B-type natriuretic peptide (BNP) is a cardiac neurohormone which has a principal effect on the kidney to signal both natriuresis and diuresis. Both BNP and renal function are prognostic indicators of survival in patients with congestive heart failure (CHF). However, the relationships between BNP, renal function, and CHF as an emergency diagnosis, are not completely understood. The correlation between BNP and estimated glomerular filtration rate (eGFR) is approximately r = -0.20. At an eGFR < 60 ml/min/1.73 m2, the optimum cutpoint for BNP to diagnose CHF rises to approximately 200 pg/ml. At this cutpoint the area under the receiver operating characteristic curve is 0.81, indicating that BNP is of diagnostic value in this group. Importantly, the precursor molecule N-terminal proBNP has a stronger correlation with eGFR of approximately -0.60, and is influenced by the age-related decline in renal function above the lower bounds of normal of < 60 ml/min/1.73 m2. Because BNP is a principal messenger from the heart to the kidneys, and because it is influenced by renal filtering function, parenchymal mass, and tubular function, BNP can be leveraged in assisting in the diagnosis and management of combined heart and renal failure.

BACKGROUND

Brain natriuretic peptide (BNP) has been widely used for the diagnosis and prognostic evaluation of chronic heart failure (CHF). In the present study, we performed association study of single nucleotide polymorphisms (SNPs) surrounding the natriuretic peptide precursor B (NPPB) gene with plasma BNP levels in 2970 adult Japanese.

RESULTS

Association analysis between SNPs of the NPPB gene and plasma BNP revealed significant associations of the 8 SNPs surrounding the entire NPPB gene with plasma BNP levels. For instance, as to SNP rs198389 (T-381C), plasma BNP levels among the three genotypic categories, i.e., 2189 homozygous T-allele carriers (BNP 26.4+/-0.6pg/ml), 697 heterozygous carriers (35.0+/-1.1pg/ml), and 52 homozygous C-allele carriers (46.0+/-4.1pg/ml) indicated a co-dominant effect of the minor C-allele on elevating plasma BNP levels (P<0.0001). Linkage disequilibrium (LD) analysis among the 8 SNPs revealed that the region consisted of two, 5' major and 3' minor, LD blocks. Haplotype-based association analysis demonstrated that plasma BNP levels were associated closely with the haplotypes-1 and -2 of the major LD block.

CONCLUSIONS

These results suggest that genetic variation at the primary locus NPPB gene, represented by definition of risk haplotypes, may be an important determinant of plasma BNP levels.

OBJECTIVE

Acute dyspnea is a common cause for emergency department visits. The aim of this study was to evaluate the prognostic value of established and novel biomarkers in patients with acute dyspnea.

METHODS

We measured 10 biomarkers [B-type natriuretic peptide (BNP), midregional pro-A-type natriuretic peptide (MR-proANP), midregional-proadrenomedullin (MR-proADM), copeptin, C-terminal endothelin-1 precursor fragment (CT-proET-1), soluble ST2 (sST2), chromogranin A (CgA), adiponectin, proguanylin, and prouroguanylin] in 251 consecutive patients with acute dyspnea presenting to the emergency department of a tertiary care hospital. Outcome measure was all-cause mortality at 1 year.

RESULTS

At baseline decedents (n=62) had significantly higher median plasma concentrations of all 10 biomarkers than survivors (n=189). Applying univariate Cox proportional-hazard regression analyses, all biomarkers were significant outcome predictors displaying risk ratios (RR) from 1.4 to 2.4 (per 1 SD increase in log transformed values). In multivariate Cox proportional-hazard regression analysis, however, only MR-proANP (RR 1.6; 95% CI, 1.1-2.2; p=0.008), sST2 (RR 1.7; 95% CI, 1.3-2.3; p<0.001), and CgA (RR 1.5; 95% CI, 1.2-1.9, p<0.001) were independently associated with 1-year mortality. We provide a possible explanation for the complementary prognostic value of those three biomarkers in our cohort, where coincidence of heart failure and inflammatory pulmonary disease was common and also related to worse outcome.

CONCLUSIONS

Our evaluation of biomarkers in patients with acute dyspnea suggests that MR-proANP, sST2, and CgA are strong, independent and complementary outcome predictors. MR-proANP is considered a specific marker of cardiac stretch, sST2 might reflect both inflammation and cardiac stretch, and CgA obviously indicates neuroendocrine activation in various diseases.

B-type Natriuretic Peptide (BNP) is a circulating hormone primarily produced by the myocardium in response to volume overload and increased filling pressure. BNP acts to increase natriuresis and to decrease cardiac load and blood pressure. The appearance of active BNP hormone in the bloodstream is preceded by the proteolytic cleavage of its precursor, proBNP. The products of proBNP processing, BNP and the N-terminal fragment of proBNP (NT-proBNP), have been extensively shown to be powerful biomarkers of heart failure (HF) and risk assessments for cardiovascular complications. In contrast to the clinical utility of proBNP-derived peptides, knowledge of posttranslational proBNP maturation and molecular aspects of its processing are far from being completely comprehended. A clear understanding of proBNP processing mechanisms in normal and diseased states appears to be required to improve our understanding of HF development and the clinical significance of both proBNP and proBNP-derived peptides. The aim of the present review is to summarize the available data in the field of human proBNP maturation and processing and to discuss potential clinical implications.

OBJECTIVE

Structural heart diseases including atrial fibrillation are precursors for ischemic stroke. Plasma B-type natriuretic peptide (BNP) has been reported to be increased in patients with several types of structural heart diseases. However, the predictive value of plasma BNP for ischemic stroke remains unknown. We have studied the predictive ability of plasma BNP for future development of stroke in community dwelling adults.

METHODS

Subjects of this community-based study were recruited from the general population (n=13,466). Plasma BNP levels and cardiovascular risk factors were determined at baseline. The incidence of ischemic stroke in the cohort was identified from regional stroke registry data. A multivariate Cox regression analysis was performed to analyze the relationship between plasma BNP levels and the risk of stroke.

RESULTS

During a mean follow-up period of 2.8 years, 102 participants (65 males, 37 females) experienced a first ischemic stroke. In men, after adjustment for classical cardiovascular risk factors and atrial fibrillation, the hazard ratio (HR) for ischemic stroke was significantly elevated in the highest plasma BNP quartile (HR=2.38; 95% CI=1.07-5.29). In women, the relationship between plasma BNP levels and risk of ischemic stroke was of marginal significance after adjusting for the presence or absence of atrial fibrillation (HR=3.03; 95% CI=0.84-10.92, P=0.09).

CONCLUSIONS

Elevated plasma BNP levels predict the risk of ischemic stroke within men from the general population.

BACKGROUND

Serial measurements of neurohormones have been shown to improve prognostication in the setting of acute heart failure (HF) or chronic HF without therapeutic intervention. We investigated the prognostic role of serial measurements of emerging neurohormones and BNP in a cohort of chronic HF patients undergoing increases in HF-specific therapy.

METHODS

In this prospective study we included 181 patients with chronic systolic HF after an episode of hospitalization for worsening HF. Subsequently, HF therapy was gradually increased in the outpatient setting until optimized. We measured copeptin, midregional proadrenomedullin, C-terminal endothelin-1 precursor fragment, midregional proatrial natriuretic peptide, and B-type natriuretic peptide before and after optimization of HF therapy. The primary endpoint was all-cause mortality at 24 months.

RESULTS

Angiotensin-converting enzyme/angiotensin receptor blocker and beta-blockers were increased significantly during the 3-month titration period (P < 0.0001 for both). In a stepwise Cox regression analysis adjusted for age, sex, glomerular filtration rate, diabetes mellitus, and ischemic HF, baseline and follow-up neurohormone concentrations were predictors of the primary endpoint as follows (baseline hazard ratios): copeptin 1.92, 95% CI 1.233-3.007, P = 0.004; midregional proadrenomedullin 2.79, 95% CI 1.297-5.995, P = 0.009; midregional proatrial natriuretic peptide 2.05, 95% CI 1.136-3.686, P = 0.017; C-terminal endothelin-1 precursor fragment 2.24, 95% CI 1.133-4.425, P = 0.025; B-type natriuretic peptide 1.46, 95% CI 1.039-2.050, P = 0.029.

CONCLUSIONS

In pharmacologically unstable chronic HF patients, baseline values and follow-up measures of copeptin, midregional proadrenomedullin, C-terminal endothelin-1 precursor fragment, midregional proatrial natriuretic peptide, and B-type natriuretic peptide were equally predictive of all-cause mortality. Relative change of neurohormone values was noncontributory.

Endothelin-1 (ET-1) at high concentrations has marked antidiuretic and antinatriuretic activities, whereas its precursor, big endothelin-1 (big ET-1), has surprisingly potent diuretic and natriuretic actions. The mechanisms underlying the excretory effects of big ET-1 have not been fully elucidated. To explore these mechanisms, we examined the effects of a highly selective ET(B) antagonist (A-192621.1), a calcium channel blocker (verapamil), a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester [L-NAME]), and a cyclooxygenase inhibitor (indomethacin) on the systemic and renal actions of big ET-1 in anesthetized rats. An intravenous bolus injection of incremental doses of big ET-1 (0.3, 1. 0, and 3.0 nmol/kg) produced a significant hypertensive effect that was dose dependent and prolonged (from 113+/-7 mm Hg to a maximum of 148+/-6 mm Hg). The administration of big ET-1 induced marked diuretic and natriuretic responses (urinary flow rate increased from 8.5+/-1 to 110+/-14 microL/min, and fractional excretion of sodium increased from 0.38+/-0.13% to 7.51+/-1.24%). Glomerular filtration rate and renal plasma flow significantly decreased only at the highest dose of big ET-1. Pretreatment with A-192621.1 (3 mg/kg plus 3 mg. kg(-1). h(-1)) significantly abolished the diuretic (17+/-5 microL/min to a maximum of 19+/-3 microL/min) and natriuretic (0. 29+/-0.1% to a maximum of 1.93+/-0.37%) responses induced by big ET-1. Moreover, A-192621.1 potentiated the decline in glomerular filtration rate and renal plasma flow and the increase in mean arterial blood pressure produced by the low doses of big ET-1. Similar to A-192621.1, pretreatment with a nitric oxide synthase inhibitor (L-NAME, 10 mg/kg plus 5 mg. kg(-1). h(-1)) significantly and comparably reduced the diuretic and natriuretic actions of big ET-1 and augmented the hypoperfusion/hypofiltration and systemic vasoconstriction induced by high doses of the peptide. Pretreatment with verapamil (2 mg. kg(-1). h(-1)) slightly inhibited the diuretic/natriuretic effects of the high-dose big ET-1 and completely prevented the increase in mean arterial blood pressure provoked by the peptide. Unlike verapamil and L-NAME, only indomethacin administration was associated with significant natriuretic/diuretic responses and did not influence the pressor effect and renal actions of big ET-1. Taken together, these results suggest that big ET-1-induced diuretic and natriuretic responses are mediated mainly by stimulation of nitric oxide production coupled to ET(B) receptor subtype activation.

BACKGROUND

Endothelin-1 is elevated in heart failure (HF) and after acute myocardial infarction (AMI) and gives prognostic information on mortality. Another part of its precursor, C-terminal pro-endothelin-1 (CT-proET-1), is more stable in circulation and ex vivo. We investigated the cardiovascular prognostic value post-AMI of CT-proET-1 and compared it to N-terminal pro-B-type natriuretic peptide (NTproBNP), a marker of death and HF.

METHODS

We measured plasma CT-proET-1 and NTproBNP in 983 consecutive post-AMI patients (721 men, mean age 65.0 +/- [SD] 12.2 years), 3 to 5 days after chest pain onset.

RESULTS

There were 101 deaths and 49 readmissions with HF during follow-up (median 343, range 0-764 days). C-terminal pro-endothelin-1 was raised in patients with death or HF compared to survivors (median [range] [pmol/L], 119.0 [14.0-671.0] vs 73.0 [4.6-431.0], P < .0001). Using a Cox proportional hazards logistic model, log CT-proET-1 (HR 6.82) and log NTproBNP (HR 2.62) were significant independent predictors of death or HF (along with age, sex, history of AMI, and therapy with beta-blockers). The areas under the receiver operating curve for CT-proET-1, NTproBNP, and the logistic model with both markers were 0.76, 0.76, and 0.81 respectively. Findings were similar for death and HF as individual end points.

CONCLUSIONS

The endothelin system is known to be activated post AMI. C-terminal pro-endothelin-1 is a powerful predictor of adverse outcome, along with NTproBNP. CT-proET-1 may represent a clinically useful marker of prognosis after AMI.

OBJECTIVE

Multiorgan involvement renders patients with AL amyloidosis particularly susceptible to treatment toxicity. The introduction of autologous stem cell transplantation (ASCT) represented a major advancement, but was associated with unacceptable toxicity in high-risk patients. Thus, efforts were made to improve the eligibility criteria for ASCT and to design novel, more effective, conventional-dose regimens. This review focuses on the role of ASCT and conventional-dose therapy in light of advances in risk stratification and patient monitoring.

RESULTS

The possibility of directly measuring the amyloidogenic precursor, the circulating free light chain (FLC), improved monitoring response to therapy. Cardiac biomarkers, N-terminal pro-natriuretic peptide type-B (NT-proBNP) and troponins (cTn) allow the most accurate prognostic stratification and direct the choice of therapy. Serial measurement of NT-proBNP, cTn and FLC are used to rapidly assess treatment efficacy. Bortezomib and immune-modulatory drugs are going to play a major role in conventional-dose therapy and as adjuvant treatment after ASCT.

CONCLUSIONS

The choice between ASCT and conventional-dose chemotherapy is based on accurate risk assessment. Tight monitoring of hematologic and cardiac response is the cornerstone of treatment. Upcoming randomized trials will redefine the role of available therapies, assisting in the choice of the growing number of active regimens.

We previously demonstrated that secreted forms of the Alzheimer's beta-amyloid precursor protein (sAPP) elevate cyclic GMP (cGMP) in primary neuronal cultures and that this effect is responsible for the modulation of neuronal calcium homoeostasis by sAPP. We have investigated further the mechanism by which sAPP elevates cGMP. Inhibition of the formation of nitric oxide or carbon monoxide did not affect the ability of sAPP to lower rapidly intraneuronal calcium levels or elevate cGMP, suggesting that sAPP does not activate a soluble (cytosolic) guanylate cyclase. A dose-dependent stimulation of cGMP formation by sAPP was observed in brain membrane preparations. The stimulation was also dependent on the presence of ATP. These data suggest that sAPP activates a membrane-associated guanylate cyclase, perhaps similar to those present in the receptors for the natriuretic peptides and sperm motility factors.

Thus far, five molecules comprise the natriuretic peptide family (NPF): ANP, urodilatin, BNP, CNP and DNP. Precursor hormones for ANP, BNP and CNP are encoded by a different gene. Final peptides are ligands for A, B and C receptors, acting the latter as a clearance receptor besides neutral endopeptidase (EC 24.11). cGMP acts as a second messenger. Natriuretic peptides (NP) have well-known functions such as natriuretic, antihypertensive and reduction of plasma renin-aldosterone concentrations. An antiinflammatory ANP potential and a pro-apoptotic action in rats endothelial cells of different NP have been described. Unlike adults, NP show a different distribution during ontogeny and a different pattern of excretion under different stimuli. Noncompetitive immunoassays have become more suitable than competitive ones for routine measurement of NP with recent advances in speed of measurement. BNP and pro-BNP are emerging as useful tools in diagnosis, management and prognosis of heart disease. Preliminary data support a role of NP in the therapy of congestive heart failure. Finally, potential therapeutic compounds of NP in different pathologies are updated with an important focus on vasopeptidase inhibitors. These are capable of strengthening NP and inhibiting renin-angiotensin system at the same time, as potential useful molecules in cardiovascular therapy.

Chromogranin A (CgA), a major component of the chromaffin granules, is co-stored and co-released with catecholamines. It is also expressed in extra-adrenal sites, including the heart. In the rat, CgA localizes in atrial myoendocrine cells, associated with Atrial Natriuretic Peptide (ANP), and in the conduction system. In the human heart it is present in the ventricular myocardium, co-localized with B-type NP (BNP). CgA is the precursor of several biologically active peptides generated by proteolytic processing also in the heart. Two of them, vasostatin-1 (VS-1) and catestatin (Cst), inhibit cardiac contraction and relaxation, counter-regulate beta-adrenergic and endothelinergic stimulation, and protect the heart against ischemia/reperfusion damages. Recently, clinical studies have suggested CgA to be involved also in cardiovascular pathologies. High plasma CgA levels were found in hypertension, chronic and acute heart failure, myocardial infarction, decompensated and hypertrophic heart, and acute coronary syndromes. These alterations correlate with those of conventional cardiovascular biomarkers, such as NP and endothelin-1 (ET-1), and have prognostic relevance, being indicative of both severity of the disease and mortality. Accordingly, the current knowledge indicates CgA as a multifaceted peptide in cardiovascular homeostasis. Whether the influence elicited by the protein on both normal and failing heart is beneficial and/or detrimental, as well as its implication in the cardiac neuroendocrine scenario is under intense investigation. This review will focus on: i) the involvement of CgA and its derived peptides in the mechanisms which sustain cardiac function and compensation, ii) CgA clinical relevance, and iii) its putative value as a clinical biomarker.

Functional studies indicate that natriuretic peptides have direct effects on Leydig cells of the testis. In this report, we demonstrate local synthesis of one member of the natriuretic peptide family, C-type natriuretic peptide (CNP), in Leydig cells of human testes. Using RT-PCR assays, messenger RNA (mRNA) for the CNP precursor was detected in human testis and found to be prominently expressed in Leydig cells. Immunohistochemical analyses revealed CNP to be almost exclusively associated with Leydig cells. Distinct differences in the staining intensity-including cells without detectable staining-suggest a heterogeneity of CNP expression within the Leydig cells. Moreover, the presence of transcripts for the CNP receptor, a particulate guanylate cyclase, termed GC-B, was demonstrated by RT-PCR in human testis and in isolated Leydig cells. The expression of this receptor in human testis membranes could be confirmed by affinity labeling with 125I-labeled CNP. These findings demonstrate, for the first time, the production of a natriuretic peptide in human Leydig cells. The occurrence of CNP and its receptor in the human testis points to a local role of the peptide, presumably acting in an auto- or paracrine manner to modulate organ-specific functions.

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.

OBJECTIVE

The purpose of this study was to assess the prognostic value of admission and discharge mid-regional pro-adrenomedullin (sAM) levels in non-ST-elevation myocardial infarction (MI) and identify values to aid clinical decision making. N-terminal pro-B-type natriuretic peptide and GRACE (Global Registry of Acute Coronary Events) score were used as comparators.

BACKGROUND

sAM is a stable precursor of adrenomedullin.

METHODS

We measured plasma sAM on admission and discharge in 745 non-ST-elevation MI patients (514 men, median age 70.0 +/- 12.7 years). The primary end point was a composite of death, heart failure, hospitalization, and recurrent acute MI over mean follow-up of 760 days (range 150 to 2,837 days), with each event assessed individually as secondary end points.

RESULTS

During follow-up, 120 (16.1%) patients died, and there were 65 (8.7%) hospitalizations for heart failure and 77 (10.3%) recurrent acute MIs. Both admission and discharge levels were increased (median 0.81 nmol/l [range 0.06 to 5.75 nmol/l] and 0.76 nmol/l [range 0.25 to 6.95 nmol/l], respectively) compared with established normal ranges. Multivariate adjusted Cox regression models revealed that both were associated with the primary end point (hazard ratio: 9.75 on admission and 7.54 on discharge; both p < 0.001). Admission sAM was particularly associated with early (<30 days) mortality (c-statistic = 0.90, p < 0.001), and when compared with N-terminal pro-B-type natriuretic peptide and GRACE score, it was the only independent predictor of this end point. Admission sAM >1.11 nmol/l identified those at highest risk of death (p < 0.001). Patients with above-median admission sAM may benefit from revascularization.

CONCLUSIONS

sAM level is prognostic for death or heart failure. Admission levels are a strong predictor of early mortality and, when >1.11 nmol/l, complements the GRACE score to improve risk stratification.

Using RIAs for the N- and C-terminal fragments of the human atrial natriuretic polypeptide (ANP) precursor gamma ANP, that is gamma ANP-(1-25), and alpha ANP [gamma ANP-(99-126)], we studied the secretion of gamma ANP-derived peptides from the heart in normal subjects and patients with heart disease, chronic renal failure, and cirrhosis. We detected gamma ANP-(1-25)-like immunoreactivity (-LI) in plasma from normal subjects (n = 17) in considerable amounts [mean, 510 +/- 62 (+/- SE) pg/mL (174 +/- 21 pmol/L)]; the mean plasma alpha ANP-LI level at the same time in these subjects was 32.8 +/- 4.4 pg/mL (10.7 +/- 1.4 pmol/L). Gel permeation chromatographic analysis of plasma samples from normal subjects and patients with heart disease and chronic renal failure revealed two major components; one was alpha ANP, and the other was the 10K N-terminal gamma ANP fragment (N-peptide) resulting from the removal of alpha ANP (3K) from gamma ANP (13K). In addition, gamma ANP (13K), which possessed both gamma ANP-(1-25)-LI and alpha ANP-LI, and beta ANP, an antiparallel dimer of alpha ANP, were detected in some patients as minor components. A significant positive correlation between plasma levels of the N-terminal gamma ANP fragment and alpha ANP (P less than 0.01) and almost equal step-ups in the coronary sinus plasma levels of the N-terminal gamma ANP fragment and alpha ANP suggest that they are cosecreted in equimolar amounts. The high molar ratio of plasma gamma ANP-(1-25)-LI to alpha ANP-LI (17.4 +/- 1.4) in normal subjects and the significantly higher ratio in patients with chronic renal failure (36.9 +/- 7.1; P less than 0.01) suggest the slower clearance of the N-terminal gamma ANP fragment than alpha ANP and a role for the kidney in its degradation. Since the molar ratio of plasma gamma ANP-(1-25)-LI to alpha ANP-LI in patients with cirrhosis (20.7 +/- 2.7) was similar to that in normal subjects, it is unlikely that the N-terminal gamma ANP fragment is metabolized by the liver. In patients with heart disease, plasma gamma ANP-(1-25)-LI and alpha ANP-LI levels were higher in those with cardiac decompensation and were positively correlated with right atrial pressure, pulmonary arterial pressure, and pulmonary capillary wedge pressure, indicating cosecretion of the N-terminal gamma ANP fragment and alpha ANP in response to atrial stretch.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Age, sex, and renal function contribute to variations in plasma concentrations of B-type natriuretic peptide (BNP) and its molecular precursor (proBNP). Recent studies indicate that anemia may also affect proBNP concentrations in patients with heart failure or stroke. However, the impact of hemoglobin status on proBNP concentrations has not been established in the general population.

METHODS

In the 4th examination in the Copenhagen City Heart Study, we performed a nested case-control study of 6238 individuals from a Danish general population. Of these, 3497 randomly selected participants also underwent an echocardiographic examination. The population was stratified into groups depending on health and hemoglobin status. Correlations between hemoglobin and proBNP concentrations were examined by simple and multiple regression analyses, adjusted for variables known to influence the proBNP plasma concentration.

RESULTS

The mean proBNP concentration was increased 1.7-fold in the group with anemia vs the nonanemic group [mean (SD) 42 (45) pmol/L vs 25 (29) pmol/L, P <0.0001, n = 5892]. Multiple regression analysis confirmed an independent effect of hemoglobin on proBNP concentrations. In a selected subgroup without signs or symptoms of heart disease (n = 2855), lower hemoglobin concentrations, defined as <120 g/L in women and <130 g/L in men, were associated with increased circulating proBNP concentrations, but the contribution to the overall variation in proBNP concentrations was modest.

CONCLUSIONS

Because moderate anemia is associated with a 1.7-fold increase in proBNP concentrations, hemoglobin concentrations should be taken into consideration in patients with nonspecific symptoms of heart disease and increased proBNP concentrations.

Iron in association with reactive oxygen species (ROS) is highly toxic, aggravating oxidative stress reactions. Increased iron not only plays an important role in the progression of hereditary hemochromatosis (HH) but also in common liver diseases such as chronic hepatitis C. The underlying mechanisms of hepatitis C virus (HCV)-mediated iron accumulation, however, are poorly understood. We introduce an in vitro-targeted approach to identify ROS/iron-regulated genes in patients with HCV using a genome-wide DNA microarray. The sensitivity of the 32,231 complementary DNA clone-carrying microarray was approximately 20% as estimated by detecting target genes of the genome-wide transcription factor hypoxia inducible factor 1alpha. Upon in vitro challenge to iron and oxidative stress, 265 iron-related and 1326 ROS-related genes could be identified in HepG2 cells; 233 significantly regulated genes were found in patients with mild (HCV) or severe (HH) iron deposition. Notably, 17 of the in vitro-selected genes corresponded to the genes identified in patients with HCV or HH. Among them, natriuretic peptide precursor B (NPPB) was the only iron-regulated gene identified in vitro that was differentially regulated between HCV and HH. Reverse-transcription polymerase chain reaction confirmed most of the microarray-identified genes in an even larger group of patients (n = 12). In patients with HCV, these included genes that are associated with RNA processing (MED9/NFAT, NSUN2), proliferation, differentiation, hypoxia, or iron metabolism (ISG20, MIG6, HIG2, CA9, NDRG1), whereas none of the nine known iron-related genes showed significant differences between HCV and HH.

CONCLUSIONS

Although high-density microarray technology is less suitable for routine liver diagnosis, its use in combination with prior in vitro selection is a powerful approach to identify candidate genes relevant for liver disease.

Neuropeptides regulate cell-cell signaling and influence many biological processes in vertebrates, including development, growth, and reproduction. The complex processing of neuropeptides from prohormone proteins by prohormone convertases, combined with the evolutionary distance between the chicken and mammalian species that have experienced extensive neuropeptide research, has led to the empirical confirmation of only 18 chicken prohormone proteins. To expand our knowledge of the neuropeptide and prohormone convertase gene complement, we performed an exhaustive survey of the chicken genomic, EST, and proteomic databases using a list of 95 neuropeptide and 7 prohormone convertase genes known in other species. Analysis of the EST resources and 22 microarray studies offered a comprehensive portrait of gene expression across multiple conditions. Five neuropeptide genes (apelin, cocaine-and amphetamine-regulated transcript protein, insulin-like 5, neuropeptide S, and neuropeptide B) previously unknown in chicken were identified and 62 genes were confirmed. Although most neuropeptide gene families known in human are present in chicken, there are several gene not present in the chicken. Conversely, several chicken neuropeptide genes are absent from mammalian species, including C-RF amide, c-type natriuretic peptide 1 precursor, and renal natriuretic peptide. The prohormone convertases, with one exception, were found in the chicken genome. Bioinformatic models used to predict prohormone cleavages confirm that the processing of prohormone proteins into neuropeptides is similar between species. Neuropeptide genes are most frequently expressed in the brain and head, followed by the ovary and small intestine. Microarray analyses revealed that the expression of adrenomedullin, chromogranin-A, augurin, neuromedin-U, platelet-derived growth factor A and D, proenkephalin, relaxin-3, prepronociceptin, and insulin-like growth factor I was most susceptible (P-value<0.005) to changes in developmental stage, gender, and genetic line among other conditions studied. Our complete survey and characterization facilitates understanding of neuropeptides genes in the chicken, an animal of importance to biomedical and agricultural research.

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (approximately 25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (approximately 144 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.

Recent studies reported that many different biochemical forms of B-type-related peptides circulate in human blood. In particular, a significant amount of the prohormone peptide (i.e., proBNP108) can be detected in plasma of patients with heart failure. These data indicate that the posttranslational maturation processing of the B-type natriuretic peptide (BNP) precursor may not be efficient in heart failure. The aim of this chapter is to describe the biochemical pathways of proBNP108 maturation and to discuss the pathophysiological relevance of alteration of the posttranslational maturation mechanisms in heart failure. An impaired cardiac endocrine function was proposed to explain the altered electrolyte and fluid homeostasis occurring in chronic heart failure. Recent studies demonstrated that a great part of BNPs assayed by immunoassay methods in healthy subjects and in patients with cardiovascular disease is devoid of biological activity. These findings suggest that an alteration in posttranslational maturation of BNP precursor may promote the resistance to biological action of BNP in patients with heart failure at a prereceptor level. These studies also open a new and more complex scenario regarding the circulating BNPs. The active hormone (i.e., BNP1-32) may be produced even in vivo from the circulating precursor proBNP108 by plasma enzyme degradation, such as the soluble form of corin, possibly able to process the circulating intact precursor of natriuretic hormones. As a future perspective, the simultaneous measurement of the proBNP1-108 and the active peptide BNP1-32 with more specific methods could allow a more accurate estimation of both production/secretion of B-type-related peptides from cardiomyocytes and the true activity of the cardiac endocrine function.

Peptides derived from the precursor of A- and B-type natriuretic peptides (ANP and BNP) are powerful clinical markers of cardiac hypertrophy and dysfunction. It is known that many stimuli affecting the intracellular calcium concentration also induce ANP and BNP secretion. It was our intention to study the mechanisms by which calcium regulates the secretion of ANP and BNP. The effects of pacing and calcium-calmodulin kinase II activity on natriuretic peptide secretion were studied in isolated perfused rat atria and cultured rat neonatal cardiomyocytes. In isolated rat atrium pacing induced an increase in diastolic, systolic, and averaged intracellular free calcium concentration and a frequency-dependent increase in the secretion of both ANP and BNP. The molar ratio of the secreted natriuretic peptides (ANP to BNP) remained nearly constant ( approximately 1000) at all the pacing frequencies tested (1, 3, 6, and 8 Hz). Calmodulin kinase II inhibitor KN-93 (3 mum) did not affect intracellular free calcium concentration but showed a frequency-dependent inhibitory effect on ANP and BNP secretion without a change in ANP to BNP ratio. In the neonatal cardiomyocytes, KN-93 (3 mum) suppressed the secretion and gene expression of both ANP and BNP. Overexpression of constitutively active (T286D) or nuclear (delta(B)) calcium-calmodulin kinase II induced an increase in ANP and BNP gene expression. The results indicate that the calcium-dependent secretion and gene expression of A- and B-type natriuretic peptides are similarly regulated by calmodulin kinase II-dependent mechanisms. This is a plausible mechanism contributing to exercise-induced natriuretic peptide secretion and the augmented secretion in heart dysfunction due to impaired calcium handling.

Hydrogen peroxide (H2O2) causes cell damage via oxidative stress. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that can protect cardiomyocytes against oxidative stress. In this study, we investigated whether the heme precursor 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) could protect cardiomyocytes from H2O2-induced hypertrophy via modulation of HO-1 expression. HL-1 cells pretreated with/without 5-ALA and SFC were exposed to H2O2 to induce a cardiomyocyte hypertrophy model. Hypertrophy was evaluated by planar morphometry, (3)H-leucine incorporation, and RT-PCR analysis of hypertrophy-related gene expressions. Reactive oxygen species (ROS) production was assessed by 5/6-chloromethyl-2',7'-ichlorodihydrofluorescein diacetate acetylester. HO-1 and nuclear factor erythroid 2-related factor 2 (Nrf2) protein expressions were analyzed by Western blot. In our experiments, HL-1 cells were transfected with Nrf2 siRNA or treated with a signal pathway inhibitor. We found several results. 1) ROS production, cell surface area, protein synthesis, and expressions of hypertrophic marker genes, including atrial natriuretic peptide, brain natriuretic peptide, atrial natriuretic factor, and β-myosin heavy chain, were decreased in HL-1 cells pretreated with 5-ALA and SFC. 2) 5-ALA and SFC increased HO-1 expression in a dose- and time-dependent manner, associated with upregulation of Nrf2. Notably, Nrf2 siRNA dramatically reduced HO-1 expression in HL-1 cells. 3) ERK1/2, p38, and SAPK/JNK signaling pathways were activated and modulate 5-ALA- and SFC-enhanced HO-1 expression. SB203580 (p38 kinase), PD98059 (ERK), or SP600125 (JNK) inhibitors significantly reduced this effect. In conclusion, our data suggest that 5-ALA and SFC protect HL-1 cells from H2O2-induced cardiac hypertrophy via activation of the MAPK/Nrf2/HO-1 signaling pathway.

Natriuretic peptides (NP) are released from the heart in response to pressure and volume overload. The biologic properties of NPs include counterregulation of the rennin-angiotensin-aldosterone pathway and a decrease in sympathetic tone resulting in diuresis, natriuresis, and vasodilation. Natriuretic peptides help to maintain fluid balance and blood pressure in a healthy physiologic range. The B-type natriuretic peptide (BNP) and its N-terminal precursor (NTpBNP) have become important diagnostic biomarkers of cardiovascular diseases (CVDs) in adults. Although many studies suggest that BNP also is a reliable test for diagnosing significant CVDs in children, data are lacking on whether additional use of BNP increases diagnostic accuracy and predicts prognosis. This comprehensive review describes the utility of BNP and NTpBNP for various CVDs of the neonatal and pediatric age groups. Because BNP is not a stand-alone test, it should not replace history, physical examination, or clinical judgment, but it has a clear value in adding details to the whole story for children, thus enabling the front-line physicians to make a diagnosis, especially in the acute care setting.