. After 4 weeks a different regulation of ARs was observed. A(1)R mRNA expression was significantly lower in the infarct regions than in controls (C = 0.75 ± 0.2, BZ = 0.05 ± 0.2, RZ = 0.07 ± 0.02 p = 0.0025, p = 0.0016, C vs. BZ and RZ, respectively). Conversely A(3)R was higher in infarct areas (C = 0.94 ± 0.2, BZ = 2.85 ± 0.5, RZ = 3.48 ± 1.0, p = 0.048 C vs. RZ). No significant differences were observed for A(2A)R (C = 1.58 ± 0.6, BZ = 0.42 ± 0.1, RZ = 1.37 ± 0.6) and A(2B)R (C = 1.66 ± 0.2, BZ = 1.54 ± 0.5, RZ = 1.25 ± 0.4). A(3)R expression was confirmed by immunohistochemical analysis and was principally localized in cardiomyocytes. TNF-α mRNA results were: C 0.41 ± 0.25; BZ 1.60 ± 0.19; RZ 0.17 ± 0.04. The balance between A(1)R and A(3)R as well as between A(2A)R and A(2B)R was consistent with adaptative retaliatory anti-ischemic adenosinergic changes in the infarcted heart with preserved LV function.

Insulin resistance is a major determinant of myocardial blood flow impairment in anginal patients.

PURPOSE: In patients with chest pain, stress-induced myocardial perfusion abnormalities are often the result of depressed myocardial blood flow (MBF) reserve. We investigated the relative contribution of cardiovascular risk factors and coronary atherosclerosis to MBF abnormalities in anginal patients. METHODS: We studied 167 patients with typical (n = 100) or atypical (n = 67) chest pain who underwent quantitative evaluation of MBF by PET at rest and after dipyridamole infusion, and quantitative coronary angiography (invasive or by 64-slice CT). Patients with left ventricular (LV) dysfunction (ejection fraction <45 %) were excluded. Coronary atherosclerosis of ≥50 % was defined as obstructive. RESULTS: At rest median MBF was 0.60 ml min-1 g-1, and after dipyridamole infusion median MBF was 1.22 ml min-1 g-1. MBF reserve was <2 in 77 of 167 patients (46 %). Coronary atherosclerosis was present in 67 patients (40 %), 26 with obstructive disease. In a univariate analysis several variables were associated with reduced MBF at rest, including male gender, coronary atherosclerosis and elevated LV end-diastolic diameter, and during hyperaemia, including male gender, insulin resistance (IR), smoking habit, LV ejection fraction and end-diastolic diameter. In a multivariate analysis, after adjustment for LV function and for pharmacological treatments, male gender was the only independent predictor of reduced MBF at rest (P < 0.001), while male gender (P = 0.003), IR (P = 0.033) and coronary atherosclerosis (P < 0.001) remained the only independent predictors of reduced hyperaemic MBF. IR (P = 0.043) and coronary atherosclerosis (P = 0.005) were the only predictors of depressed MBF reserve. Coronary atherosclerosis, male gender and IR showed additive effects on hyperaemic MBF. CONCLUSION: In patients with chest pain and normal LV systolic function, IR, male gender and coronary atherosclerosis are independent and additive determinants of impaired hyperaemic MBF.

Adenosine receptor expression and gene reference evaluation in human leukocytes.

BACKGROUND: Peripheral blood mononuclear cells and isolated polymorphonuclear neutrophilis were used to evaluate gene expression studies. Unfortunately, there are many methodological problems related to these cellular models, limiting their use. The aim was to evaluate a fast and easy procedure for the extraction of total RNA from leukocytes obtained from human whole blood (WB) < 10 mL; to determine adenosine receptor (AR) mRNA expression in WB samples of normal subjects and to establish the most stable reference genes for data normalization. METHODS: mRNA expression was performed by Real-Time PCR. RESULTS: The most stably expressed genes were TPT1, EEF1A, and RPL13A. Similar levels of mRNA expression were observed for A2aR, A2bR, and A3R while lower levels were measured for A1R (p = 0.02 A1R vs. A2aR; p = 0.04 A1R vs. A3R). CONCLUSIONS: Our study represents an important and useful starting point for future investigations devoted to evaluate the expression of ARs in human diseases.

High peripheral levels of h-FABP are associated with poor prognosis in end-stage heart failure patients with mechanical circulatory support.

AIM: To associate the time-course of h-FABP and N-terminal pro B-type natriuretic peptide (NT-proBNP)after left ventricular assist device (LVAD) implantation to outcome in end-stage heart failure patients. MATERIALS & METHODS: Patients (n = 14, NYHA class III/IV; left ventricular ejection fraction <25% were enrolled; ten survived up to 1 month after LVAD (survivors) and four died of multiorgan failure within 2 weeks (nonsurvivors). Blood samples were obtained at admission; at 4, 24 and 72 h; and at 1 and 4 weeks after LVAD. RESULTS: h-FABP significantly increases after surgery, decreasing since 72 h in all patients. At 72 h all survivor patients present h-FABP lower than the median value. N-terminal pro B-type natriuretic peptide is not associated with patient outcome at any time. CONCLUSION: High h-FABP levels, indicating the presence of more severe myocardial damage, are associated with a poor prognosis in patients with LVAD, suggesting that an early cardiac injury marker could improve the prediction of clinical outcome.

Impact of normalization strategy on cardiac expression of pro-inflammatory cytokines: evaluation of reference genes in different human myocardial regions after Left Ventricular Assist Device support.

OBJECTIVE: New device therapies have expanded the strategies for treating heart failure (HF) patients. Unloading of the heart with a left ventricular assist device (LVAD) can lead to the reversal of many remodeling changes whose underlying mechanisms are not yet completely known. Molecular analysis might play a role in obtaining further insight into the regulatory mechanisms of this process. A critical step in an RT-PCR study is the selection of reference genes for data normalization. This study aimed to determine an optimal combination of stably expressed reference genes in different regions of the human heart in order to study the effects of LVAD implants on cardiac remodeling, and in particular to check their reliability on the evaluation of pro-inflammatory cytokine expression. DESIGN AND METHODS: We validated nine of the most commonly used reference genes in human myocardium samples obtained at heart transplantation from patients with LVAD implant (n=30 from a total of six patients) and from heart transplant (HT from a total of seven patients) recipients as controls (n=35). Samples from both left (LV) and right (RV) ventricles were analyzed. The normalization strategy was tested by analyzing mRNA expression of IL-6, IL-8 and TNF-α, whose protein levels were measured by immunometric assay. RESULTS: The most stable gene combinations changed according to the experimental groups (the LVAD and HT groups and the different myocardial regions). Considering all the cardiac samples as a whole, the three most stably expressed genes were PPIA, RPL13A, and YWHAZ (M=0.70). Using the best normalization strategy, a significant increase in IL-6, IL-8 mRNA expression was observed in LVAD samples compared to HT (p<0.0001). Similar results were obtained by protein analysis. CONCLUSIONS: Our results underline the importance of always selecting reference genes for the specific system studied. The most appropriate normalization strategy is of pivotal importance for understanding the molecular mechanisms associated with the pathophysiology of HF, such as inflammation.