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.

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.

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.

Right heart failure chronically stimulates heat shock protein 72 in heart and liver but not in other tissues.

OBJECTIVES: During cardiac failure several ontogenically developed adaptional mechanisms are activated. Among these, heat-shock proteins (HSP) are expressed in response to stress. The aim of the present study was to investigate the HSP72 protein expression in lungs, liver, cardiac and skeletal muscles during congestive heart failure (CHF). METHODS: CHF was induced in Sprague-Dawley rats by a single intraperitoneal injection of monocrotaline (50 mg/kg). Two groups of animals emerged: a CHF group (n = 10) with right ventricular hypertrophy, pleural and peritoneal effusions, and an Hypertrophy group (n = 12) with right ventricular hypertrophy without CHF. The data for each group were compared with those of control (saline infused) age-matched rats. Lungs, liver, right and left ventricles, soleus, extensor digitorum longus and tibialis anterior muscles were excised and analyzed for HSP72 concentration by Western blot analysis using a specific monoclonal antibody. Noradrenaline levels in the heart were also measured using HPLC. RESULTS: The CHF group showed: (1) reduced right (0.460 +/- 0.090 vs 0.830 +/- 0.070 nmol/ventricle, P < 0.01) and left (1.10 +/- 0.09 vs 2.10 +/- 0.130 nmol/ventricle, P < 0.001) ventricular content of noradrenaline compared to the control; (2) significant activation of HSP72 concentration in right and left ventricles (39.4 +/- 1.6 vs 5 +/- 0.9% and 13 +/- 1.2 vs 3.5 +/- 0.6%, P < 0.001 both) and in the liver (39.8 +/- 11 vs 6 +/- 2%, P < 0.001); (3) no modification in HSP72 concentration in lungs and all of the peripheral muscles considered. The Hypertrophy group showed: (1) unchanged total noradrenaline tissue content as compared to the control; and (2) unmodified HSP72 concentration in all tissues analyzed. CONCLUSIONS: The present study demonstrates that CHF, but not compensatory hypertrophy, is a specific stimulus for chronic HSP72 induction in the heart and liver. On the contrary, CHF does not affect HSP in lungs and peripheral muscles. HSP 72 induction represents an intracellular marker of stress reaction which can persist chronically.

Estrogen regulation of prodynorphin gene expression in the rat adenohypophysis: effect of the antiestrogen tamoxifen.

Prodynorphin (Prodyn)-derived peptides are synthesized in a subset of gonadotrope cells and released concomitantly with LH and FSH, and their levels in the rat adenohypophysis are influenced by the gonadal steroid environment. In several hormonal systems, factors that affect peptide levels may modulate the transcription of messenger RNA (mRNA) encoding for the target gene. Therefore, the present study was designed to investigate the effects of gonadal ablation and estrogen replacement on changes in steady state levels of anterior pituitary Prodyn mRNA and on the transcription rate in the adult female rat. The antiestrogen tamoxifen was employed for further exploring the relationships between estrogens and dynorphin (dyn)-related peptides. Adopting a solution hybridization-ribonuclease protection assay, steady state levels of Prodyn mRNA doubled in 2-week ovariectomized (OVX) rats, in parallel with a 3-fold increase in immunoreactive dyn-A-(1-17)-like material (irdyn-A). Estradiol (E2) replacement through sc SILASTIC implants for 1, 3, 7, and 14 days, which produces serum E2 levels between 25-35 pg/ml, restored in a time-dependent manner mRNA and peptide concentrations to values in sham-OVX rats. A significant decrease was observed after 3 days, and after 7 days, the effect was maximal. Tamoxifen (250 micrograms/kg.day, sc) administered simultaneously antagonized the action of E2 on Prodyn gene expression. Tamoxifen administered without E2 for 7 or 14 days significantly raised anterior pituitary levels of Prodyn mRNA and ir-dyn-A. To establish whether E2 and tamoxifen exert their effects on adenohypophyseal Prodyn mRNA by influencing the transcriptional activity of this gene, an in vitro transcriptional elongation assay was performed on nuclei from the anterior pituitary. The transcriptional rate of the Prodyn gene was significantly increased in 2-week OVX rats. Prodyn mRNA synthesis was suppressed in OVX rats exposed to E2, an effect antagonized by tamoxifen administered concomitantly. The antiestrogen administered alone for 14 days further elevated the transcriptional rate of Prodyn mRNA induced by gonadal ablation. In conclusion, E2 down-regulated the synthesis of Prodyn-derived peptides in adenohypophyseal cells. The antiestrogen tamoxifen antagonized the effect of E2 and, when chronically administered to OVX rats, further elevated the postcastrational rise in Prodyn gene expression.

Hypertension, aging, and myocardial synthesis of heat-shock protein 72.

We determined the temperature-induced synthesis of the 72-kD heat-shock protein (hsp72) in hearts of normotensive and spontaneously hypertensive rats (SHR) subjected to whole-body hyperthermia (42.0 +/- 0.5 degrees C for 15 minutes). The animals were studied at three different ages: young (2 months), adult (6 months), and old (18 months). The hsp72 was determined by Western blot analysis using a monoclonal antibody. The results were calculated densitometrically as a percentage of a commercial standard. Young SHR responded to hyperthermic stress with increased synthesis of hsp72 compared with age-matched normotensive rats (298.8 +/- 70.0% versus 88.3 +/- 25.5%). This trend was maintained in adult rats (118.1 +/- 31.0% versus 54.8 +/- 21.3%) but not in old rats (65.3 +/- 29.4% versus 43.6 +/- 15.1%). Aging caused a reduction of hsp72 expression in response to hyperthermic stress in both SHR (4.6-fold) and normotensive rats (twofold). These data show that hearts of young and adult SHR respond to heat shock with enhanced synthesis of hsp72. This abnormal response, attenuated by aging, is independent of the presence and degree of hypertension or hypertrophy and is potentially linked to the genetic determination of the disease.

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.

Dynorphin A-(1-17) and dynorphin B are released from in vitro superfused rat hypothalami. Effects of depolarizing agents and ovariectomy.

We measured the release of immunoreactive (ir) dynorphin (dyn) A-(1-17) and dyn B from the rat hypothalamus by an in vitro superfusion technique. The system was validated on the basis of the recovery and stability of radiolabeled peptides added to the superfused hypothalami. These were detected as authentic peptides by reverse-phase high-performance liquid chromatography (rp-HPLC) only in the presence of a cocktail of peptidase inhibitors added to the superfusion medium. We observed spontaneous release of ir-dyn B, evaluated by a validated radioimmunoassay in the superfusates, that was increased by potassium and veratridine depolarization. It was calcium-dependent and tetrodotoxin-sensitive. We could not evaluate ir-dyn A-(1-17) directly in the superfusates, because the peptidase inhibitors added to the medium significantly altered the tracer-antibody reaction. To obviate this problem, pooled superfusate samples were purified on C18 cartridges and assayed by rp-HPLC. Rp-HPLC analysis of superfusates revealed two molecular forms with the same retention time as authentic dyn A-(1-17) and dyn B which were four times higher in K(+)-stimulated fractions. We could not detect dyn A-(1-32), comprising dyn A-(1-17) and dyn B, even though this peptide is recognized by the antibodies used in this study and is detected in acetic acid extracts of the rat hypothalamus. The spontaneous and K(+)-evoked release of ir-dyn A-(1-17) and ir-dyn B were significantly higher in 2-week ovariectomized rats, in parallel with the increase of their content in the anterior hypothalamus preoptic area.

Ca2+ channel blocking activity of lacidipine and amlodipine in A7r5 vascular smooth muscle cells.

Inhibition of the K(+)-stimulated increase in cytosolic free Ca2+ by a series of 1,4-dihydropyridines was evaluated in A7r5 vascular smooth muscle cells loaded with the fluorescent Ca2+ indicator fura-2 acetoxymethyl ester. The IC50 of the drugs, added to suspended cells 3 min before 150 mM KCl, gave the following order of potency: lacidipine (2.76 nM) > nitrendipine (3.81 nM) > amlodipine (4.56 nM) > nifedipine (10.08 nM). A7r5 cells were also exposed to the 1,4-dihydropyridines, at their IC50, for 25 min, and then repeated washout cycles were performed before adding KCl. The Ca2+ channel blocking activity of nifedipine and nitrendipine gradually diminished, disappearing after four washout cycles 25, 55, 115 and 175 min after drug treatment. Amlodipine and lacidipine displayed slow onset and offset of antagonism, their activity becoming stronger with time, in spite of the repeated washes. [3H]Lacidipine was avidly and promptly entrapped in A7r5 cells and was not removed by washout. However, its potency as a Ca2+ channel blocker was not directly related to the amount of drug locked in the cell since it increased with time, indicating that lacidipine binds to the lipid bilayer of the cell membrane and then gradually diffuses towards a specific binding site. This model can, therefore, predict the Ca2+ blocking properties of 1,4-dihydropyridines with slow onset and offset of antagonism and could be employed to evaluate compounds selective for vascular smooth muscle.

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.

Heterogeneity of immunoreactive dynorphin B-like material in human, rat, rabbit and guinea-pig heart.

Immunoreactive dynorphin B-like material (ir-dyn B) was detected in acetic acid extracts of human atrial specimens and of rat, rabbit and guinea-pig atria and ventricles by a validated radioimmunoassay. Levels were high in rabbit atrium (66.76 +/- 7.04 pmol/g) but lower and superimposable in human and rat atria (28.18 +/- 3.20 and 30.22 +/- 2.45 pmol/g, respectively). Gel permeation chromatography revealed ir-dyn B eluting close to column exclusion and in forms with an apparently higher molecular weight than authentic dyn B in human and rat samples. In contrast, almost all the immunoreactivity from rabbit and guinea-pig acetic extracts eluted as a single peak in the region of standard dyn B. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions of this peak showed up a molecular form with the same retention time as authentic dyn B and a second minor peak of unknown immunoreactive material eluting three fractions earlier. Digestion with carboxypeptidase B excluded the hypothesis that this latter could be dyn B-Arg14. Therefore, it might be a metabolite of endogenous dyn B recognized by the antibody used in this study.

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.

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.

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.