Distinguishable DNA methylation defines a cardiac-specific epigenetic clock.

BACKGROUND: The present study investigates whether epigenetic differences emerge in the heart of patients undergoing cardiac surgery for an aortic valvular replacement (AVR) or coronary artery bypass graft (CABG). An algorithm is also established to determine how the pathophysiological condition might influence the human biological cardiac age. RESULTS: Blood samples and cardiac auricles were collected from patients who underwent cardiac procedures: 94 AVR and 289 CABG. The CpGs from three independent blood-derived biological clocks were selected to design a new blood- and the first cardiac-specific clocks. Specifically, 31 CpGs from six age-related genes, ELOVL2, EDARADD, ITGA2B, ASPA, PDE4C, and FHL2, were used to construct the tissue-tailored clocks. The best-fitting variables were combined to define new cardiac- and blood-tailored clocks validated through neural network analysis and elastic regression. In addition, telomere length (TL) was measured by qPCR. These new methods revealed a similarity between chronological and biological age in the blood and heart; the average TL was significantly higher in the heart than in the blood. In addition, the cardiac clock discriminated well between AVR and CABG and was sensitive to cardiovascular risk factors such as obesity and smoking. Moreover, the cardiac-specific clock identified an AVR patient's subgroup whose accelerated bioage correlated with the altered ventricular parameters, including left ventricular diastolic and systolic volume. CONCLUSION: This study reports on applying a method to evaluate the cardiac biological age revealing epigenetic features that separate subgroups of AVR and CABG.

PHOX2A and PHOX2B genes are highly co-expressed in human neuroblastoma.

The detection of PHOX2B mutations in a small proportion of patients affected with either familial or sporadic neuroblastoma (NB), has arisen interest on the possible pathogenic role of this gene in the disease determination. In this light, we have carried out a quantitative expression analysis of PHOX2B and its paralogue PHOX2A on a panel of NB cell lines and NB tumour samples to identify a possible differential expression between NB cells and their normal counterpart (adrenal medulla cells). Our results revealed that both PHOX2A and PHOX2B are over-expressed in tumour samples and NB cell lines. Particularly, the expression levels of the two genes in NB cell lines show a highly significant correlation, suggesting their possible synergistic role or a coordinated expression regulation. Furthermore, PHOX2 gene over-expression in NB tumours and cell lines suggests these genes may be widely involved in NB development through either a direct mechanism of up-regulation or a failure in maintaining proper transcript levels after embryonic development.

Therapeutic modulation of the nitric oxide: all ace inhibitors are not equivalent.

The properties of the angiotensin-converting enzyme (ACE) inhibitors have largely been attributed to a class effect. However, this opinion is now increasingly challenged in view of the findings from recent clinical trials, which have demonstrated differential effects of ACE inhibitors, in particular with respect to secondary cardiovascular prevention outcomes. In this experimental study, Sprague-Dawley rats were treated with five different ACE inhibitors (enalapril, perindopril, quinapril, ramipril, and trandolapril) at equihypotensive doses. All ACE inhibitors increased endothelial nitric oxide synthase (eNOS) protein expression and activity in the aorta (both P<0.0001 versus vehicle) and in cardiac myocytes (both P<0.05 versus vehicle). A highly significant effect was observed with perindopril when compared with vehicle in the modulation of eNOS protein expression and activity in aorta (22.52+/-1.09 versus 9.12+/-0.57 AU microg(-1) protein and 1.59+/-0.03 versus 0.77+/-0.02 pmol l(-1) citrulline min(-1)mg protein(-1), respectively) and in cardiac myocytes (17.64+/-0.94 versus 11.30+/-0.59 AU microg(-1) protein and 0.93+/-0.02 versus 0.62+/-0.03 pmol l(-1) citrulline min(-1)mg protein(-1), respectively). On the basis of the eNOS protein expression in the rat aorta, the other ACE inhibitors had similar, but lower effects. Indeed, the rank of potency - based both on eNOS protein expression and activity - was perindopril>trandolapril approximately quinapril approximately ramipril approximately enalapril (P<0.05 perindopril versus trandolapril and ramipril and P<0.01 perindopril versus enalapril, respectively). Levels of circulating nitrite/nitrate, the end-metabolites of nitric oxide, were also significantly affected by ACE inhibition, with the same order of potency. Our findings provide further evidence in favor of differential effects associated with ACE inhibitor therapy and suggest that the clinical benefits associated with these drugs may not solely reflect a class effect extending their benefit beyond blood pressure-lowering effect.

Chromogranin A in heart failure; a novel neurohumoral factor and a predictor for mortality.

BACKGROUND: In chronic heart failure, several hormonal systems are activated with diagnostic and prognostic implications. We tested the hypotheses that serum Chromogranin-A (CgA) -- a 49 kDa acid protein present in the secretor granules of neuroendocrine cells -- is increased in chronic heart failure and that CgA levels are a predictive factor for mortality. METHOD AND RESULTS: In 160 patients with chronic heart failure, we measured serum CgA and other neuroendocrine hormones. The results showed that CgA is increased in chronic heart failure and the increase is related to the clinical severity of the syndrome: CgA levels in New York Heart Failure (NYHA) class II (median 146.9 ng x ml(-1), inter-quartiles 108.3-265.5) were significantly higher (P<0.05) than in class I (median 109.7 ng x ml(-1), inter-quartiles 96.7-137.6), and significantly lower (P<0.05) than in class III (median 279.0 ng x ml(-1), inter-quartiles 203.6-516.1). Class IV patients showed the highest serum levels of CgA (median 545.0 ng. ml(-1), inter-quartiles 231.8-1068.3), being statistically significantly different from class III patients (P<0.001). The association between survival and some recognized variables of prognostic significance, including CgA was also studied. The results showed that ejection fraction, noradrenaline, atrial natriuretic peptide, NYHA class and CgA were significant univariate prognosticators; however, in the multivariate analysis by the Cox proportional-hazard model, CgA and NYHA class were the only independent predictive factors for mortality (P<0.005, RR=1.22, 95% CI=1.06-1.41 and P=0.04, RR=1.58, 95% CI=1.02-2.46, respectively). CONCLUSIONS: CgA is a pro-hormone, precursor of several active fragments likely to exert biological effects in chronic heart failure. CgA serum levels are increased in patients with chronic heart failure and are a predictive factor for mortality.

Role of bradykinin and eNOS in the anti-ischaemic effect of trandolapril.

1. Angiotensin converting enzyme (ACE) inhibitors are under study in ischaemic heart diseases, their mechanism of action being still unknown. 2. The anti-ischaemic effect of trandolapril and the possible involvement of a bradykinin-modulation on endothelial constitutive nitric oxide synthase (eNOS) in exerting this effect, were investigated. 3. Three doses of trandolapril, chronically administered in vivo, were studied in isolated perfused rat hearts subjected to global ischaemia followed by reperfusion. 4. Trandolapril has an anti-ischaemic effect. The dose of 0.3 mg kg(-1) exerted the best effect reducing diastolic pressure increase during ischaemia (from 33.0+/-4.5 to 14.0+/-5.2 mmHg; P<0.05 vs control) and reperfusion (from 86.1+/-9.4 to 22.2+/-4.1 mmHg; P<0.01 vs control), improving functional recovery, counteracting creatine phosphokinase release and ameliorating energy metabolism after reperfusion. 5. Trandolapril down-regulated the baseline developed pressure. 6. Trandolapril increased myocardial bradykinin content (from 31.8+/-6.1 to 54.8+/-7.5 fmol/gww; P<0.05, at baseline) and eNOS expression and activity in aortic endothelium (both P<0.01 vs control) and in cardiac myocytes (from 11.3+/-1.5 to 17.0+/-2.0 mUOD microg protein(-1) and from 0.62+/-0.05 to 0.80+/-0.06 pmol mg prot(-1) min(-1); both P<0.05 vs control). 7. HOE 140 (a bradykinin B(2) receptor antagonist) and NOS inhibitors counteracted the above-reported effects. 8. There was a negative correlation between myocyte's eNOS up-regulation and myocardial contraction down-regulation. 9. Our findings suggest that the down-regulation exerted by trandolapril on baseline cardiac contractility, through a bradykinin-mediated increase in NO production, plays a crucial role in the anti-ischaemic effect of trandolapril by reducing energy breakdown during ischaemia.

Ace-inhibition with quinapril modulates the nitric oxide pathway in normotensive rats.

Angiotensin-converting enzyme (ACE) inhibitors exert some cardiovascular benefits by improving endothelial function. We evaluated the effects of chronic treatment with quinapril (Q) on the l -arginine/nitric oxide (NO) pathway in normotensive rats under baseline and inflammatory conditions. The role of bradykinin was also investigated. The animals received for 1 week either the ACE-inhibitor Q (1 and 10 mg/kg/day), the B(2)receptor antagonist HOE 140, Q+HOE 140, or no drug. At the end of chronic treatment, rats underwent either a 6-h placebo or an E. coli endotoxin challenge. The following measurements were made: (i) endothelial and inducible NO synthase (eNOS and iNOS) protein expression; (ii) eNOS/iNOS activity; (iii) serum levels of nitrite/nitrate and tumour necrosis factor (TNF)- alpha; (iv) NO in the expired air (eNO). Q increased baseline aortic eNOS protein expression (up to 99%, P<0.001) and activity (l -citrulline synthesis up to 94%, P<0.01; serum nitrite/nitrate up to 55%, P<0.05). HOE 140 partially reversed Q-induced upregulation of eNOS (P<0.05). Moreover, Q counteracted LPS effects, i.e. increased the impaired eNOS pathway and limited iNOS induction (up to 94 and 24%, respectively), and reduced the increased nitrite/nitrate and TNF- alpha serum levels as well as eNO (up to 25, 38 and 28%, respectively, P<0.01 for all comparisons). HOE 140 did not influence Q effects on iNOS during endotoxaemia. In conclusion, in (patho)physiological conditions in rats, Q up-regulated eNOS with a bradykinin-mediated mechanism, while downregulated iNOS with a possible TNF- alpha -mediated mechanism.

Endothelial dysfunction in chronic heart failure: some new basic mechanisms.

Endothelial dysfunction contributes to the maintenance of peripheral vasoconstriction and abnormal vascular compliance in chronic heart failure. Endothelial dysfunction results in an imbalance between vasodilation and vasoconstriction, particularly when adjustments in blood flow are required. Recently, new factors have been recognized to determine endothelial dysfunction: a) disturbances of the L-arginine/nitric oxide pathway, either at the enzymatic or substrate level; b) increased synthesis of endothelin-1; c) microvessel structural remodeling; d) increased adhesive properties to blood cell components; and e) apoptotic cell injury. The understanding of the complex interplay among these factors is the basis for development of new targeted strategies to correct endothelial dysfunction in chronic heart failure.

Circulating levels of tumour necrosis factor-alpha and its soluble receptors are increased in the blood of patients with amyotrophic lateral sclerosis.

An immunologic pathogenesis for amyotrophic lateral sclerosis (ALS) has been recently proposed. We tested the whole tumour necrosis factor (TNF) system in the serum of 51 ALS patients at different stages of the disease and 36 healthy controls. Antigenic TNF-alpha and its soluble receptors (sTNF-Rs), measured by ELISA, were significantly higher in ALS patients than in healthy controls. However, biologically active TNF-alpha, corresponding to the sTNF-Rs-unbound trimeric TNFalpha molecule and assayed by its cytotoxic activity on a sensitive cell line, was similar between ALS patients and healthy controls. Neither antigenic TNF-alpha, bioactive TNF-alpha nor sTNF-Rs correlated with disease severity, disease duration, or weight loss. In conclusion, we reported an activation of the TNF system in ALS. The role of this activation in the pathogenesis of the disease remains elusive.

Endothelial cells in culture: a model for studying vascular functions.

Vascular endothelium - lining the inner side of blood bessels - is one of the largest secretory tissues of the body. Therefore, understanding the cellular and molecular biology of the endothelial cells is essential for the development of new approaches for both the prevention and therapy of cardiovascular diseases. To this aim, in vitro cultures of endothelial cells provide a valuable technical resource. This review focuses on some of the critical phases of the endothelial cells culturing methodology such as: i) isolation and growing of endothelial cells; ii) identification of endothelial cells by morphological, biochemical and cellular markers; iii) studying endothelial cells in function of vascular pharmacology, vasomotor tone, vessel remodelling (angiogenesis/apoptosis), blood haemostasis, inflammatory reactions, and molecular engineering. Practical suggestions for culturing endothelial cells are presented while pros and cons of each method are discussed.

Serum from patients with severe heart failure downregulates eNOS and is proapoptotic: role of tumor necrosis factor-alpha.

BACKGROUND: Cytokine activation and endothelial dysfunction are typical phenomena of congestive heart failure (CHF). We tested the hypothesis that incubating human umbilical vein endothelial cells with serum from patients with CHF will downregulate endothelial constitutive nitric oxide synthase (eNOS) and induce apoptosis. METHODS AND RESULTS: We studied 21 patients with severe CHF. Levels of tumor necrosis factor-alpha (TNF-alpha) and several neuroendocrine parameters were assessed. eNOS was measured by Western Blot analysis and apoptosis by optical microscopy and flow cytometry. We observed (1) eNOS downregulation (difference versus healthy subjects at 24 hours [P<0.05] and 48 hours [P<0.001]), (2) nuclear morphological changes typical of apoptosis; and (3) a high apoptotic rate with propidium iodide (increasing from 2.1+/-0.4% to 11.3+/-1.2% at 48 hours; P<0.001 versus healthy subjects) and annexin V. An anti-human TNF-alpha antibody did not completely counteract these effects. A strong correlation existed between eNOS downregulation and apoptosis (r = -0.89; P<0.001). CONCLUSIONS: Serum from patients with severe CHF downregulates eNOS expression and increases apoptosis. High levels of TNF-alpha likely play a role, but they cannot be the only factor responsible.

Induction of functional inducible nitric oxide synthase in monocytes of patients with congestive heart failure. Link with tumour necrosis factor-alpha.

AIMS: We studied the induction of monocytic inducible nitric oxide synthase expression and the tumour necrosis factor-alpha system in patients with congestive heart failure. METHODS AND RESULTS: Forty-three congestive heart failure patients and 15 healthy subjects were studied. Antigenic tumour necrosis factor-alpha and its soluble receptors, measured by ELISA, were increased in chronic heart failure and the increase was related to the clinical severity of the syndrome (tumour necrosis factor-alpha from 8.2+/-5.2 in NYHA class II to 18.2+/-7.2 in class III and 26.9+/-13.2 pg. ml(-1)in class IV, P<0.0001 classes III and IV vs class II; soluble tumour necrosis factor receptor I from 1.0+/-0.2 in class II to 2.3+/-1.1 in class III and 5.5+/-3.2 ng. ml(-1)in class IV, P<0.0001 classes III and IV vs class II; soluble tumour necrosis factor receptor II from 2.7+/-0.7 in class II to 4.9+/-1.9 in class III and 8.4+/-5.0 ng. ml(-1)in class IV, P<0.002 classes III and IV vs class II). Monocytic inducible nitric oxide synthase assessed by Western blot, was expressed only in congestive heart failure patients (13 out of 43). The association among monocytic inducible nitric oxide synthase expression, tumour necrosis factor-alpha system activation, neurohormones and other clinical parameters was studied. The univariate logistic regression showed that inducible nitric oxide synthase expression was strictly associated with NYHA class (P<0.05), antigenic tumour necrosis factor-alpha (P<0.01) and its soluble receptors (P<0.05). The multivariate analysis showed that antigenic tumour necrosis factor-alpha was the only predictor for monocytic inducible nitric oxide synthase expression (P<0.05, RR=2.75, CI 1. 34-5.43). CONCLUSIONS: Inducible nitric oxide synthase is expressed in circulating monocytes of patients with severe congestive heart failure. This phenomenon is linked to the activation of the tumour necrosis factor-alpha system.

The blood perfused isolated heart: characterization of the model.

We have characterized the aerobic blood-perfused isolated heart model evaluating the hemodynamics and metabolism of both the blood donor animal and the isolated organ. Anaesthesia of the blood donor with sodium pentobarbital (30 mg/kg) increases arterial concentration of non esterified fatty acids (NEFA) from 80 +/- 6 to 452 +/- 70 microM; p < 0.01. Injection of 1,000 U/kg heparin causes a second significant increase from 452 +/- 70 to 1012 +/- 104 microM; p < 0.01. Insertion of the perfusion circuit, without the isolated heart, causes a reduction in blood pressure of the blood donor and a significant increase in norepinephrine from 277 +/- 44 to 634 +/- 130 pg/ml; p < 0.05. Two hours of aerobic perfusion of the isolated heart inserted in the perfusion circuit, decreases arterial pressure of the blood donor with a concomitant increase of plasma norepinephrine from 475 +/- 150 to 841 +/- 159 pg/ml; p < 0.05. Developed pressure, oxygen consumption, glucose and NEFA uptake of the isolated heart remain constant during two hours of aerobic perfusion, NEFA being the preferred substrate. Tissue content of high energy phosphates at the end of the perfusion is high and similar to that observed "in vivo". Despite this, there is a release of lactate and CPK from the isolated heart. We conclude that: 1) the model allows accurate measurement of hemodynamics and metabolism of both the isolated heart and the blood donor animal; 2) the perfusion procedure modifies the substrates concentration of the blood donor animal which, in turn, results in the preferential NEFA utilization of the isolated heart. These changes do not affect the functional parameters of the perfused heart.

High-dose heparin impairs nitric oxide pathway and vasomotion in rats.

BACKGROUND: Platelet-activating effects have been reported with high-dose heparin in acute thrombotic disorders. Recent studies have shown that increased platelet aggregation is due to reduced nitric oxide (NO) production in endothelial cells cultured in the presence of high-dose heparin. The aim of this study was to determine whether heparin can affect the NO pathway and the regulation of the vascular tone in vivo. METHODS AND RESULTS: Anesthetized and mechanically ventilated Sprague-Dawley rats were treated with high-dose heparin. After 4 hours, the endothelial constitutive NO synthase (ecNOS) protein content in the aorta decreased (36% reduction, P<0.05), as detected by immunoblotting, and NO-dependent vascular reactivity was impaired. In fact, the increase in mean arterial blood pressure after inhibition of ecNOS with NG-nitro-L-arginine methyl ester (30 mg/kg) was smaller in heparin-treated animals than in controls (+26. 9+/-4.8 versus +48.3+/-9.1 mm Hg, P<0.05), and further infusion of the biological ecNOS substrate L-arginine (0.5 g/kg) was ineffective in reversing systemic vasoconstriction (-1% versus 28% vasodilatation, P<0.001). CONCLUSIONS: High-dose heparin can significantly affect vascular reactivity in vivo by downregulation of ecNOS protein expression.

Detection of nitric oxide in exhaled air of different animal species using a clinical chemiluminescence analyser.

The aim of the present study was to evaluate the nitric oxide (NO) concentrations present in end-expired gas (FENO) of different animal species under basal and stimulated conditions using a clinical chemiluminescence analyser, which has been developed for measurement of single exhalations in humans. Anaesthetised, tracheotomised and artificially ventilated guinea pigs, rats and rabbits were prepared for recording systemic blood pressure and FENO. Stable levels of FENO were detected in expired air over a 1-h observation period in the three animal species tested. Rabbits exhibited the highest concentrations and output (FENO 12.9+/-1.0 ppb, VNO 9.0+/-0.7 nl min-1), followed by guinea pigs (FENO 6.2+/-0.70 ppb, VNO 1.7+/-0.19 nl min-1) and rats (FENO 0.9+/-0.01 ppb, VNO 0.25+/-0.00 nl min-1). L-arginine (1 g kg-1 i.v.) evoked significant increments in VNO in guinea pigs and rabbits but was ineffective in rats. However, L-arginine showed a direct effect on blood pressure in all the animal species tested, causing a rapid fall in the mean arterial blood pressure (MABP; 38, 48 and 50% decrease in rabbits, guinea pigs and rats, respectively; P<0.05). An inhibitor of endogenous NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME, 20 mg kg-1 i.v.), decreased both basal and L-arginine-induced VNO in guinea pigs and rabbits, but was ineffective in rats. L-NAME increased MABP in all the animal species tested (58% in guinea pigs, 43% in rats and 18% in rabbits; P<0.05). The results indicate that it is possible to detect NO in the exhaled air of different animal species using a clinical chemiluminescence analyser and that different species exhibit striking differences in the levels of basal and stimulated NO output.