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Background: Inflammatory mechanisms are associated with worse prognosis in end-stage heart failure (ESHF) patients who require left ventricular assist device (LVAD) support. Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles describe patient condition at pre-implant and outcome. This study assessed the relationship among inflammation patterns and INTERMACS profiles in LVAD recipients., Method: Thirty ESHF patients undergoing LVAD implantation as bridge to transplant were enrolled. Blood and urine samples were collected pre-operatively and serially up to 2 weeks post-operatively for assessment of inflammatory markers (plasma levels of interleukin [IL]-6, IL-8, IL-10, and osteopontin, a cardiac inflammatory-remodeling marker; and the urine neopterin/creatinine ratio, a monocyte activation marker). Multiorgan function was evaluated by the total sequential organ failure assessment (tSOFA) score. Outcomes of interest were early survival, post-LVAD tSOFA score, and intensive care unit (ICU) length of stay., Results: Fifteen patients had INTERMACS profiles 1 or 2 (Group A), and 15 had profiles 3 or 4 (Group B). At pre-implant, only IL-6 levels and the IL-6/IL-10 ratio were higher in Group A vs B. After LVAD implantation, neopterin/creatinine ratio and IL-8 levels increased more in Group A vs B. Osteopontin levels increased significantly only in Group B. The tSOFA score at 2 weeks post-LVAD and ICU duration were related with pre-implant IL-6 levels., Conclusions: The INTERMACS profiles reflect the severity of the pre-operative inflammatory activation and the post-implant inflammatory response, affecting post-operative tSOFA score and ICU stay. Therefore, inflammation may contribute to poor outcome in patients with severe INTERMACS profile., (Copyright © 2012 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Obesity is a complex pathology with interacting and confounding causes due to the environment, hormonal signaling patterns, and genetic predisposition. At present, the Zucker rat is an eligible genetic model for research on obesity and metabolic syndrome, allowing scrutiny of gene expression profiles. Real-time PCR is the benchmark method for measuring mRNA expressions, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. In the Zucker rat model, no specific reference genes have been identified in myocardium, kidney, and lung, the main organs involved in this syndrome. The aim of this study was to select among ten candidates (Actb, Gapdh, Polr2a, Ywhag, Rpl13a, Sdha, Ppia, Tbp, Hprt1 and Tfrc) a set of reference genes that can be used for the normalization of mRNA expression data obtained by real-time PCR in obese and lean Zucker rats both at fasting and during acute hyperglycemia. The most stable genes in the heart were Sdha, Tbp, and Hprt1; in kidney, Tbp, Actb, and Gapdh were chosen, while Actb, Ywhag, and Sdha were selected as the most stably expressed set for pulmonary tissue. The normalization strategy was used to analyze mRNA expression of tumor necrosis factor α, the main inflammatory mediator in obesity, whose variations were more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance of having three stably expressed reference gene sets for use in the cardiac, renal, and pulmonary tissues of an experimental model of obese and hyperglycemic Zucker rats.

The prototypic long pentraxin PTX3 is a novel vascular inflammatory marker sharing similarities with the classic short pentraxin (C-reactive protein). PTX3 is rapidly produced and released by several cell types in response to local inflammation of the cardiovascular system. Plasma PTX3 levels are very low in normal conditions and increase in heart failure (HF) patients with advancing NYHA functional class, but its exact role during HF pathogenetic mechanisms is not yet established. No data about PTX3 cardiac expression in normal and pathological conditions are currently available, either in human or in large-size animals. Of the latter, the pig has a central role in "in vivo" clinical settings but its genome has not been completely sequenced and the PTX3 gene sequence is still lacking. The aim of this study was to sequence the PTX3 in Sus scrofa, whose sequence is not yet present in GenBank. Utilizing our knowledge of this sequence, PTX3 mRNA expression was evaluated in cardiac tissue of normal (n=6) and HF pigs (n=5), obtained from the four chambers. To sequence PTX3 gene in S. scrofa, the high homology between Homo sapiens and S. scrofa was exploited. Pig PTX3 mRNA was sequenced using polymerase chain reaction primers designed from human consensus sequences. The DNA, obtained from different RT-PCR reactions, was sequenced using the Sanger method. S. scrofa PTX3 mRNA, 1-336 bp, was submitted to GenBank (ID: GQ412351). The sequence obtained from pig cardiac tissue shared an 84% sequence identity with human homolog. The presence of PTX3 mRNA expression was detected in all the cardiac chambers sharing an increase after 3 weeks of pacing compared to controls (p=0.036 HF right atrium vs. N; p=0.022, HF left ventricle vs. N). Knowledge of the PTX3 sequence could be a useful starting point for future studies devoted to better understanding the specific role of this molecule in the pathogenesis of cardiovascular diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The adenosinergic system is essential in the mediation of intrinsic protection and myocardial resistance to insult; it may be considered a cardioprotective molecule and adenosine receptors (ARs) represent potential therapeutic targets in the setting of heart failure (HF). The aim of the study was to test whether differences exist between mRNA expression of ARs in the anterior left ventricle (LV) wall (pacing site: PS) compared to the infero septal wall (opposite region: OS) in an experimental model of dilated cardiomyopathy. Cardiac tissue was collected from LV PS and OS of adult male minipigs with pacing-induced HF (n = 10) and from a control group (C, n = 4). ARs and TNF-α mRNA expression was measured by Real Time-PCR and the results were normalized with the three most stably expressed genes (GAPDH, HPRT1, TBP). Immunohistochemistry analysis was also performed. After 3 weeks of pacing higher levels of expression for each analyzed AR were observed in PS except for A(1)R (A(1)R: C = 0.6±0.2, PS = 0.1±0.04, OS = 0.04±0.01, p<0.0001 C vs. PS and OS respectively; A(2A)R: C = 1.04±0.59, PS = 2.62±0.79, OS = 2.99±0.79; A(2B)R: C = 1.2±0.1, PS = 5.59±2.3, OS = 1.59±0.46; A(3)R: C = 0.76±0.18, PS = 8.40±3.38, OS = 4.40±0.83). Significant contractile impairment and myocardial hypoperfusion were observed at PS after three weeks of pacing as compared to OS. TNF-α mRNA expression resulted similar in PS (6.3±2.4) and in OS (5.9±2.7) although higher than in control group (3.4±1.5). ARs expression was mainly detected in cardiomyocytes. This study provided new information on ARs local changes in the setting of LV dysfunction and on the role of these receptors in relation to pacing-induced abnormalities of myocardial perfusion and contraction. These results suggest a possible therapeutic role of adenosine in patients with HF and dyssynchronous LV contraction.

Chronic heart failure (HF) is associated with increased systemic (plasma) and reduced local (myocardial) adenosine levels. The final biological action of adenosine in a particular organ or cell population may depend on the relative degree of expression and signaling efficiency of individual adenosine receptor (AR) subtypes. The aim of this study was to determine the myocardial expression of ARs, in the different chambers of failing versus normal minipig hearts. Cardiac tissue was collected from minipigs without (n = 5) and with HF (n = 5). ARs, adenosine deaminase, and tumor necrosis factor-α (TNF-α) mRNA expression were evaluated by real time-polymerase chain reaction. ARs were expressed in all cardiac regions. After 3 weeks of pacing, the only significant change was observed in A2BR mRNA expression in the left ventricle (P = 0.02), with a similar trend for A3R, A2AR, and A1R. A trend toward higher expression of mRNA adenosine deaminase in the myocardium of pigs with HF was observed. TNF-α mRNA expression was higher after HF in all cardiac chambers (left ventricle: P = 0.009), and a significant correlation was observed between TNF-α and A2BR (r = 0.80, P < 0.0001). In this study, A2BR mRNA resulted in being overexpressed in the left ventricle of pigs with HF as well as TNF-α expression possibly testifying a link between AR expression, inflammation, and HF.

An impaired ability to store fatty acids (FA) in subcutaneous adipose tissue (SAT) may be implicated in the pathogenesis of obesity-related diseases via overexposure of lean tissues and production of free radicals from FA oxidation (FAO). We studied regional FA metabolism in skeletal muscle and adipose tissue in humans and investigated the long-term effects of the FAO inhibitor trimetazidine on glucose and FA metabolism. Positron emission tomography (PET) and [(11)C]palmitate were used to compare FA metabolism in SAT and skeletal muscle between eight obese and eight nonobese subjects (BMI ≥/< 30 kg/m(2)). A subgroup of nine subjects underwent a 1-mo trimetazidine administration. PET with [(11)C]palmitate and [(18)F]fluorodeoxyglucose, indirect calorimetry, and MRI before and after this period were performed to characterize glucose and FA metabolism, fat masses, skeletal muscle triglyceride, and creatine contents. Obesity was characterized by a 100% elevation in FAO and a defect in the FA esterification rate constant (P < 0.05) in skeletal muscle. FA esterification was reduced by ~70% in SAT (P < 0.001) in obese vs. control subjects. The degrees of obesity and insulin resistance were both negatively associated with esterification-related parameters and positively with FAO (P < 0.05). Trimetazidine increased skeletal muscle FA esterification (P < 0.01) and mildly upregulated glucose phosphorylation (P = 0.066). Our data suggest that human obesity is characterized by a defect in tissue FA storage capability, which is accompanied by a (potentially compensatory) elevation in skeletal muscle FAO; trimetazidine diverted FA from oxidative to nonoxidative pathways and provoked an initial activation of glucose metabolism in skeletal muscle.

Real-time PCR is the benchmark method for measuring mRNA expression levels, but the accuracy and reproducibility of its data greatly depend on appropriate normalization strategies. Though the minipig model is largely used to study cardiovascular disease, no specific reference genes have been identified in porcine myocardium. The aim of the study was to identify and validate reference gene to be used in RT-PCR studies of failing (HF) and non-failing pig hearts. Eight candidate reference genes (GAPDH, ACTB, B2M, TBP, HPRT-1, PPIA, TOP2B, YWHAZ) were selected to compare cardiac tissue of normal (n=4) and HF (n=5) minipigs. The most stable genes resulted: HPRT-1, TBP, PPIA (right and left atrium); PPIA, GAPDH, ACTB (right ventricle); HPRT-1, TBP, GAPDH (left ventricle). The normalization strategy was tested analyzing mRNA expression of TNF-α, which is known to be up-regulated in HF and whose variations resulted more significant when normalized with the appropriately selected reference genes. The findings obtained in this study underline the importance to provide a set of reference genes to normalize mRNA expression in HF and control minipigs. The use of unvalidated reference genes can generate biased results because also their expression could be altered by the experimental conditions., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The role of myocardial apoptosis during the development of heart failure (HF), in the absence of coronary artery stenosis, is still debated. The aim of the study was to evaluate whether (similar to functional impairment) the activation of myocardial apoptosis follows a regional pattern in an established model of pacing-induced HF. HF was induced in adult male minipigs by rapid and sustained left ventricular (LV) epicardial pacing (n=8; n=5 healthy controls). Progressive regional derangement of the contractile function and perfusion was assessed by magnetic resonance imaging as LV end-systolic wall thickening (LVESWT) and relative upslope of signal intensity (LVRUSI, %) in the anterior/anterior-lateral (pacing site, PS) and infero-septal LV region (opposite site, OS). LV tissue from PS and OS was analyzed for biomarkers of cell apoptosis and injury. After 21 days of LV pacing, LVESWT was lower in the PS compared to OS (7.6±3.7 vs 24.16±3.6%, p<0.05), and LV ejection fraction was 24.0±3.7 (p<0.05 vs control). The mRNA expression of caspase (Casp)-3 was significantly higher in the PS of HF hearts than in controls (1.28±0.125 vs 0.82±0.10), but not Casp-9. Bcl-2 and Hsp72 expression was significantly increased in PS compared to control (0.90±0.60 vs 0.63±0.033; 0.72±0.10 vs 0.28±0.098), in the presence of a TNF-α level increased by 50.7%. The regional myocardial apoptotic index, assessed by TUNEL, was unchanged in HF. In conclusion, the activators and inhibitors of cell apoptosis are equally expressed without affecting the survival of cardiomyocytes and the magnitude of regional myocardial dysfunction during development of non-ischemic HF., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Reduced plasma adiponectin has been associated with abnormal lipid profile, reduced left ventricle (LV) function, and the extent of coronary atherosclerosis in coronary artery disease. The aim of this study was to assess these relationships in patients with dilated cardiomyopathy (DCM) without overt heart failure. Plasma adiponectin was measured in 55 DCM patients (age, 59 ± 12 years; male, 36; body mass index [BMI], 26.9 ± 0.49 kg/m²; LV ejection fraction, 39.8% ± 1.3%; New York Heart Association class I-II) and in 40 age- and BMI-matched healthy controls. In a subset of 25 patients, myocardial blood flow (MBF) was measured at rest and during intravenous dipyridamole (0.56 mg/kg in 4 minutes) by positron emission tomography and ¹³N-ammonia as a flow tracer. Adiponectin was 6.6 ± 0.34 μg/mL in controls and 10.9 ± 0.85 μg/mL in DCM patients (P < .001), where it was related inversely with BMI (P = .009) and directly with brain natriuretic peptide (P = .017), high-density lipoprotein (HDL) cholesterol (P = .002), and MBF dipyridamole (P = .020). Adiponectin lesser than median value in patients was associated with higher total to HDL cholesterol ratio (4.8 ± 0.24 vs 3.9 ± 0.18, P = .009) and lower MBF reserve (1.76 ± 0.16 vs 2.43 ± 0.19, P = .01). These results could suggest that down-regulation of the adiponectin levels and reduced HDL cholesterol have a key role in causing impaired coronary function and myocardial perfusion in DCM., (© 2011 Elsevier Inc. All rights reserved.)

C-type natriuretic peptide (CNP) plasma levels are extremely low and a pre-analytical phase is necessary to assay plasma CNP concentrations. Amino-terminal CNP (NT-proCNP) circulates at higher concentrations than CNP, allowing a direct assay and the use of smaller amounts of plasma. Aim of this study was to evaluate the analytical performance of a direct NT-proCNP assay and to measure its plasma levels in heart failure (CHF), diabetes and chirrosis patients. NT-proCNP and CNP were measured in 130 CHF, 19 patients with diabetes, 24 with hepatic cirrhosis and 73 controls. Plasma NT-proCNP was higher in all the clinical conditions studied (controls:45.5 ± 1.84 pg/ml, CHF:67.09 ± 7.36, diabetes:51.5 ± 5.75 cirrhosis:78.4 ± 19.9; p = 0.034, p = 0.04 controls vs. CHF and cirrhosis, respectively) and in CHF NT-proCNP concentration showed a significant increase as a function of clinical severity. By comparison of ROC curves, CNP assay resulted better associated with disease than NT-proCNP assay in all the different clinical conditions probably due to different release and clearance. The determination of NT-proCNP adds a piece of information to better understanding the molecular mechanisms at the basis of CNP action in different diseases. Due to its higher analytical feasibility, this determination could become widespread in clinical biochemistry laboratories and serve as a complementary marker of disease conditions., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The cardiac resynchronization therapy (CRT), based on correction of electro-mechanical dyssynchrony by biventricular pacing in patients with severe chronic HF unresponsive to optimal medical treatment and left ventricular conduction disturbances, has been developed. The determination of plasma adrenomedullin (ADM) levels before implantation could provide important additional information to reduce the high percentage (30%) of patients not responding to treatment despite the use of increasingly sophisticated methods for selecting candidates. The case described illustrates the importance of basal ADM plasma levels in predicting the clinical and functional improvement after treatment with CRT.

Heart failure is characterized by a chronic inflammatory status, with high circulating levels of inflammatory cytokines significantly correlated with deterioration of functional capacity, cardiac performance, and coronary flow reserve--the latter occurring even with normal systemic endothelial function. Impaired coronary flow reserve in heart failure is poorly related to systemic inflammation levels and somewhat matched by a reduction in myocardial contractile reserve. Both coronary flow and myocardial functional reserve can be imaged noninvasively and can be useful clinically for disease severity titration, diagnostic anticipation, and prognostic stratification. Coronary microcirculatory dysfunction can be a trigger of disease and a potential target for therapeutic intervention in heart failure patients. Clinical observational studies showed a striking beneficial effect of endogenous adenosine accumulation on symptoms, exercise capacity, and left ventricular function in chronic heart failure, but this needs to be confirmed in prospective randomized large-scale trials., (© 2010 New York Academy of Sciences.)

Objectives: Radiofrequency (RF) ablation of arrhythmias induces myocardial damage and release of biomarkers. This study aimed to assess the kinetics of heart-type fatty acid-binding protein (h-FABP), a cytosolic protein released after myocardial injury incurred by both atrial and ventricular RF ablation, compared to other markers of myocardial injury., Design and Methods: h-FABP, cTnI, CK-MB(mass) and myoglobin were evaluated in 30 patients with atrial or ventricular tachyarrhythmias before, immediately after and at 3, 6 and 24h after the procedure., Results: h-FABP increased immediately after the procedure in all subjects (6.6 ± 1.2 μg/L vs 2.7 ± 0.3, p<0.001) but increased significantly only in ventricular ablations. The peak of h-FABP significantly correlates with the values of time for mean power of RF application in both the entire patient cohort and in ventricular ablations., Conclusions: h-FABP may be an early parameter for monitoring RF-induced lesions and the site of ablation was relevant for biomarker increase., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The importance of biomarker assay such as cardiac troponins and H-FABP is assuming a pivotal role not only in the diagnosis and follow-up of patients with acute coronary syndromes. Radiofrequency (RF) ablation represents a widely used method for the non pharmacologic treatment of arrhythmias.We report a case of a patient complaining of life-threatening arrhythmias treated by RF in whom temporal changes of cardiac biomarkers was determined after the procedure.

Background: Increase in adrenomedullin (ADM) plasma levels in congestive heart failure (HF) patients is due to many cardiac and systemic factors, particularly to greater fluid retention and to activation of sympathetic nervous system. Aim of this study was to assess the role of plasma ADM levels in HF patients treated by cardiac resynchronization therapy (CRT)., Methods: 50 patients, mean age 70 years, 34 male, New York Heart Association (NYHA) Class III-IV HF, left ventricular ejection fraction (LVEF) < 35%, underwent CRT. All patients were in sinus rhythm and with complete left bundle branch block (QRS duration 138 +/- 6 msec). A complete echoDoppler exam, blood samples for brain natriuretic peptide (BNP), and ADM were obtained from 2 to 7 days before implantation., Results: At 16 +/- 6 months follow-up, >or=1 NYHA Class improvement was observed in 38 patients. However, a >10% reduction in end-systolic dimensions (ESD) was reported in 21 patients (Group I): -16.6 +/- 1.8%; in the remaining 29 patients ESD change was almost negligible: -2.0 +/- 1.03% (Group II), P < 0.0001. The two groups were comparable for age, sex, cause of LV dysfunction, therapy, QRS duration at baseline, preimplantation ESD, LVEF%, and BNP. Significantly higher pre implantation ADM levels were present in Group I than in Group II (27.2 +/- 1.8 pmol/l vs 17.9 +/- 1.4, P = 0.0003)., Conclusions: Significantly higher ADM levels indicate a subgroup of patients in whom reverse remodeling can be observed after CRT. Patients with lower ADM basal values before CRT could represent a group in whom the dysfunction is so advanced that no improvement can be expected.

Pharmacological treatments able to activate natriuretic receptors (NPRs) and inhibit cardiac remodelling in heart failure (HF) patients, are currently under investigation. To better understand the therapeutic potential of the NPRs activation is necessary to dispose of experimental models devoid of confounding effects. The pig constitutes an animal model largely used but its genome is not completely sequenced. Aims of this study were to sequence NPR-A and NPR-C in Susscrofa and to evaluate ANP, BNP and NPRs mRNA expression in cardiac tissue of normal and HF minipigs in order to have a starting point for future studies devoted to check new potential drugs. Cardiac tissue was collected from adult male minipigs without (n=4) and with HF (n=5). Pig NPR-A (179bp) and NPR-C (203bp) mRNA were partially sequenced (GenBank n.: FJ518622, FJ518621). Compared to control, ANP and BNP gene expression resulted higher in all the cardiac chambers of HF heart. This increase is associated to a down-regulation of NPR-A and an up-regulation of NPR-C in HF. These sequences will provide a new tool to investigate the role of natriuretic peptides and of their receptors under physiological and pathological conditions and their response to therapeutic interventions., (Copyright 2010 Elsevier B.V. All rights reserved.)

Aim: The increased myocardial production and the elevated plasma concentrations of C-type natriuretic peptide (CNP) in heart failure patients suggest its involvement in pathophysiological cardiac remodeling. The cardiovascular action of CNP seems to be mainly mediated by natriuretic peptide receptor (NPR)-B but the importance of CNP/NPR-B signaling in heart is not yet well characterized. The aim of this study was to assess the cardiac mRNA expression of CNP and NPR-B together with those of BNP and NPR-A in order to evaluate the relative importance of these peptides and of their receptors in cardiovascular system., Methods: The expression of mRNA coding for CNP, NPR-B, BNP and NPR-A was investigated in myocardial tissue (BALB/c mice, N=5) by use of RT-PCR. NPR-A and NPR-B expression were also evaluated in left ventricle of male adult minipigs without (N=5) and with pacing-induced heart failure (HF, N=5)., Results: The proposed method allowed to detect the expression of mRNA coding for CNP and NPR-B in myocardial tissue confirming the presence of these effectors in the heart. These data also indicate that CNP mRNA expression is lower with respect to that of BNP (CNP/GAPDH= 0.117+/-0.035 vs. BNP/GAPDH=0.247+/-0.066) and that NPR-B is the predominant subtype receptor in the heart (Mouse: NPR-A/GAPDH=0.244+/- 0.028; NPR-B/GAPDH=0.657+/-0.022; p=0.0008; Pig: NPR-A/GAPDH=3.06+/-1.75, NPR-B/GAPDH= 14.3+/-3.6, p=0.0028; HF Pig: NPR-A/GAPDH= 4.29+/-0.93, NPR-B/GAPDH=7.9+/-1.1, p=0.0043)., Conclusion: In the present study, we provided the first evidence of a higher mRNA expression in cardiac tissue of NPR-B with respect to NPR-A indicating that CNP specific receptor (NPR-B) is the predominant biological receptor in mouse and pig myocardial tissue.

Background: Adiponectin, present in different multimeric forms in circulation, is increasingly used in clinical settings as a cardiometabolic marker. The development of several commercially available enzyme-linked immunosorbent assays (ELISA) has allowed for the widespread measurement of adiponectin in research as well as in clinical practice. The comparative performance of these assays is thus an issue of major relevance., Methods: The analytical performance of four different ELISAs (LINCO, B-Bridge, SPIbio and ALPCO) was evaluated. Samples from 102 cardiac patients and 40 healthy subjects were tested using LINCO and ALPCO. The latter is able to measure the concentrations of multimers. For the multimer assay, an error propagation study was performed. A subset of subjects was tested by all four ELISAs for comparison purposes., Results: Bland-Altman plots revealed differences between the results of the four ELISAs, mainly for ALPCO. However, a strong correlation between the different ELISAs (ALPCO, r=0.83; B-Bridge, r=0.98; SPIbio, r=0.91 vs. LINCO) was observed. Total adiponectin measured by LINCO showed a better association with cardiac disease [receiver-operating characteristic (ROC) curves] than total and high molecular weight adiponectin measured by ALPCO., Conclusions: The results of this study contribute to a better understanding of the methodological features of the several ELISAs, and help in the evaluation and comparison of the relative results.

In patients with dilated cardiomyopathy (DCM) abnormal myocardial blood flow (MBF) has been associated with coronary microvascular dysfunction. The aim of this study was to test the hypothesis that osteopontin (OPN) plasma levels could be associated with the activation of the renin-aldosterone system (RAS) in these patients and be involved in mediating myocardial and coronary damage. In 66 patients with idiopathic left ventricular dysfunction of variable severity the plasma levels of OPN were correlated with biomarkers of systemic metabolism, RAS activation, myocardial dysfunction and with clinical indexes of left ventricle (LV) function and perfusion obtained by 2D-echocardiography and PET. As compared to controls, patients showed a significant increase of inflammatory markers (OPN: 508+/-30.8ng/ml vs. 426.9+/-16.4, p<0.05 and interleukin (IL)-6: 1.71+/-0.29pg/ml vs. 0.38+/-0.03pg/ml, p<0.001) and of indexes of cardiac damage. OPN levels were significantly correlated with the extent of microvascular dysfunction (MBF at rest: p=0.01; during dipyridamole: p=0.0003) and with plasma renin activity (PRA) (r=0.26, p=0.04). Both in patients with milder or more severe LV dysfunction lower MBF values were associated with higher OPN levels and PRA. These results suggest a interdependent role of RAS and vascular inflammation in cardiomyopathy., (2009 Elsevier Ltd. All rights reserved.)

In humans, the Apelin gene is located on chromosome Xq25-26.1 and it encodes a 77-aminoacid prepropeptide. Considerable sequence homology exists across different mammalian species. Apelin is emerging as an important regulator of cardiovascular homeostasis but, at present, few data from humans are available and further studies are necessary to better define its role in cardiovascular pathophysiology. The role and function of Apelin in cardiovascular system could be reliably investigated in experimental models devoid of confounding effects reflecting only the natural history of the disease. The pig constitutes a model largely used in experimental pathology where it has a central role in "in vivo" clinical settings. Sus Scrofa genoma is not completely sequenced and Apelin gene is still lacking. Aim of this study was to sequence the Apelin in Sus Scrofa for future applications to molecular biology studies. Using the guanidinium thyocyanate-phenol-chloroform method, we extracted total RNA from samples obtained from heart of mouse and from atrium and ventricle of normal pigs. Pig Apelin mRNA was sequenced using polymerase chain reaction primers designed from mouse consensus sequences. A partial sequence of Sus Scrofa Apelin mRNA, 1-201 pb, was submitted to GenBank (accession number FJ362603). The bands obtained from pig cardiac tissue shared a 99% sequence identity with Mus musculus and 90% with Rattus norvegicus. The knowledge of Apelin sequence can be an useful starting point for future studies devoted to better understand the possible alterations of Apelin mRNA expression in different cardiac diseases.

High-frequency pacing of the left ventricle (LV) free wall causes a dyssynchronous pattern of contraction that leads to progressive heart failure (HF) with pronounced differences in regional contractility. Aim of this study was to evaluate possible changes in brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) mRNA expression in the anterior/anterior lateral region (pacing site, PS) as compared to the infero-septal region (opposite site, OS) and to explore possible association between the contractiling pattern and biomarker expression. Cardiac tissue was collected from minipigs with pacing-induced HF (n=8) and without (control, n=6). The samples were selectively harvested from the anterior left ventricular (LV) wall, PS, and from an area remote to the pacing-site, OS. BNP and CNP mRNA expression was evaluated by semi-quantitative polymerase chain reaction (PCR). A significant difference in BNP expression was found in the PS between HF animals and controls (BNP/GAPDH: 0.65+/-0.11 vs. 0.35+/-0.04, p=0.02), but not in the OS (BNP/GAPDH: 0.36+/-0.05, ns vs. controls). CNP expression was not different compared to controls, although higher levels were observed in the PS and in the OS with respect to the controls (CNP/GAPDH: controls 0.089+/-0.036, PS 0.289+/-0.23, OS 0.54+/-0.16). This finding was in tune with an increase of CNP tissue concentration (controls: 0.69+/-0.13; PS=1.56+/-0.19; OS=1.70+/-0.42 pg/mg protein; p=0.039 controls vs. OS). Higher BNP mRNA expression in the PS is consistent with a reduction in contractile function in this region, while higher CNP mRNA expression in the OS suggests the presence of concomitant endothelial dysfunction in the remote region.

In this study the effects of nitric oxide (NO) on intimal hyperplasia (IH) were evaluated in an ex-vivo model of human saphenous vein (SV). SV segments were cultured in conditions able to reproduce IH (FCS), or in medium alone (RPMI), or in presence of a NO donor (NO). Osteopontin (OPN) and Interleukin (IL)-6 were determined in the medium at different culture times and in the tissue, at the end of experiment. OPN and IL-6 release in medium was increased in FCS with respect to RPMI (OPN: 13.9+/-2.9 vs. 2.3+/-0.8 microg/ml, p=0.0011; IL-6: 304.2+/-64.7 vs. 42.0+/-10.1 ng/ml, p<0.0006) as well as intima thickness, that positively correlated with OPN production (r=0.81). In tissue OPN was higher in FCS (82.0+/-30.3 ng/mg protein) than in RPMI (13.8+/-4.2, p=0.0051) and at baseline (3.7+/-0.7, p=0.018). NO reduces IH progression (25%) and both OPN and IL-6 expression (OPN/GAPDH: undetectable baseline; 0.27+/-0.06 RPMI; 0.89+/-0.28 FCS; 0.09+/-0.05 NO; p=0.026 FCS vs. baseline, p=0.018 vs. RPMI, p=0.005 vs. NO). The beneficial NO effect on IH reduction appears to be mediated by the indirect inhibition of OPN production. NO could modulates the initial inflammatory signals that induces the OPN over-production with the related cascade of events leading to IH.

C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n=5) and with pacing-induced HF (n=5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9+/-10.4 pg/ml vs.16.7+/-1.1, p=0.013, mean+/-S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7+/-1.9 pg/mg protein; LA: 8.7+/-3.8; RV: 1.07+/-0.33; LV: 0.93+/-0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8+/-9.9 pg/mg protein vs. 0.9+/-0.17, p=0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.

Density, affinity, and subtype distribution of endothelin-1 (ET-1) binding sites were determined in rat cardiac tissue as a function of age in order to evaluate the association of alterations in the endothelin receptor system and aging in the heart. A significant decrease in the receptor subtype ET-A, which represents 70% to 80% of the total receptor population in cardiac tissue of 3- and 12-month-old rats, was observed in 24-month-old rats with respect to the younger groups. These findings indicate an alteration in ET-1 cardiac receptors associated with aging, mainly due to a variation in the receptor subtype distribution.

Background: C-type natriuretic peptide (CNP) is structurally related to cardiac natriuretic peptides and is currently considered as an endothelium-derived hyperpolarizing factor. Endothelial dysfunction, commonly observed in chronic heart failure (HF) patients is positively affected by physical training., Methods: To evaluate the effect of aerobic physical training on the expression of CNP, 90 HF patients on optimal pharmacological treatment (age 62+/-2 years, mean+/-SEM), randomly assigned in a 3 : 1 ratio to either control group (C, 19 patients) or home-based aerobic exercise-training program group (T, 71 patients), completed the protocol. Plasma assay of CNP, brain natriuretic peptide or B-type natriuretic peptide (BNP), and norepinephrine; echocardiogram; and cardiopulmonary-stress test were performed in all patients at enrollment and after 9 months., Results: At baseline, in both groups, CNP plasma level was significantly related to BNP (R=0.50), ejection fraction (R=0.43), and peak oxygen uptake (VO2, R=0.43, all P<0.001). After 9 months, trained patients showed an improvement in peak VO2 (P<0.001) and ejection fraction (P<0.05), whereas norepinephrine (P<0.05), BNP (P<0.001), and CNP (P<0.001) decreased. No changes occurred in group C. In group T, the decrease in CNP was significantly related to the increase in peak VO2 (R=0.31, P<0.01), and the relation between CNP and BNP was preserved at the end of the program (R=0.41, P<0.001)., Conclusion: Clinical and functional improvement after physical training in HF patients is associated with a decrease in adrenergic activation and in both CNP and BNP concentration. Changes in CNP plasma concentration after physical training might reflect an improvement in endothelial function.

C-type natriuretic peptide (CNP) significantly increases in chronic heart failure (CHF) patients as a function of clinical severity. Aim of this study was to evaluate in CHF patients the relationship between circulating CNP concentrations and echo-Doppler conventional indices of left ventricular (LV) function as well as less load independent parameters as dP/dt. LV ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD) and LV dP/dt were evaluated together with plasma CNP levels in 38 patients with CHF and in 63 controls. CNP levels resulted significantly higher in CHF patients than in controls (7.19+/-0.59 pg/ml vs. 2.52+/-0.12 pg/ml, p<0.0001). A significant correlation between dP/dt and CNP levels (r=-0.61, p<0.0001) was observed. A good correlation with EF (r=-0.55, p<0.001) and a less significant relation with LVEDD (r=0.316, p<0.05) were also reported. When patients were divided according to dP/dt values a very significant difference in CNP levels was observed: Group I (<600, n=25) vs. Group II (>600, n=13): 8.46+/-0.69 and 4.75+/-0.75 pg/ml, respectively, p<0.001. This is the first study that reports a correlation between CNP and dP/dt in CHF patients, thus suggesting a possible role on cardiac contractility.

Fat is now considered as an endocrine organ that produces a lot of molecules having biological activity, called adipocytokines. Among these, adiponectin, a 247 amino acid protein produced abundantly and specifically by adipose tissue, besides its effects on glucose metabolism, plays important protective function against cardiovascular diseases. Circulating levels lower than those of healthy control subjects have found to be associated to conditions such as obesity, diabetes and cardiovascular diseases. In animal experimental models, administration of adiponectin has been shown to have beneficial effects against the development of obesity-related vascular diseases, including atherosclerosis. In humans, circulating levels can be raised by life style modification (weight loss or exercise training) or pharmacological treatments. Adiponectin is present in the human plasma in different isoforms: a large multimeric structure of high molecular weight and in a trimer and examer form, whereas the monomeric form is found only in the adipose tissue. The biological activities of the different multimers are not yet fully known, although the different isoforms appear to have different functional importance following the different diseases. This paper reports the main biological features of adiponectin in order to highlight its possible role as diagnostic/prognostic marker in cardiovascular diseases. Particular attention is paid to practical considerations relative to the analytical determination of this protein in humans.

Little information is available on peripheral levels of Hsp72, Hsp60, and anti-Hsp60 antibodies in patients with left ventricular (LV) dysfunction due to non-atherosclerotic cardiac disease. In this study, serum Hsp72, Hsp60 and anti-Hsp60 antibodies, IL-6, and C-reactive protein (CRP) were measured in 44 healthy controls and in 82 patients with angiographically normal coronary arteries (LV ejection fraction [EF] > or = 50%, n=22; -35% to <50%, n=32; <35%, n=28). Patients with more severe disease (more depressed myocardial blood flow at rest and during dipyridamole, indicative of coronary microvascular impairment) showed more elevated circulating Hsp60 and auto-antibodies, Hsp72, and CRP levels. IL-6 was increased progressively as a function of severity of LV dysfunction. Anti-Hsp60 antibodies, Hsp72, and IL-6 were significantly correlated with brain natriuretic peptide (BNP) levels and LV end-diastolic dimensions (LVEDD) values. IL-6 tended to be related with Hsp72 in particular in patients with more severe disease (r = 0.45, P = 0.021). Hsp60 and Hsp72 activation and inflammatory markers were correlated with the extent of cardiac and microvascular dysfunction in patients with angiographycally normal coronary arteries. These results suggest a pathogenic role of infective-metabolic insult and inflammatory reaction in the development of vascular and myocardial damage in patients with heart failure even in the absence of overt coronary artery disease.

The cardiovascular actions of the C-type natriuretic peptide (CNP) are mainly mediated by the interaction with natriuretic peptide receptor-B (NPR-B). The aim of this study was to identify the sequence of NPR-B in Sus Scrofa, which is not present in GenBank, to verify the expression of NPR-B in the different cardiac chambers of normal pigs and evaluate its homology with murine and human species. Using the guanidinium thyocyanate-phenol-chloroform method, we extracted total RNA from samples obtained from heart of mouse and from the atrium, ventricle, and septum of normal pigs. Pig NPR-B mRNA was sequenced using polymerase chain reaction primers designed from mouse consensus sequences. Sus Scrofa natriuretic peptide receptor 2 mRNA, 1-396 bp, was submitted to GenBank (accession number DQ487044). The presence of NPR-B at mRNA level was detected in all the cardiac chambers; moreover, the bands obtained from pig cardiac tissue shared a 93% sequence homology with a region of the mouse NPR-B and a 95% sequence homology with Homo sapiens. Therefore, NPR-B sequencing provides a new tool to investigate the role of CNP under physiological and pathological conditions in the experimental and clinical setting.

C-type natriuretic peptide (CNP) is expressed in the vascular endothelium. It is not known whether CNP is specifically increased in patients with idiopathic left ventricular systolic dysfunction (ILVDys) with or without overt heart failure, and whether in these patients it is related with indicators of myocardial and/or endothelial/microvascular impairment. We determined plasma CNP levels in 51 ILVDys and in 60 controls. We observed a significant increase in patients with (7.0+/-0.9 pg/ml) or without (6.1+/-0.53 pg/ml) overt heart failure (p<0.001) in respect to controls (2.5+/-0.12 pg/ml). CNP was significantly correlated with LVEF (p<0.001), end-diastolic dimension (p<0.05), ANP (p<0.001) and BNP (p<0.001), interleukin-6 (p<0.001), total cholesterol (p<0.05), low-density lipoprotein (p=0.05), ratio total cholesterol/ high-density lipoprotein (p=0.05) and, in a subgroup of patients, with abnormal vasodilating capacity of the coronary microcirculation. In conclusion, CNP is activated in patients with LV dysfunction but without coronary artery disease, independently of the presence of overt heart failure and in tune with the extent of myocardial functional involvement. In these patients CNP is also related with both systemic and coronary indicators of endothelial/microvascular damage.

Background: Matrix metalloproteases (MMPs) play an important role in cardiovascular remodeling by degrading the extracellular matrix. The aim of this study was to compare two different methods for MMP-2 and MMP-9 concentration and activity determination., Methods: MMP-2 and -9 levels were measured by immunometric and enzymatic assays to determine total and active levels. The two procedures differ in assay principle and in the extent of cross-reactions with interfering substances present in biological samples. Both human serum and culture medium from an ex vivo human model of intimal hyperplasia were checked., Results: All methods were able to detect MMP-2 and -9 with similar levels of sensitivity, reproducibility and accuracy, and furnished positively related results, although significantly different, in both types of sample. Both systems were able to detect changes in MMP production such as the time-course of MMP-2 and -9 release by cultured saphenous vein associated with intima hyperplasia progression., Conclusions: Our data indicate that different values for MMP concentrations can be obtained using different analytical methods, even if they are intrinsically reliable. This suggests that methodological differences should be taken into account when comparing MMP results from different studies.

C-type natriuretic peptide (CNP) is a peptide produced by the vascular endothelium with vasodilative properties. It shares structural and physiological properties with the atrial and brain natriuretic peptides (ANP and BNP), whose central role in the pathophysiology of heart failure (CHF) is firmly established. The role of CNP, first isolated from porcine brain, has not been yet completely determined. The transcription of the gene, that in man is located on chromosome 2, is regulated by factors such as tumor necrosis factor and interleukin-1. Two mature forms of the peptide exist: CNP-53, that predominates in tissues and CNP-22, found mainly in plasma. As recently found, CNP is produced directly in the myocardium and an increase in plasma levels of this peptide and of its precursor was observed in CHF. The aim of this review was to examine the current literature relating to cardiovascular functions of CNP and in particular to its role in CHF. In fact, CNP may represent an important new local autocrine and endocrine mediator in CHF although further evaluations are required to define its full pathophysiological role in this disease.

Background: C-type natriuretic peptide production by the heart in patients with chronic heart failure has recently been demonstrated by measuring the difference in C-type natriuretic peptide plasma levels between the aortic root and coronary sinus samples. To assess this previous relevant observation, we re-evaluated the cardiac production of C-type natriuretic peptide, thus increasing the total number of patients studied. In light of the difficulty in recruiting patients with these characteristics, this kind of study can be performed with a small number of subjects., Methods: Cardiac production of C-type natriuretic peptide was evaluated in a subset (n = 6) of patients, referred to our institute for the evaluation of chronic heart failure. C-type natriuretic peptide was measured in the coronary sinus and in aorta blood collected during right and left heart catheterization using a specific immunometric assay, after solid-phase extraction of plasma samples., Results: Significantly higher C-type natriuretic peptide levels were found in the coronary sinus than in aorta blood (7.8 +/- 1.3 versus 6.1 +/- 1.5 pg/ml, P = 0.034) and our results were in accordance with previous ones. Analysing as a whole the data gathered in the two studies, a noteworthy significant increase was observed (n = 15, P = 0.002) between the C-type natriuretic peptide concentrations in the two sites., Conclusions: The results of the present study strengthen the hypothesis that C-type natriuretic peptide is produced by the heart in patients with chronic heart failure.

Background: Increased circulating adrenomedullin (AM) concentration has been reported in congestive heart failure (HF) and considered as a possible marker of cardiac dysfunction., Hypothesis: The study was undertaken to assess the relationship between circulating AM concentration and left ventricular (LV) functional state, estimated by echo-Doppler techniques in patients with mild to moderate HF and different degrees of LV dysfunction., Methods: Plasma AM, B-type natriuretic peptide (BNP), and N-terminal (NT) proBNP levels were measured in 55 patients with HF (New York Heart Association [NYHA] I n = 8, II n = 26, III n = 21) and in 20 controls; dP/dt was calculated by the Doppler tracing of the mitral regurgitation jet., Results: The study was completed in 51 patients. Adrenomedullin levels were higher than in controls (19.2 +/- 1.4 vs. 13.3 +/- 0.7, p < 0.005) and elevated in proportion to NYHA functional class. B-type natriuretic peptide and NT-proBNP were 344 +/- 67 vs. 12 +/- 2 pg/ml and 2196 +/- 623 vs. 52 +/- 4 pg/ml, respectively (p < 0.0001); dP/dt was better related to AM (r = 0.582, p < 0.001) than to the other peptides. Adrenomedullin was significantly (p < 0.001) different between patients grouped according to the dP/dt cut-off predictive of event-free survival., Conclusions: The combination of depressed contractility and increased AM may provide a clue for further characterization of the severity of LV dysfunction in HF, independent of baseline LV ejection fraction.

Background: [corrected] C-type natriuretic peptide (CNP), secreted by the endothelium and the heart, is structurally related to atrial and brain natriuretic peptides, but its clinical significance in chronic heart failure (CHF) is controversial., Aim: To investigate the role of CNP in CHF, plasma CNP levels were determined in a prospective series of 133 patients with CHF (age 64 +/- 1 years, left ventricular ejection fraction (EF), 31.5 +/- 0.7%, mean +/-S.E.M.) and in 21 age-matched healthy subjects., Methods and Results: CNP was measured by a radioimmunoassay (sensitivity: 0.41+/-0.009 pg/tube) after a preliminary solid-phase extraction. Plasma level of CNP in healthy subjects was 2.7 +/- 0.2 pg/ml and significantly increased in CHF, as a function of clinical severity: 4.9 +/- 0.7 pg/ml in NYHA class I; 7.0 +/- 0.4 pg/ml in class II (p < 0.001 vs. controls); 9.6 +/- 0.7 pg/ml in class III (p < 0.001 vs. controls and class I and II), and 11.8 +/- 2.0 pg/ml in class IV (p < 0.001 vs. controls, class I and II; Fisher's test after ANOVA). A significant relation was also found between CNP plasma levels and EF (R = 0.40, p < 0.001)., Conclusion: Plasma CNP elevation is related to clinical and functional disease severity. These findings suggest a pathophysiological role for this peptide that, for its vasorelaxing activity, could influence the endothelial vasomotor response in CHF.

Heat shock proteins (Hsps) are induced by stressful stimuli and have been shown to protect cells and organs from such stresses both in vitro and in vivo, and play a positive role in lifespan determination. An attenuated response to stress is characteristic of senescence and no Hsp induction is observed upon exposure to stress and no protective effect of a mild stress is observed in cells from aged individuals. The artificial over-expression of Hsps, can produce a protective effect against a variety of damaging stimuli in cells from aged rats or aged humans, in whom cardiovascular disease is a major cause of morbidity in older age. Here, we show that aging significantly decreases the levels of Hsp27, Hsp60, Hsp72 and Hsc70 in right atrium and left ventricle of the rat heart, both at level of protein and of mRNA. Two different caloric restriction regimens have been found to counteract in part the decrease in the levels of Hsp expression in the aged heart tissue as well as the tendency to an increase of the levels of carbonyl in cardiac proteins. Our data suggest that cardiac Hsp levels may be a determinant of longevity in rodents, and that generation of new regimens of caloric restriction may eventually show how to improve modulation of cardiac aging.

C-Type natriuretic peptide (CNP) is a member of the family of natriuretic peptides with vasodilatory properties, and is produced and secreted by endothelial cells. It seems to play a central role in the paracrine vasomotor control of tone and to be important in several clinical conditions characterized by endothelial dysfunction. We evaluated the analytical performance of a commercially available radioimmunoassay for CNP after a preliminary extraction with Sep-Pak C18. Its analytical reliability was checked by determination of CNP plasma levels in healthy subjects (n = 23) and in patients with different diseases, likely characterized by endothelial dysfunction, such as chronic heart failure (n = 133) and cirrhosis (n = 84). The extraction yield was 78+/-3%. Accuracy of the radiommunological determination was evaluated by dilution (45-370 microL of extracted plasma) and recovery tests (>80%). Between- and within-assay variabilities were < or = 10% and analytical sensitivity was 0.41+/-0.015 pg/tube. Plasma CNP in patients with chronic heart failure and with cirrhosis was significantly raised compared to controls (p<0.0001 and p = 0.001, respectively). The sensitivity, accuracy and variability levels of the method proposed for CNP assay was suitable for reliable detection of changes in CNP plasma levels in the clinical setting.

Background: The evaluation of big-endothelin (ET)-1 plasmatic concentrations may serve as another noninvasive marker in patients with cardiovascular diseases, based on the assumption that it may reflect endothelin overproduction more accurately than circulating ET-1. For this reason the analytical performance of an immunoenzimatic assay for plasma Big-ET-1, with or without a preliminary step of extraction, was evaluated., Methods and Results: Sensitivity for direct assay was 0.15+/-0.010 fmol/ml (n=27), inter-assay variability, evaluated at 2 different concentrations, resulted 2.5+/-0.062, CV%=5.6 (n=5) and 1.47+/-0.09 fmol/ml, CV%=12.8 (n=4), respectively, while intra-assay variability was 0.89+/-0.022 fmol/ml, CV%=5.6. Sensitivity for the assay with extraction resulted 0.71+/-0.104 (n=6) fmol/ml and intra-assay variability was 3.6+/-0.13 fmol/ml, CV=7.4% (n=4). The comparison between the 2 procedures, performed on 107 plasma samples at different peptide concentrations, showed a close agreement between the results of the 2 procedures for Big-ET-1 values higher than 1 fmol/ml., Conclusion: The main limitation of the direct assay is due to possible interference effects while the correct evaluation of extraction yield of the individual samples is the main drawback of the procedure with extraction. These effects become important when assaying samples with low levels of analyte. The direct assay of plasma Big-ET-1, easier to perform, more rapid and less expensive, could be the choice method.

Background: Overexpression of heat shock proteins (Hsps) is associated to myocardial protection and it has been suggested that they could be a marker of cardiac preservation in conditions such as extracorporeal circulation. Aim of this study was to evaluate if cardioplegic arrest can modify the expression of Hsps in the heart and if this alteration is associated to cardiac preservation., Method: The levels of Hsp 27, Hsp 60, and both the constitutive and the inducible form of Hsp 70 were measured in the cardiac tissue from right atrium of pediatric patients before and after aortic cross-clamping (ACC) during cardiopulmonary bypass surgery for correction of congenital heart disease (n=20). The quantitative evaluation of Hsps was made by Western blotting analysis after tissue extraction and protein separation. Hsp 72 mRNA expression was also evaluated in pre- and post-ACC samples of eight subjects by semiquantitative RT-PCR. Peripheral levels of Troponin I, Myoglobin, LDH, CK, CK-MB were measured in basal conditions and at 12 and 24h after cardiosurgery as markers of heart damage., Results: The cardioplegic arrest did not significantly modify the mean levels of all the Hsps measured. Hsp 72 levels increased after cardioplegia in the 40% of the patients and all Hsps in the 28% of subjects. The patients whose levels of Hsps are increased after cardioplegia are associated with lower post-surgery concentrations of all the markers of cardiac injury., Conclusions: This observation suggests a relationship between the increase of Hsps and the reduction of cardiac injury.

In order to study the influence of gender on circulating levels of cardiac natriuretic hormones (CNHs) in heart failure, we measured the plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) by means of highly sensitive and specific IRMA methods in 239 consecutive patients (age 64.7 +/- 11.6 years, range 21-89 years; 170 men and 69 women) with cardiomyopathy. There was different response of CNH according to gender in patients with heart failure, as indicated by the ratio between the individual CNH values of patients and the gender-specific cut-off values. Indeed, the mean ratio for ANP found in men (3.6 +/- 3.6) was significantly higher (p = 0.0075) than that found in women (2.4 +/- 2.1). The mean ratio for BNP was on average 2.3 fold higher (15.9 +/- 27.1 in men and 6.9 +/- 6.8 in women, p = 0.0084). Moreover, age, ejection fraction, and disease severity independently and significantly contributed to regression with both ANP (R = 0.612, F = 39.969, p < 0.0001) and BNP (R = 0.656, F = 49.957, p < 0.0001) values, while gender did not. In conclusion, our study suggests a different, gender-specific activation of the CNH system in this clinical condition, although age, ejection fraction and disease severity seem to be more powerful predictors than gender of circulating levels of ANP and BNP in patients with heart failure.

In order to study the relationships between sex hormones, aging, and circulating levels of cardiac natriuretic peptides and to define reference values for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) assays, we measured the plasma levels of cardiac natriuretic peptides in a large group of healthy adults divided according to age and sex. We studied 216 healthy subjects of both sexes (109 men and 107 women) with age ranging from 20 to 77 years (mean 43.2+/-14.8 years). All subjects were non-obese and had normal arterial blood pressure; they were free from acute diseases, including asymptomatic heart disease. Highly sensitive and specific IRMA methods were used to measure plasma ANP and BNP. The mean ANP value in healthy adult subjects of both sexes was 17.8+/-10.9 pg/ml with no significant difference between men (16.7+/-10.0 pg/ml) and women (18.8+/-11.7 pg/ml). The mean BNP value in healthy adult subjects of both sexes was 9.9+/-9.0 pg/ml with a significant difference (p<0.0001) between men (7.7+/-7.1 pg/ml) and women (12.2+/-10.2 pg/ml). There was a weak linear relationship between age and either ANP (r=0.350, p<0.0001) or BNP (r=0.254, p=0.0002) values. When the circulating levels of cardiac natriuretic hormones, and age and sex were analyzed by multiple stepwise regression analysis, both age and sex significantly and independently contributed to the regression. Our study indicates independent positive effects of aging and female sex hormones on ANP and BNP levels in healthy adult subjects. These effects should be taken into account in the calculation of appropriate reference values for cardiac natriuretic hormones.

The mechanisms that mediate the cardioprotective action of steroid hormones in postmenopausal women are poorly understood. To study the inter-relationship between female steroid hormones and cardiac natriuretic peptides, plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured in postmenopausal women, both before and after oestrogen replacement therapy. A total of 22 healthy postmenopausal women (mean age 51.9+/-4.6 years) were enrolled in the study; all had been postmenopausal for at least 1 year and all reported climacteric symptoms accompanied by increased levels of follicle-stimulating hormone (>30 m-i.u./ml) and luteinizing hormone (>20 m-i.u./ml), and a reduction in oestradiol (<25 pg/ml). All women were given hormone replacement therapy with transdermal oestradiol, either patch (50 microg/24 h) or gel (1 mg/day), cyclically combined with oral dihydrogesterone (10 mg/day for 12 days/month, on days 19-30 of the month). ANP and BNP were measured directly in plasma samples with specific and sensitive immunoradiometric assays before and after hormone replacement therapy (transdermal oestradiol combined with oral dihydrogesterone). Body weight, arterial blood pressure and echocardiographic examination values did not change after hormone replacement therapy. As expected, serum oestradiol increased significantly and gonadotropins decreased as an effect of the hormone replacement therapy. On average, both ANP and BNP had increased significantly after 3 months of hormone replacement therapy [ANP: before treatment, 17.6+/-9.6 pg/ml; after, 23.6+/-5.6 pg/ml (P=0.0173); BNP: before treatment, 12.6+/-10.2 pg/ml; after, 19.8+/-14.0 pg/ml (P<0.0001)]. Our study indicates that hormone replacement therapy for a period of 3 months induces a rise in the circulating levels of cardiac natriuretic hormones in postmenopausal women. Our data also suggest the working hypothesis that cardiac natriuretic peptides may play an important role in mediating the cardioprotective effects of female steroid sex hormones in women throughout life.

Nitrates act as donors of nitric oxide (NO), a molecule with a recognized potential for genotoxicity. In order to assess whether chronic long-term nitrate therapy may increase genotoxicity, we evaluated chromosomal damage in peripheral lymphocytes of 27 ischaemic patients undergoing chronic nitrate treatment for vertical line4 years (7.9 +/- 3.1, mean +/- SD) and 18 age- and sex-matched subjects without any previous nitrate treatment. At the same time, after treatment in vitro with 0-20 microM sodium nitroprusside as NO donor, micronucleus induction and cell proliferation were also evaluated using blood from six different healthy donors. The results showed that the frequency of structural chromosomal aberrations was not significantly higher in the drug-treated group than the control [2.1 +/- 1.4 versus 1.6 +/- 1.2 (mean +/- SD); P = 0.23]. The frequency of micronucleated lymphocytes was higher in the nitrate group than in the control group (6.5 +/- 4.6 versus 3.5 +/- 2.9, P=0.01). In vitro treatment indicated a dose-dependent increase in the frequency of micronucleated lymphocytes with increasing SNP concentrations. Cytotoxicity and cell cycle delay, with a statistically significant difference with respect to control culture, were also observed. Our results suggest a possible genotoxic activity of nitrate therapy. Further studies focusing on the possible link between nitrate therapy and genotoxicity are warranted at this point.

Background: Three specific receptors for the cardiac natriuretic peptide system have been identified to date. Down-regulation of the biologically active binding sites (i.e. NPR-A and NPR-B) could explain the blunted response to cardiac natriuretic hormones observed in heart failure (HF), but not the increased metabolic clearance rate. Variations in the ratio between biological and clearance (NPR-C) receptors in target tissue may explain this increase., Aim: The aim of this study was to investigate the regulation of NPR-C receptors on platelets, in patients with HF., Methods: Eighteen patients with HF (NYHA class: I-II, n=8; III-IV, n=10) and 18 age-matched healthy subjects were studied. The affinity constant (K(d)) and density (B(max)) of binding sites were derived by saturation assays on platelet suspensions using 125I-ANP as radioligand., Results: B(max) increased as a function of the severity of disease: 21.3+/-3.3 fmol/10(9) cells in class III-IV, 11.7+/-2.2 in class I-II, and 11.6+/-1.1 in controls, respectively (P=0.0179 for class III-IV vs. controls and P=0.0451 vs. NYHA I-II)., Conclusions: The increase in density of 'clearance' receptors in severe HF is theoretically consistent with the reduction in cardiac natriuretic peptide biological activity, as well as the increase in metabolic clearance rate. This suggests that clearance receptor blockade may be of potential therapeutic value in HF.

Endothelin (ET)-1 is a potent vasoconstrictor peptide produced in the myocardium that can exert important effects on cardiac myocyte growth and phenotype; cardiac natriuretic peptides (ANP and BNP) are known to act as physiological antagonists of ET-1. In this study a comparative determination of ET-1 receptors and of the local productions of ET-1 and of ANP and BNP was made in different sites of failing and nonfailing hearts. Tissue from right and left atrium, right and left ventricle and interventricular septum from seven adult heart transplant recipients with end-stage idiopathic dilated cardiomyopathy (functional class III and IV, with ejection fraction < 35%) and from four postmortem subjects without cardiac complications was analyzed. In failing hearts we observed a tendency to increase of density of binding sites, most evident in left ventricle (62.6+/-22.6 fmol/mg protein vs. 29.0+/-3.3, mean +/- SEM, p = ns). A prevalence of ET-A subclass, observed in all samples, resulted more pronounced in failing hearts where this increase, found in all the cardiac regions, was more evident in left ventricle (p = 0.0007 vs nonfailing hearts). The local concentrations of ET-1, ANP and BNP resulted significantly increased in failing hearts with respect to controls in all sides of the heart. In failing hearts we have observed a tendency to increase in endothelin receptor density mainly due to a significant upregulation of ET-A subtype and a parallel increase of the tissue levels of ANP, BNP and ET-1 indicating an activation of these systems in heart failure.

The clinical relevance of measuring plasma brain natriuretic peptide (BNP) is well-known, especially in patients with heart failure. Recently, a new method for measuring BNP, called TRIAGE, has been developed which can be used for point-of-care testing of patients with congestive heart failure. The aim of the present study is to compare the analytical performance of this fully-automated method to that of an immunoradiometric assay (IRMA), routinely used to measure BNP. The TRIAGE method is a non-competitive immunofluorometric assay which uses two different binding phases, specific for two different epitopes of the BNP amino acid chain, to form a sandwich with the specific ligand (i.e., BNP). A polyclonal antibody is included in the fluorescent immunoassay reagents which are contained in the assay devices and a monoclonal antibody is immobilized in the detection lane of the assay device. The imprecision of the TRIAGE method was approximately 12% for BNP concentrations in the normal range and about 18% for BNP concentrations above the normal range. The mean reading time of the TRIAGE method was 14.5 +/- 8.6 min. A close linear relationship was found between the BNP values measured with the two methods (TRIAGE = 24.6+0.933 IRMA; r = 0.932, n = 83). The TRIAGE method is indicated for BNP assay in ambulatory and coronary or emergency units, where usually only a few samples (preferentially whole blood samples) must be measured in a short time. The IRMA method should be preferred for pathophysiological studies, requiring the highest degree of precision and sensitivity for simultaneous measurement of several stored plasma samples or tissue extracts.

Endothelin (ET) is a peptide composed of 21 amino acids, derived from a larger precursor, the big-endothelin, by action of the endothelin-converting enzyme (ECE) family; three isoforms of endothelin, named ET-1, ET-2 and ET-3, have been identified. Endothelin-1 is generated mainly by vascular endothelial cells and exerts various important biological actions, mediated by two receptor subtypes, ET-A and ET-B, belonging to the G protein-coupled family that have been identified in various human tissues such as the cardiac tissue. Endothelin-1 is a potent vasoconstrictive agent, has inotropic and mitogenic actions, modulates salt and water homeostasis and plays an important role in the maintenance of vascular tone and blood pressure in healthy subjects. Endothelin-1, as well as ET-A and ECE-1, also has an important role in cardiovascular development, as observed by the variety of abnormalities related to neural crest-derived tissues in mouse embryos deficient of a member of the ET-1/ECE-1/ET-A pathway. Various evidence indicates that endogenous endothelin-1 may contribute to the pathophysiology of conditions associated with sustained vasoconstriction, such as heart failure. In heart failure, elevated circulating levels of both endothelin-1 and big-endothelin-1 are observed; in failing hearts an activation of the endothelin system is found: tissue level of ET-1 is increased with respect to non-failing hearts as well as receptor density, due mainly to an upregulation of the ET-A subtype, the prevalent receptor subclass in cardiac tissue. Finally, studies in both humans and animal models of cardiovascular disease show that inhibition of the endothelin function (anti-endothelin strategy) is associated with an improvement of haemodynamic conditions; these observations indicate that endothelin receptor antagonists or endothelin-converting enzyme inhibitors may constitute a novel and potentially important class of agents for the treatment of this disease., (Copyright 2001 Academic Press.)

Increased levels of cardiac natriuretic peptides in patients undergoing hemodialysis may be a marker of cardiomyopathy and in consequence may be suitable prognostic indicators for the risk of development of cardiac disease. We measured plasma levels of ANP, BNP, proANP(1-98) and proBNP(1-76)-related peptides with some competitive and non-competitive immunoassay methods in patients with renal failure on chronic hemodialysis in order to compare the analytical performances of these methods and to evaluate the clinical usefulness of each assay for patients with chronic renal failure. ANP and BNP values significantly decreased after hemodialysis (on average, ANP by 36% and BNP by 16%); while all proANP and proBNP values tended to increase, but only proANP(1-30) (by 14.4%) and Nt-proBNP (by 9.5%) significantly. Although significant correlations were found among all the circulating levels of cardiac peptides studied, N-terminal pro-peptides correlated better among themselves than with ANP and BNP; ANP was only slightly correlated with all the other peptides, the only exception being BNP. Only BNP levels significantly increased according to the degree of ventricular hypertrophy and/or ventricular function in patients with chronic renal failure. The ANP assay is preferable in physiological and clinical studies for the rapid changes in atrial pre-load. BNP would be more useful in the follow-up of cardiac complications in patients with end-stage renal disease on regular hemodialysis. The assays of N-terminal proANP(1-98)-and proBNP(1-76)-related peptides proved to be of limited use, because they were not able to detect acute changes in pre-load during hemodialysis and were less useful than BNP levels as markers of ventricular hypertrophy and/or functional cardiac impairment.

Natriuretic peptide binding sites on platelets have been hypothesized to act as clearance receptors; however, there is no clear definition of the function of this receptor. The aim of the study was: 1) to characterize natriuretic peptide receptors in human platelets by original competition study; 2) to evaluate a possible age modulation of these binding sites, since a delayed clearance of ANP in the elderly has been observed. The binding of 125I-ANP to intact platelets was completely inhibited by h-ANP, h-BNP, h-CNP and c-ANP, the selective ligand of the clearance receptor. IC50 values were 0.089+/-0.029, 0.703+/-0.104 and 1.19+/-0.13, 3.84+/-0.04 nmol/l, mean+/-SE, respectively (p<0.001 for IC50 value of h-ANP compared to the other natriuretic peptides). This observation on the receptor selectivity of natriuretic peptides in human platelets provides new evidence for the presence of the clearance receptor on platelets. In control subjects the Kd was 34.6+/-4.0 pmol/l and Bmax 13.6+/-0.92 fmol/10(9) platelets (mean+/-SE), (no.=46, mean age 41.7+/-2.1 years). Bmax was significantly reduced in older subjects (no.=25, mean age 53.2+/-1.5 years) with respect to the younger group (no.=21, mean age 28.0+/-0.87 years): 11.4+/-1.1 vs 16.1+/-1.4 fmol/10(9) cells, p=0.0096, respectively; moreover, a significant inverse relationship between Bmax and the subject's age was observed. This observation suggests a possible reduction of the natriuretic peptide clearance with aging, associated to a significant increase of plasma levels of natriuretic peptides.

Background: Cardiac natriuretic hormones (CNHs) are a family of related peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and other peptides derived from the N-terminal portion of the proANP and proBNP peptide chains. Assays for cardiac natriuretic peptides have been proposed to help assess clinical conditions associated with expanded fluid volume. In particular, the assays can be useful for distinguishing healthy subjects from patients in different stages of heart failure. Measurements of these hormones have also been considered for prognostic indicators of long-term survival in patients with heart failure and/or after acute myocardial infarction. The different CNHs differ in their production/secretion patterns and have different clearance rates. Furthermore, there are numerous proposed assay configurations for each of these hormones, and it is not clear which assay provides the best pathophysiological and/or clinical information., Approach: Here we review recent studies concerning the competitive (such as RIA, enzyme immunoassay, or luminescence immunoassay) and noncompetitive immunoassays (such as two-site IRMA, ELISA, or immunoluminometric assay) for the different cardiac natriuretic peptides to compare the analytical characteristics and clinical relevance of assays for the different CNHs and the different assay formats., Content: Developing sensitive, precise, and accurate immunoassays for cardiac natriuretic peptides has been difficult because of their low concentrations (on average, approximately 3-6 pmol/L) in healthy subjects and because of their structural, metabolic, and physiological characteristics. Competitive assays have historically suffered from lack of sensitivity and specificity for the biologically active peptides. These usually require tedious extraction procedures prior to analysis. Recently, immunometric assays have been developed that have improved sensitivity and specificity; it appears these will be the methods of choice., Summary: To date, there is no consensus on the best assay procedure of cardiac natriuretic peptides. To facilitate widespread propagation of determination of these hormones in routine clinical practice, it will be necessary to study the new generation of noncompetitive immunometric methods that are less time-consuming and more sensitive and specific. Although several studies suggest that BNP exhibits better clinical utility than the other CNHs, more studies examining multiple CNHs in the same cohorts of patients will be necessary.