Assays Specific for BNP1-32 and NT-proBNP Exhibit a Similar Performance to Two Widely Used Assays in the Diagnosis of Heart Failure.

BACKGROUND: Secretion of cardioprotective B-type natriuretic peptide 1-32 (BNP1-32) is increased proportionately with cardiac dysfunction, but its measurement in plasma is difficult. Therefore, less specific BNP and amino-terminal proBNP (NT-proBNP) assays that detect the precursor molecule proBNP alongside BNP or NT-proBNP metabolites were developed to reflect BNP1-32 secretion and are now mandated in the diagnosis of heart failure (HF). We compared the diagnostic performance of 2 widely used clinical assays: the Roche proBNPII assay, and Abbott BNP assay, against our recently developed in-house assays that measure either intact BNP1-32 or NT-proBNP. METHODS: EDTA plasma samples obtained from patients presenting with breathlessness (n = 195, 60 [31%] with clinically adjudicated HF) were assayed using the Roche NT-proBNP and our specific in-house BNP1-32 and NTBNP assays. A subset (n = 75) were also assessed with the Abbott BNP assay. RESULTS: Roche NT-proBNP was highly correlated with BNP1-32 and NTBNP (Spearman rho = 0.92 and 0.90, respectively, both Ps < 0.001), and all 3 assays similarly discriminated acute HF from other causes of breathlessness (ROC analysis areas under the curve 0.85-0.89). The Abbott BNP assay performed similarly to the other assays. Roche NT-proBNP and BNP1-32 assays had similar sensitivity (83% and 80%), specificity (83% and 84%), positive (70% and 71%) and negative (91% and 90%) predictive values, and accuracy (both 83%) at their optimal cutoffs of 1536 and 12 ng/L, respectively. CONCLUSIONS: Since all assays exhibited similar performance in the diagnosis of HF, currently mandated assays provide a reliable proxy for circulating concentrations of active BNP1-32 in HF diagnosis.

ProBNP That Is Not Glycosylated at Threonine 71 Is Decreased with Obesity in Patients with Heart Failure.

BACKGROUND: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Plasma concentrations of B-type natriuretic peptide (BNP) or its amino terminal congener (NT-proBNP) are used for HF diagnosis and risk stratification. Because BNP concentrations are inexplicably lowered in obese patients, we investigated the relationship between proBNP glycosylation, plasma NT-proBNP, and body mass index (BMI) in HF patients. METHODS: Three assays were developed to distinguish between total proBNP (glycosylated plus nonglycosylated proBNP), proBNP not glycosylated at threonine 71 (NG-T71), and proBNP not glycosylated in the central region (NG-C). Intraassay and interassay CVs were <15%; limits of detection were <21 ng/L; and samples diluted in parallel. RESULT: Applying these assays and an NT-proBNP assay to plasma samples from 106 healthy volunteers and 238 HF patients determined that concentrations [median (interquartile range)] of proBNP, NG-T71, and NT-proBNP were greater in HF patients compared with controls [300 (44-664), 114 (18-254), and 179 (880-3459) ng/L vs 36 (18-229), 36 (18-175), and 40 (17-68) ng/L, respectively; all P < 0.012]. NG-C was undetectable in most samples. ProBNP concentrations in HF patients with BMI more or less than 30 kg/m2 were not different (P = 0.85), whereas HF patients with BMI >30 kg/m2 had lower NT-proBNP and NG-T71 concentrations (P < 0.003) and higher proBNP/NT-proBNP and proBNP/NG-T71 ratios (P = 0.001 and P = 0.02, respectively) than those with BMI <30 kg/m2. CONCLUSIONS: Increased BMI is associated with decreased concentrations of proBNP not glycosylated at T71. Decreased proBNP substrate amenable to processing could partially explain the lower NT-proBNP and BNP concentrations observed in obese individuals, including those presenting with HF.

High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure.

BACKGROUND: Urocortin 2 (Ucn2) has powerful hemodynamic, renal, and neurohormonal actions and likely participates in normal circulatory homeostasis and the compensatory response to heart failure (HF). A validated assay for endogenous circulating Ucn2 would facilitate investigations into Ucn2 physiology and elucidate its derangement and potential as a biomarker in heart disease. METHOD: We developed a chemiluminescence-based sandwich ELISA to measure plasma N-terminal (NT)-proUcn2 in non-HF patients (control; n = 160) and HF patients with reduced (HFREF; n = 134) and preserved (HFPEF; n = 121) left ventricular ejection fraction (LVEF). RESULTS: The ELISA had a limit of detection of 8.47 ng/L (1.52 pmol/L) and working range of 23.8-572 ng/L. Intra- and interassay CV and total error were 4.8, 16.2, and 17.7%, respectively. The median (interquartile range) plasma NT-proUcn2 concentration in controls was 112 (86-132) ng/L. HFREF, HFPEF, and all HF plasma concentrations were significantly increased [117 (98-141) ng/L, P = 0.0007; 119 (93-136) ng/L, P = 0.0376, and 119 (97-140) ng/L, P = 0.001] compared with controls but did not differ significantly between HFREF and HFPEF. NT-proUcn2 was modestly related to age (r = 0.264, P = 0.001) and cardiac troponin T (r = 0.258, P = 0.001) but not N-terminal pro-B-type natriuretic peptide, body mass index, LVEF, or estimated glomerular filtration rate. On multivariate analysis, plasma NT-proUcn2 was independently and inversely related to 2-year mortality in HF. CONCLUSIONS: The validated ELISA measured human NT-proUcn2 in plasma and showed modest but significant increases in HF patients compared with controls. In HF, the unusual inverse relationship between plasma NT-proUcn2 and 2-year mortality portends potential prognostic value but requires further corroboration.

B-type natriuretic peptide forms within the heart, coronary sinus, and peripheral circulation in humans: evidence for degradation before secretion.

BACKGROUND: The B-type natriuretic peptides (BNP and N-terminal pro-BNP) are secreted by the heart and, in the case of BNP, serve to maintain circulatory homeostasis through renal and vascular actions and oppose many effects of the renin-angiotensin system. Recent evidence suggests that in patients with severe heart failure, circulating immunoreactive BNP is made up mainly of metabolites that may have reduced bioactivity. We hypothesized that BNP may be degraded before it even leaves the heart. METHODS: Peripheral venous plasma plus atrial and ventricular tissue, obtained from explanted hearts at the time of transplantation, were collected from 3 patients with end-stage heart failure. In a separate study, plasma was collected from the coronary sinus and femoral artery of 3 separate patients undergoing cardiac catheterization. Plasma C18 reverse-phase extracts were separated on reverse-phase HPLC, and the collected fractions were subjected to RIAs with highly specific antisera directed to the amino- and carboxy-terminal ends of BNP(1-32). RESULTS: ProBNP, BNP(1-32), and 2 major BNP metabolites were present in atrial and ventricular tissue, where BNP(1-32) represented 45% and 70% of total processed BNP, respectively. Neither BNP(1-32) nor the 2 metabolites were detected in peripheral venous plasma. Nor was BNP(1-32) detected in matching coronary sinus and femoral artery plasma from the 3 patients undergoing cardiac catheterization. CONCLUSIONS: BNP(1-32) is partly degraded within the hearts of patients with end-stage heart failure, and even in patients with relatively well-preserved left ventricular systolic function, only BNP metabolites enter the systemic circulation.

Plasma corin levels provide minimal prognostic utility incremental to natriuretic peptides in chronic systolic heart failure.

BACKGROUND: Corin is a serine protease that cleaves pro-atrial and pro-B-type natriuretic peptides into biologically active hormones. The relationship between soluble plasma corin levels, plasma natriuretic peptide levels, myocardial structure and performance, and long-term clinical outcomes in the setting of chronic systolic heart failure has not been described. METHODS AND RESULTS: In 126 patients with chronic systolic heart failure (left ventricular ejection fraction

Clinical significance of endogenous vasoactive neurohormones in chronic systolic heart failure.

BACKGROUND: Neurohormonal activation is a pathophysiological hallmark of acute and chronic heart failure (HF). The clinical significance of more recently discovered endogenous vasoactive hormones has not been well-characterized. METHODS AND RESULTS: In 154 subjects with stable, chronic systolic HF (New York Heart Association Class I-IV, left ventricular [LV] ejection fraction or=12.1 pM (HR: 2.02, 95% CI: 1.08-3.93, P = .029) and ET-1 >or=2.29 pM (HR: 2.52, 95% CI: 1.24-5.03, P = .011) remained significant independent risk factors for adverse clinical events. CONCLUSION: Higher levels of plasma levels of UCN-1 and ET-1 but not UT-II were associated with worse LV diastolic performance and poorer long-term clinical outcomes in patients with chronic systolic HF.

Renal and cardiac function for long-term (10 year) risk stratification after myocardial infarction.

Aims To determine whether combined renal and cardiac function after acute myocardial infarction (MI) predicts 10 year mortality and heart failure (HF). Methods and results Estimated glomerular filtration rate (eGFR), plasma amino terminal pro-brain natriuretic peptide (NT-proBNP), and radionuclide ventriculography were obtained in 1063 patients with MI between 24-96 h of symptom onset. Mortality and HF were documented over follow-up of 9.3 years. Estimated GFR, NT-proBNP, and left ventricular ejection fraction (LVEF) each independently predicted 10 year mortality. Reduced eGFR (below 60 mL/min/1.73 m(2)) combined with increased NT-proBNP (above 1000 pg/mL) was associated with higher mortality rate compared with preserved eGFR together with lower NT-proBNP (60 vs. 14%, P < 0.001). Similar results for mortality were identified for eGFR combined with LVEF (dichotomized about 50%) (58 vs. 17%, P < 0.001). Corresponding analysis combining eGFR and NT-proBNP to predict HF yielded rates of 34 and 7% for high- and low-risk groups, respectively (P < 0.001). Similar risk stratification for HF was observed when combining eGFR with LVEF (35 vs. 7%, P < 0.001). Conclusion Ten year rates of mortality and HF are 5-10 times higher when lower eGFR is present together with increased NT-proBNP or depressed LVEF.

Intermedin (adrenomedullin-2): a potential protective role in human aortic endothelial cells.

BACKGROUND/AIMS: Intermedin (IMD) is a novel peptide with significant vasodilator and cardiac protective actions similar to the related peptide adrenomedullin (ADM). Unlike those of ADM the actions and expression of IMD in endothelial cells are poorly characterised. ADM expression can be increased during cardiovascular disease/stress in vitro and in vivo where it may have a role in several cardiovascular protective actions. To characterise IMD mRNA expression cultured human aortic endothelial cells (HAEC) were stressed by removing serum and bicarbonate, and the addition of hydrogen peroxide. The responses were compared to those of ADM mRNA. We also compared the effects of ADM and IMD on caspase activity and cell viability, and investigated if IMD actions could be altered by a CGRP receptor antagonist. METHODS/RESULTS: Using the cell immunoblot assay, immunoreactive IMD was shown to be secreted by HAEC. IMD mRNA expression was also detected in HAEC grown in endothelial growth media (but at markedly lower levels than that of ADM). Absence of bicarbonate, a redox-mediated regulator of endothelial response to various stresses, increased IMD mRNA and ADM mRNA expression. However IMD mRNA, but not ADM mRNA, was markedly increased over time in HAEC in conditions of cell stress including incubation with serum-free Dulbecco's modified Eagle's medium (DMEM) and in response to hydrogen peroxide (H2O2). These vigorous responses in IMD mRNA expression were further enhanced by incubation in 5% serum in DMEM without bicarbonate, but in a selective manner since ADM expression was suppressed by serum. We also observed that IMD mRNA was markedly increased and ADM mRNA suppressed in HAEC following a period of suspension and replating. Finally, we observed that IMD, like ADM, increased cell viability in HAEC in DMEM without serum but only IMD reduced caspase activity, perhaps via and a yet to be defined receptor system. CONCLUSION: HAECs express IMD mRNA and secrete IMD peptide. IMD mRNA expression is markedly dependent on metabolic conditions and is selectively regulated in a contrary fashion to ADM mRNA. IMD mRNA expression in endothelial cells is markedly sensitive to oxidative stress, and IMD peptide itself has antiapoptotic activity in human endothelial cells. Our data suggest that IMD has a different role to ADM and may perform a protective function in humans.

Characterization of NT-proBNP in human urine.

BACKGROUND: Urine amino-terminal probrain natriuretic peptide (NT-proBNP) concentrations may exclude the presence of heart failure and provide insight into renal clearance mechanisms for human NT-proBNP. We characterized the molecular forms of urine NT-proBNP detected by immunoassay. METHODS: Urine from patients with heart failure was subjected to HPLC and analyzed using immunoassays specific toward different epitopes of NT-proBNP. We assessed urine NT-proBNP immunoreactivity in healthy subjects and patients with heart failure. RESULTS: Size-exclusion chromatography of heart failure urine identified no NT-proBNP immunoreactivity coeluting with NT-proBNP(1-76); multiple immunoreactive NT-proBNP fragments were present. The absence of intact urinary NT-proBNP was supported by reversed-phase HPLC. Urine NT-proBNP immunoreactivity was higher in patients with acute [median 192 (interquartile range 108-1445) pg/mg creatinine] and chronic [52 (15-118) pg/mg creatinine] heart failure than in healthy subjects [4.2 (2.6-5.8) pg/mg creatinine] (P < 0.001). In 40 patients with heart failure, urine NT-proBNP immunoreactivity correlated with plasma NT-proBNP (r = 0.72, P < 0.001) and inversely with left ventricular ejection fraction (r = -0.33, P = 0.04). CONCLUSIONS: Our findings clarify previous reported relationships of urine NT-proBNP-like immunoreactivity with plasma NT-proBNP concentrations and the diagnosis of heart failure. As urine NT-proBNP immunoreactivity is not intact NT-proBNP(1-76), but rather reflects assorted metabolites, the diagnostic performance of NT-proBNP assays in urine may be assay specific, necessitating validation of biomarker performance on an assay-by-assay basis.

Urotensin II immunoreactivity in the human circulation: evidence for widespread tissue release.

BACKGROUND: The sources of secretion and clearance of plasma urotensin II (UII) in the human circulation remain uncertain and may be relevant to understanding the role of UII in human physiology and cardiovascular disease. METHODS: In 94 subjects undergoing clinically indicated cardiac catheterization, we collected blood samples from arterial and multiple venous sites to measure transorgan gradients of plasma UII immunoreactivity. RESULTS: Net UII release occurred (in descending order of proportional transorgan gradient) across the heart, kidney, head and neck, liver, lower limb, and pulmonary circulations (P < 0.01). Although no specific clearance site was localized, the absence of an overall subdiaphragmatic aorto-caval peptide gradient indicated that there were lower body segment sites of UII clearance as well as secretion. The proportional increase in UII immunoreactivity was significantly correlated across all sites of net peptide release within an individual (P < or = 0.05). In univariate analyses, mixed venous UII concentrations were correlated with diagnosis of acute coronary syndrome and femoral artery oxygen tension and inversely with systolic blood pressure and body mass index. Diagnosis of acute coronary syndrome and body mass index were independent predictors of mixed venous UII immunoreactivity in multivariate analysis. No correlates of net cardiac UII release were identified. CONCLUSIONS: UII is secreted from the heart and multiple other tissues into the circulation. Related increments in UII immunoreactivity across multiple tissue sites suggest that peptide release occurs via a shared mechanism. Increased UII immunoreactivity is observed in subjects with acute coronary syndrome.

Transforming growth factor-beta blockade down-regulates the renin-angiotensin system and modifies cardiac remodeling after myocardial infarction.

After myocardial infarction (MI), the heart may undergo progressive ventricular remodeling, resulting in a deterioration of cardiac function. TGF-beta is a key cytokine that both initiates and terminates tissue repair, and its sustained production underlies the development of tissue fibrosis, particularly after MI. We investigated the effects of a novel orally active specific inhibitor of the TGF-beta receptor 1 (SD-208) in an experimental model of MI. Mice underwent ligation of the left coronary artery to induce MI and were subsequently treated for 30 d after infarction with either SD-208 or a vehicle control. Blockade of TGF-beta signaling reduced mean arterial pressure in all groups. SD-208 treatment after MI resulted in a trend for reduced ventricular and renal gene expression of TGF-beta-activated kinase-1 (a downstream modulator of TGF-beta signaling) and a significant decrease in collagen 1, in association with a marked decrease in cardiac mass. Post-MI SD-208 treatment significantly reduced circulating levels of plasma renin activity as well as down-regulating the components of the cardiac and renal renin-angiotensin system (angiotensinogen, angiotensin converting enzyme, and angiotensin II type I receptor). Our findings indicate that blockade of the TGF-beta signaling pathway results in significant amelioration of deleterious cardiac remodeling after infarction.

Plasma amino-terminal pro C-type natriuretic Peptide in the neonate: relation to gestational age and postnatal linear growth.

CONTEXT: C-type natriuretic peptide (CNP) plays an essential role in endochondral bone growth. Insight into CNP's paracrine actions is possible using plasma measurements of the amino-terminal pro C-type natriuretic peptide (NTproCNP). Whether correlations of NTproCNP with linear growth, as found in children and lambs, apply in neonates is unknown. OBJECTIVES: Our objective was to determine the effects of prematurity, gender, and antenatal steroids on plasma NTproCNP at birth, and serial changes in hormone concentrations, linear growth, and markers of bone turnover in the first month of postnatal life. DESIGN AND SETTING: This is a prospective study of newborn infants admitted to an intensive care unit. SUBJECTS: A total of 48 infants (four gestation groups) were enrolled. Umbilical cord samples were also obtained from 39 healthy term infants. MAIN OUTCOME MEASURES: Plasma NTproCNP and CNP were measured in cord plasma. In enrolled neonates, serial measurements of hormone concentrations and markers of bone turnover were related to tibial growth velocity as measured by knemometry. RESULTS: Cord plasma NTproCNP was inversely related to gestational age (r = -0.35; P = 0.003) and was higher in males (P < 0.001). Plasma NTproCNP (P = 0.016) and CNP (P < 0.001) increased within the first week of life, the increase relating inversely to gestational age (r = -0.64; P < 0.001). Plasma NTproCNP at 1 wk was strongly correlated with linear growth velocity (r = 0.49; P < 0.001), and also at 2-4 wk, the relation being stronger than observed between bone turnover markers and growth velocity. CONCLUSIONS: In neonates with diverse disorders affecting growth and nutrition, plasma NTproCNP was strongly correlated with linear growth during the first 4 wk of postnatal life and may prove to be a novel marker of growth plate activity in neonates.

Regulation of endothelin-1 release from human endothelial cells by sex steroids and angiotensin-II.

It is well documented that there are gender differences in the incidence and patterns of cardiovascular disease; males have a higher incidence of cardiovascular disease than premenopausal women. We have therefore investigated whether the sex hormones, estradiol and testosterone, could directly influence the secretion of vascular peptides from human aortic endothelial cells (HAEC). Previously we have shown that testosterone can increase the number of HAECs that secrete adrenomedullin. In this study we investigated sex hormone regulation of endothelin-1 in HAEC. Several studies have observed a reduction in endothelin-1 secretion from endothelial cells in the presence of estradiol, the effect being more marked for stimulated cells. Studies on the actions of testosterone are much fewer and inconclusive. In this study we observed that estradiol did not change the number of cells secreting endothelin-1 during 4h incubation under basal conditions but decreased the number of secreting cells stimulated with angiotensin-II. Testosterone induced an increase in the number of cells secreting endothelin-1 (p=0.03). Complementary incubations revealed that testosterone up-regulated endothelin-1 mRNA at 1-3h (p<0.05). These results, together with our previous observations, indicate that angiotensin-II, testosterone and estradiol have parallel effects on the production of endothelin-1 as on adrenomedullin in HAEC. We conclude that there is potential for coordinated modulation by sex steroids and angiotensin-II of vasoactive peptide production in human endothelial cells.

Introduction of metoprolol increases plasma B-type cardiac natriuretic peptides in mild, stable heart failure.

BACKGROUND: The effect of beta-blockade on the cardiac natriuretic peptides is poorly understood but could contribute to their beneficial treatment effect and may be relevant to clinical use of plasma brain natriuretic peptide (BNP)/N-terminal pro brain natriuretic peptide (NTproBNP) measurements in risk stratification and in titration of anti-heart failure therapy. METHODS AND RESULTS: Sixteen men with mild, stable heart failure (NYHA class II to III; left ventricular ejection fraction <40%) underwent serial blood sampling for plasma natriuretic peptide levels and received infusions of atrial natriuretic peptide (ANP) and BNP before and 6 weeks after the introduction and uptitration of metoprolol or 6 weeks unchanged therapy in a randomized, parallel-group design. Plasma natriuretic peptides (BNP, NTproBNP, ANP, and NTproANP) were increased by metoprolol (P<0.01 for all). The natriuretic responses to ANP and BNP infusions were sustained with the introduction of metoprolol despite reduced renal perfusion pressure. The levels of the noninfused natriuretic peptide were increased by both ANP and BNP infusions, and this effect was enhanced by metoprolol. The early plasma half-life (t(1/2)alpha) of BNP was prolonged by metoprolol (5.6+/-0.45 to 11+/-1.3 versus 5.7+/-0.8 to 6.6+/-1.3 minutes in control subjects; P=0.019). CONCLUSIONS: Plasma cardiac natriuretic peptide levels increase significantly with the introduction of metoprolol in heart failure as a result of effects on secretion and clearance. Natriuretic responses to NP infusions are sustained with beta-blockade despite reduced renal perfusion pressure. Clinicians should be aware that the introduction of metoprolol causes a rise in plasma BNP/NTproBNP that is unrelated to deterioration in clinical status and must be considered when measurements are undertaken for risk stratification or titration of treatment.

C-type natriuretic peptide forms in the ovine fetal and maternal circulations: evidence for independent regulation and reciprocal response to undernutrition.

C-type natriuretic peptide (CNP) has a crucial role in postnatal endochondral bone growth and is rapidly responsive to changes in nutrition. Although CNP is expressed in the placenta, little is known about the regulation and role of CNP in fetal-maternal health. We hypothesized that CNP may be similarly responsive to undernutrition in the growing fetus, in which maternal nutrition is crucial to normal growth and development. We therefore studied maternal and fetal CNP and the aminoterminal (bioinactive) fragment of proCNP (NTproCNP) in 39 chronically catheterized pregnant sheep before and after a 3-d maternal fast from 121 d gestation. Maternal CNP and NTproCNP levels were higher than in the fetus (CNP 12-fold, NTproCNP 1.5-fold, both P < 0.001). The ratio of NTproCNP to CNP was higher in the fetus than the mother (53 +/- 3 vs. 8.7 +/- 0.6, P < 0.001), suggesting enhanced synthesis and/or degradation of CNP in the fetus. As in postnatal lambs, fetal plasma CNP forms fell promptly during maternal fasting. In contrast, maternal levels exhibited reciprocal and contemporaneous increase, which was reversed by refeeding. Uteroplacental production of CNP was suggested by a high venoarterial concentration gradient across the gravid uterus, and a correlation between maternal NTproCNP levels and placental weight (r(2) = 0.26, P = 0.01). These studies provide the first evidence that CNP is regulated independently in the fetus. Reciprocal increases in maternal CNP forms may reflect the response of the uteroplacental unit to substrate deficiency. CNP may have a role in maintaining fetal welfare and provides a possible marker of uteroplacental nutrient supply.

Amino-terminal propeptide of C-type natriuretic peptide and linear growth in children: effects of puberty, testosterone, and growth hormone.

CONTEXT: C-type natriuretic peptide (CNP), a paracrine factor of the growth plate, plays a key role in stimulating bone growth. The amino-terminal propeptide of CNP (NTproCNP) is produced in equimolar amounts with CNP and is measurable in plasma, providing a potential biomarker for growth plate activity and, hence, linear growth. OBJECTIVE: We explored the effects of puberty, testosterone, and GH treatment on NTproCNP levels in normal and short-statured children. DESIGN: This was a retrospective analysis of samples obtained during previous studies. SETTING: The study was conducted at a pediatric clinical research center. SUBJECTS: Children with short stature due to GH deficiency, idiopathic short stature (ISS), or constitutional delay of growth and maturation (CDGM) were studied (n = 37). A cohort of normal-statured adolescent boys was also studied (n = 23). INTERVENTIONS: Children with GH deficiency and ISS were studied before and during testosterone and/or GH treatment. Boys with CDGM and healthy controls were studied once. MAIN OUTCOME MEASURES: The main outcomes were NTproCNP levels before and during growth-promoting therapy and during pubertal growth. RESULTS: Children with short stature due to GH deficiency, ISS, or CDGM had comparable baseline levels of NTproCNP, and levels increased markedly in response to GH or testosterone treatment. In boys with CDGM, levels were comparable with height-matched controls but were less than those from age-matched controls. In healthy boys, NTproCNP appears to peak with the pubertal growth spurt. CONCLUSIONS: NTproCNP levels increase during growth-promoting therapy and are increased during puberty in boys. This novel biomarker of growth may have clinical utility in the evaluation of children with short stature and for monitoring growth-promoting therapy.

Regulation of adrenomedullin release from human endothelial cells by sex steroids and angiotensin-II.

It is well documented that there are gender differences in the incidence and patterns of cardiovascular diseases but the reasons are unclear. Sex steroids may modulate the behaviour of vascular endothelial cells, which in turn act by paracrine processes to alter adjacent vascular smooth muscle activity. We hypothesised that the sex steroids alter the percentage of vascular endothelial cells that secrete the vasodilator peptide, adrenomedullin and modify the adrenomedullin-stimulating action of angiotensin-II. The percentage of adrenomedullin-secreting human aortic endothelial cells was determined using the cell immunoblot method. Cells were incubated with selected concentrations of angiotensin-II, oestradiol and testosterone alone and sex steroids in combination with angiotensin-II. Adrenomedullin mRNA expression in endothelial cells was quantified by real-time PCR. It was observed that at 4 h, angiotensin-II increased the percentage of adrenomedullin-secreting cells in a concentration-dependent manner. Testosterone at physiological concentrations was observed to increase the number of adrenomedullin-secreting cells whilst oestradiol had no effect. Addition of testosterone to angiotensin-II resulted in less than additive increases in the number of cells secreting adrenomedullin. Oestradiol reduced the angiotensin-II-induced increase in adrenomedullin-secreting cells. Adrenomedullin mRNA expression was significantly increased by testosterone and there was also a trend for an increase in adrenomedullin mRNA expression, which occurred when cells were incubated with angiotensin-II. Our results point to a complex interplay between the sex steroids and angiotensin-II in regulating adrenomedullin production by human endothelial cells, which may contribute to gender-related differences in vascular disease in humans.

NTproBNP-guided drug treatment for chronic heart failure: design and methods in the "BATTLESCARRED" trial.

BACKGROUND: How best to decide when to introduce drugs and what doses are optimal in individual patients with chronic heart failure (CHF), is unclear. AIMS: We will determine whether titration of drug treatment according to plasma NTproBNP is superior regarding clinical outcomes to intensive standardised clinical assessment; whether either of the regimens noted above is superior to usual care; and whether age alters the relative efficacy of NTproBNP guided treatment. METHODS: We will randomise 360 patients, stratified by age, to drug treatment directed by plasma NTproBNP, to intensive standardised clinical assessment, or to usual care. The primary outcome is total mortality, and secondary outcomes include death plus hospital admission for any cardiovascular event plus episodes of outpatient decompensated heart failure. Analyses will be conducted at the end of one and two years. RESULTS: 308 patients have been recruited, the majority being in NYHA functional class II, 60.6% being >75 years. The entry plasma NTproBNP level is 238, 50-1250 pmol/l, median and range, approximately 400-11,000 pg/ml. CONCLUSION: We describe details of a study to test the potential utility of serial measurements of NTproBNP in adjusting the drug treatment of patients with CHF. Projected completion date is 2007.

Plasma urocortin 1 in sheep: regional sampling and effects of experimental heart failure.

Although urocortin 1 (Ucn-1) has been reported to circulate in human plasma and be raised in heart failure, little, if any, information is available regarding the source of circulating Ucn-1. Accordingly, we have performed trans-organ arteriovenous sampling for measurement of Ucn-1 concentration in anesthetized sheep before and after development of pacing-induced heart failure. Arterial plasma Ucn-1 levels measured 15.2 +/- 0.5 pmol/L in normal sheep and increased significantly following development of heart failure to 19.1 +/- 1.6 (p < 0.05). Small but significant positive arteriovenous gradients were observed across the hepatic and renal tissue beds in both states, with rises across the hind limb significant in normal animals and across the head in heart failure. This is the first report identifying sources of circulating Ucn-1.

Regional sampling and the effects of experimental heart failure in sheep: differential responses in A, B and C-type natriuretic peptides.

While regional plasma concentrations of the endocrine hormones atrial and brain natriuretic peptide (ANP and BNP) have been studied, there are few reports of regional changes in the largely paracrine C-type natriuretic peptide (CNP) and its amino terminal fragment NT-CNP. Accordingly, we have performed trans-organ arteriovenous sampling for measurement of plasma ANP, BNP, CNP and NT-CNP in anesthetized sheep before and after induction of experimental heart failure. ANP and BNP plasma concentrations are sourced from a single organ (the heart) and are subject to substantial extraction across most tissue beds. In contrast, our data demonstrate that multiple tissues including liver, heart, hind limb and kidney contribute to circulating CNP. Given that arteriovenous gradients for NT-CNP were similar, this is likely to represent de novo secretion. Circulating levels of CNP and NT-CNP were raised in heart failure but to a much lesser degree than ANP and BNP. There was no evidence of net extraction of CNP or NT-CNP across any tissue bed.