1. The natriuretic peptide precursor A (Nppa) and B (Nppb) genes are candidate genes for hypertension and cardiac hypertrophy in the spontaneously hypertensive rat (SHR). The purpose of the present study was to determine the role of the Nppa and Nppb genes in the development of hypertension in the SHR. 2. A cohort (n = 162) of F2 segregating intercross animals was established between strains of hypertensive SHR and normotensive Wistar-Kyoto rats. Blood pressure and heart weight were measured in each rat at 12-16 weeks of age. Rats were genotyped using 11 informative microsatellite markers, distributed in the vicinity of the Nppa marker on rat chromosome 5 including an Nppb marker. The phenotype values were compared with genotype using the computer package mapmaker 3.0 (Whitehead Institute, Boston, MA, USA) to determine whether there was a link between the genetic variants of the natriuretic peptide family and blood pressure or cardiac hypertrophy. 3. A strong correlation was observed between the Nppa marker and blood pressure. A quantitative trait locus (QTL) for blood pressure on chromosome 5 was identified between the Nppa locus and the D5Mgh15 marker, less than 2 cM from the Nppa locus. The linkage score for the blood pressure QTL on chromosome 5 was 3.8 and the QTL accounted for 43% of the total variance of systolic blood pressure, 54% of diastolic blood pressure and 59% of mean blood pressure. No association was found between the Nppb gene and blood pressure. This is the first report of linkage between the Nppa marker and blood pressure in the rat. There was no correlation between the Nppa or Nppb genes or other markers in this region and either heart weight or left ventricular weight in F2 rats. 4. These findings suggest the existence of a blood pressure-dependent Nppa marker variant or a gene close to Nppa predisposing to spontaneous hypertension in the rat. It provides a strong foundation for further detailed genetic studies in congenic strains, which may help to narrow down the location of this gene and lead to positional cloning.

Brain natriuretic peptide/natriuretic peptide precursor B (NPPB) is one of the most studied genes in relation to heart failure (HF) conditions. However, it is still unclear as to whether alternative splicing could create NPPB mRNA variants, which may be expressed in normal and diseased myocardium. We aimed to identify and characterize a novel alternatively spliced variant of porcine and human NPPB resulting from exon 2 skipping (designated as DeltaE2-NPPB). A variety of conventional molecular, biochemical and immunochemical methods were used to examine the expression and functional consequences of DeltaE2-NPPB in vitro and in vivo. The pig DeltaE2-NPPB mRNA is effectively translated into stable protein in cell-based assays but, in contrast to normally spliced NPPB, the DeltaE2-NPPB protein is not secreted into the media. Co-transfection assays demonstrate that DeltaE2-NPPB attenuates production and secretion of normally spliced NPPB, suggesting a negative feedback loop of NPPB signaling through generation of DeltaE2-NPPB. The inhibitory effects of DeltaE2-NPPB on the expression of NPPB are associated with sequence elements residing in exon 3 of DeltaE2-NPPB. In piglets, DeltaE2-NPPB gene expression is downregulated in both ventricles after birth, but it is markedly re-activated in the postnatal myocardium in experimental diastolic heart failure. In addition, we demonstrate that the exon-skipped NPPB variants are expressed in the postnatal and adult human myocardium and upregulated at end-stage HF due to dilated cardiomyopathy. Our work uncovers an important role of alternative exon skipping in the regulation of NPPB gene expression, thereby pinpointing a putative new mechanism for post-transcriptional regulation of NPPB production and secretion.

BACKGROUND

We examined whether B-type natriuretic peptide (BNP) and its precursor fragment, N-terminal proBNP (NT-proBNP), can serve as non-invasive markers of hemodynamic response to treatment in acute heart failure in a prospective observational study.

METHODS

29 unselected, consecutive patients (mean age: 61.6, 39-83 years; 25 males, 4 females) in urgent need for positive inotropic support and invasive hemodynamic monitoring by a Swan-Ganz catheter. Positive hemodynamic response to treatment was defined as>> or =25% decrease in pulmonary artery occlusion pressure after 24 h. Hemodynamics were recorded simultaneously with blood sampling for BNP and NT-proBNP testing before and 24 h after initiation of inotropic support. BNP (Bayer Diagnostics) and NT-proBNP (Roche Diagnostics) were measured by commercial immunoassays.

RESULTS

Both markers were markedly elevated. However, there were no close correlations (r<0.43, p<0.05) of BNP or NT-proBNP with hemodynamic parameters at baseline or 24 h thereafter. Only BNP showed a significant (p=0.023) decrease compared to baseline values in hemodynamic responders. The area under receiver operating characteristics curve for relative changes of BNP for the prediction of hemodynamic response was 0.76.

CONCLUSIONS

Our preliminary results indicate that BNP is more sensitive to acute hemodynamic changes than NT-proBNP. This study also highlights limitations of both markers as surrogates of hemodynamics in critically ill acute heart failure patients.

To elucidate the circulating forms of human atrial natriuretic peptide (hANP) in patients with congestive heart failure (CHF), plasma samples obtained from 36 patients with CHF were analyzed and compared with those from normal subjects. Plasma concentrations of hANP-like immunoreactivity (LI) from normal subjects and patients with mild CHF (class I), as classified by the New York Heart Association (NYHA) functional criteria, did not differ (15 +/- 1 vs. 16 +/- 1 pmol/L, mean +/- SE), whereas plasma levels of hANP-LI in patients with moderate and severe CHF significantly (P less than 0.01) increased in relation to the severity of CHF (class II, 44 +/- 4 pmol/L; class III, 116 +/- 24 pmol/L; class IV, 141 +/- 21 pmol/L). Reverse-phase HPLC and gel permeation chromatography coupled with RIA for hANP revealed that the circulating forms of hANP-LI consisted of alpha-hANP, beta-hANP, and gamma-hANP in CHF, whereas alpha-hANP predominated in normal plasma. The percentage of beta-hANP in total hANP-LI as calculated from the chromatograms by gel filtration was greater in severe CHF (NYHA class III and IV) than those in mild CHF (NYHA class I and II), and apparently exceeded those of other forms. Successful medical treatment for CHF resulted in a marked reduction of total plasma hANP-LI levels with a concomitant disappearance or reduction of beta-hANP in 14 patients examined. These data suggest that beta-hANP and gamma-hANP are secreted from the failing human heart, possibly resulting from the augmented synthesis and/or the altered processing of hANP precursor in cardiocytes, and that circulating beta-hANP may serve as a potential marker for the severity of CHF in man.

BACKGROUND

Several types of structural heart disease are important precursors for congestive heart failure or cardioembolic stroke. We have previously demonstrated that plasma B-type natriuretic peptide (BNP) measurement is useful for detection of structural heart disease in a multiphasic health screening setting. To extend our hypothesis to the general population, the utility of BNP testing for identifying structural heart disease was assessed in a general population and in subgroups divided by sex, age, and presence/absence of risk factors.

RESULTS

This cross-sectional cohort study measured plasma <em>B</em>NP concentrations in 993 randomly selected community-dwelling adults (mean age 58 years). All subjects underwent plasma <em>B</em>NP measurement and transthoracic echocardiography. Using prejudged criteria, 41 subjects were diagnosed to have some form of structural heart disease (mild left ventricular systolic dysfunction in 11, valvular heart disease in 9, hypertensive heart disease in 3, hypertrophic cardiomyopathy in 2, ischemic heart disease in 2, lone atrial fibrillation in 14). The utility of <em>B</em>NP testing was evaluated by receiver operating characteristic (ROC) analysis and by cost analysis for detection of 1 case within each subgroup of the cohort. Overall, the sensitivity and specificity of <em>B</em>NP testing for identification of structural heart disease were 61% and 92%, respectively. The area under the ROC curve was 0.77 (95% CI; 0.74-0.79). When sex-specific ROC analyses were performed, sensitivity and specificity were 61% and 91% in men, and 50% and 95% in women, respectively. Although the performance of <em>B</em>NP testing on the basis of these figures might be suboptimal, efficacy was improved in subgroups with a high prevalence of heart disease (>8%) such as the cohort aged>> or =65 years (men, area under ROC curve = 0.88; cost <US $1400: women, area under ROC curve = 0.83; cost <US $3000) as well as the cohort having cardiovascular risk factors such as hypertension or diabetes (men, area under ROC curve = 0.85; cost <US $1700: women, area under ROC curve = 0.83; cost <US $3100).

CONCLUSIONS

The present results suggest that BNP testing for structural heart disease screening in community-based populations is useful for cohorts with a high prevalence of heart disease. However, its efficacy is reduced in cohorts with a low prevalence rate.

Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic alpha-factor (hANF), we have constructed a secretion vector. B. subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 micrograms/liter. The hANF is the first human gene product to be secreted from B. subtilis using this signal peptide. We have used promoters active during vegetative growth or sporulation and hosts deficient in several extracellular proteases but some proteolysis of the secretion products still occurs. In addition, both cell growth and sporulation are adversely affected by hANF production. Possible explanations for this observation are inefficient secretion of the atrial hormone or toxicity of the precursor or mature peptide.

OBJECTIVE

To maintain the homeostasis of stem cells and prevent their ability to initiate tumorigenesis, it is important to identify and modify factors that prevent or accelerate stem cell senescence. We used microarrays to attempt to identify such factors in human amniotic fluid (HAF)-derived stem cells.

METHODS

To identify gene expression changes over a time course, we compared gene expression profiles of HAF-derived stem cells in different passages (1(st), 2(nd), 4(th), 6(th), 8(th), and 10(th)) using a Sentrix Human illumina microarray.

RESULTS

Of the 25,804 genes in the microarray chip, 1,970 showed an over 2-fold change relative to the control (the 1(st) passage)-either upregulated or downregulated. Quantitative real-time PCR validated the microarray data for selected genes: markedly increased genes were CXCL12, cadherin 6 (CDH6), and folate receptor 3 (FOLR3). Downregulated genes included cyclin D2, keratin 8, insulin-like growth factor 2 (IGF2), natriuretic peptide precursor B (NPPB) and cellular retinoic acid binding protein 2 (CRABP2). The expression pattern of the selected genes was consistent with the microarray data except for CXCL12 and IGF2. Interestingly, the expression of NPPB was dramatically downregulated along the time course; it was almost completely shut-down by the 10(th) passage. In contrast, FOLR3 mRNA expression was dramatically increased.

CONCLUSIONS

Taken together, although a function for NPPB and FOLR3 in stem cell senescence has not been reported, our results strongly suggest that NPPB and/or FOLR3 play a significant role in the regulation of stem cell senescence.

The natriuretic peptide system is suggested to be involved in the pathogenesis of salt-sensitive hypertension; a recent report indicated that disruption of the atrial natriuretic peptide precursor gene caused salt-sensitive hypertension. However, natriuretic peptide receptor (NPR)-A knockout mice did not show enhanced salt sensitivity of blood pressure. The aim of the present study was to investigate the role of NPR-C, the other receptor for atrial natriuretic peptide, in increased salt sensitivity of blood pressure. Dahl salt-sensitive (DS) and salt-resistant (DR) rats were placed on a 0.3% or 8% NaCl diet for 4 weeks. Blood pressure was elevated by salt loading only in DS rats. RNase protection assay demonstrated that NPR-C transcript level in the kidney was reduced by chronic salt loading in both DR and DS rats, whereas expression of NPR-A and NPR-B was not altered. The reduction of NPR-C mRNA in response to salt loading was enhanced in DS compared with DR rats. In situ hybridization indicated that the salt-induced NPR-C change was attributed mainly to suppressed expression of NPR-C in the podocytes. NPR-C gene expression was regulated by salt loading in a tissue-specific manner; the marked decrease in NPR-C mRNA by salt loading was seen only in the kidney. These data suggest that the exaggerated salt-induced reduction of NPR-C in the kidney of DS rats may play an important role in the pathogenesis of salt hypertension in this animal, possibly related to impaired renal sodium excretion.

Atrial natriuretic factor, originally isolated from the atrium of the heart, has been found to consist of three major groups: atrial natriuretic peptide (ANP), B-form natriuretic peptide (BNP), and C-form natriuretic peptide (CNP). In addition, ANP exists in its precursor form, pro-ANP, an active ANP with a longer peptide chain (urodilatin) and an antiparallel dimer of active ANP. Sites and production of these diverse forms of the peptides are also diverse, depending on pathologic states. Three major subtypes of ANP receptors exist; these include a clearance receptor and two types of a transmembrane receptor with guanylyl cyclase structures in their intracellular domain. The latter exists at least in two forms, one of which is found mainly in the brain. All the actions of ANP mediated by the transmembrane form of ANP receptors are mediated by cGMP generated by the guanylyl cyclase in the cytosolic domain of the receptor. Among the numerous effects of ANP, its major effects are stimulation of natriuresis and diuresis by the kidney through its hemodynamic and tubular effects. In addition, ANP causes vasodilatation and fluid volume reduction by direct actions on vascular smooth muscle cells, and inhibition of secretion of hormones, such as aldosterone, from adrenal cortex and norepinephrine from peripheral adrenergic neurons. Centrally mediated effects on the regulation of the fluid volume may also be important.

Wnt activity is a key regulator of cardiac progenitor cell self-renewal, differentiation and morphogenesis. However, Wnt inhibitory factor 1 (WIF1), a antagonists of Wnt signaling activity, its potential effects on heart development has not yet been approached by either in vivo or in vitro studies. Here, the expression of WIF1 was regulated in a different way in the dilated and hypertrophic cardiomyopathy heart from transgenic mice by mutations in cardiac troponin T, cTnT(R141W) and cTnT(R92Q). The heart tissue specific transgenic mice of WIF1 was studied using M-mode echocardiography and histologic analyses. Production levels of an array of effectors and transcription factors that impact cellular organization and tissue morphology were measured. The effects of WIF1 on β-catenin pathway could be reversed by LiCl regarding signaling pathways and effector and respondent molecules in H9c2 cells, consistent with the expression levels of c-myc, natriuretic peptide precursor type B and skeletal muscle actin α1. Among the most noteworthy findings were that WIF1 impaired the function and structure of heart, and the effects on β-catenin pathway maybe the course of the former. It is anticipated that our findings will contribute to expansion of our understanding of WIF1 biological function on heart development and possible modes of treatment of heart diseases.

BACKGROUND

Brain natriuretic peptide (BNP) acts primarily as a cardiac hormone; it is produced by the ventricle and has both vasodilatory and natriuretic actions. Therefore, the BNP gene is thought to be a candidate gene for essential hypertension (EH). The present study identified variants in the 5'-flanking region of natriuretic peptide precursor B (NPPB) gene and assessed the relationship between gene variants and EH.

METHODS

The polymerase chain reaction-single strand conformation polymorphism method and nucleotide sequencing were used to identify variants.

RESULTS

A novel variable number of tandem repeat (VNTR) polymorphism in the 5'-flanking region (-1241 nucleotides from the major transcriptional initiation site) was discovered. This VNTR polymorphism is a tandem repeat of the 4-nucleotide sequence TTTC. There were 8 alleles, ranging from 9-repeat to 19-repeat. An association study was done involving 317 EH patients and 262 age-matched normotensive (NT) subjects. The 11-repeat allele was the most frequent (88.2%); the 16-repeat allele was the second most frequent (10.5%) in the NT group. The observed and expected genotypes were in agreement with the predicted Hardy-Weinberg equilibrium values (P=0.972). Among females, the overall distribution of genotypes was significantly different between the EH and NT groups (p=0.039). The frequency of the 16-repeat allele was significantly lower in the female EH group (6.5%) than in the female NT group (12.2%, p=0.046).

CONCLUSIONS

The 16-repeat allele of the VNTR in the 5'-flanking region of NPPB appears to be a useful genetic marker of EH in females.

BACKGROUND

Blood-based biomarkers such as cardiac troponin and B-natriuretic peptides are widely used in clinical practice for the diagnosis, rule out, and risk stratification for patients with acute coronary syndromes and heart failure. Because neither these nor any other laboratory test meets all clinical needs, there are many novel biomarkers that are proposed and evaluated each year for possible implementation into clinical practice. Results of clinical trials are used as a means to validate their effectiveness and to obtain regulatory approval.

RESULTS

Novel biomarkers are discovered through a targeted approach using knowledge of the pathophysiology disease process and an untargeted approach where proteins from tissues or blood of disease patients are compared against healthy subjects or those with benign conditions. Once a candidate biomarker has been identified, it is important to understand where the protein is located and how it is released into blood. In designing trials, the requirements for Food and Drug Administration clearance and approval should be taken into consideration. There are preanalytical studies that should be considered including the preservative used to collect samples and in vivo and in vitro analyte stability. If the analyte is not stable, a surrogate marker could be used such as stable "pro" molecules (precursor proteins) may be preferred. Assay imprecision and bias, biological variation and criteria for the establishment of a reference range are important analytical attributes. The need for harmonization and commutability and correlation of results to other markers and clinical outcomes are important postanalytical attributes of novel biomarkers.

CONCLUSIONS

Inadequate adherence to these variables when conducting clinical trials reduces the quality and value of the information contained in literature reports of novel serum/plasma-based biomarkers.

OBJECTIVE

Corin is a natriuretic peptide-converting enzyme that cleaves precursor pro-B-type natriuretic peptide to active B-type natriuretic peptide (BNP) (diuretic, natriuretic, and vasodilatory properties). Increased plasma BNP is a known diagnostic and prognostic heart failure (HF) biomarker in ambulatory and surgical patients. Recent studies indicate that plasma corin is decreased significantly in chronic HF patients, yet perioperative plasma corin concentrations have not been assessed in cardiac surgical patients. The objectives of this study were to determine the effect of coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass (CPB) on plasma corin concentrations and to assess the association between change in perioperative plasma corin concentration and long-term postoperative HF hospitalization or death. It was hypothesized that plasma corin concentrations decrease significantly from preoperative baseline during postoperative days 1 to 4 and that hospitalization or death from HF during the 5 years after surgery is associated with higher relative difference (preoperative baseline to postoperative nadir) in plasma corin concentrations.

METHODS

Prospective observational pilot study.

METHODS

Two institutions: Brigham and Women's Hospital, Boston, Massachusetts and the Texas Heart Institute, St. Luke's Hospital, Houston, Texas.

METHODS

99 patients of European ancestry who underwent isolated primary CABG surgery with CPB.

METHODS

Nonemergency isolated primary CABG surgery with CPB.

RESULTS

Plasma corin concentration was assessed preoperatively and at 4 postoperative time points (postoperative days 1-4). HF hospitalization or HF death events during the 5 years after surgery were identified by review of hospital and death records. Postoperative plasma corin concentrations were significantly lower than preoperative baseline concentrations (p<0.0001). Perioperative corin concentrations were significantly higher in males than in females (p<0.0001). Fifteen patients experienced long-term postoperative HF events. Patients who experienced HF hospitalization or HF death during study follow-up had significantly higher relative difference in plasma corin concentration (preoperative baseline to postoperative nadir) than patients who did not experience HF events during study follow-up (p=0.03).

CONCLUSIONS

Plasma corin concentrations decrease significantly from preoperative concentrations after CABG surgery. HF hospitalization or HF death during the 5 years after CABG surgery with CPB is associated with larger relative decrease in plasma corin concentration from preoperative baseline. Further investigation is warranted to determine the role of corin in postoperative HF biology.

BACKGROUND

Left ventricular remodeling (LVR) after myocardial infarction is associated with an increased risk of heart failure and death. In spite of a modern therapeutic approach, LVR remains relatively frequent and difficult to predict in clinical practice. Our aim was to identify new biomarkers of LVR and understand their involvement in its development.

RESULTS

Proteomic analysis of plasma from the REVE-2 study (Remodelage Ventriculaire)-a study dedicated to the analysis of LVR which included 246 patients after a first anterior myocardial infarction-identified increased plasma levels of CLU (clusterin) in patients with high LVR. We used a rat model of myocardial infarction to analyze CLU expression in the LV and found a significant increase that was correlated with LVR parameters. We found increased CLU expression and secretion in primary cultures of rat neonate cardiomyocytes hypertrophied by isoproterenol. Silencing of CLU in hypertrophied neonate cardiomyocytes induced a significant decrease in cell size, ANP (atrial natriuretic peptide), and BNP (B-type natriuretic peptide) expression, associated with a decreased ERK (extracellular signal-regulated kinase) 1/2 activity, suggesting a prohypertrophic role of CLU. We then confirmed a significant increase of both intracellular p-CLU (precursor form of CLU) and m-CLU (mature form of CLU) in failing human hearts. Finally, the circulating levels of CLU (secreted form) were increased in patients with chronic heart failure who died from cardiovascular cause during a 3-year follow-up (n=99) compared with survivors (n=99).

CONCLUSIONS

Our results show for the first time that plasma CLU levels are associated with LVR post-myocardial infarction, have in part a cardiac origin, and are a predictor of early death in heart failure patients.

N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) has emerged as an important adjunct in the management of heart failure (HF) and other cardiovascular diseases. NT-proBNP is a 76-amino acid peptide created during cleavage of the precursor molecule, Pro B-type natriuretic peptide (ProBNP). NT-proBNP is of significant diagnostic value in patients presenting with possible HF and is an important prognostic factor in this condition and other cardiovascular diseases. Ongoing research supports the potential value of this biomarker in non-cardiovascular disease. This review will describe clinical applications of NT-proBNP in HF and a broad range of other conditions.

OBJECTIVE

To examine the neurohormonal effects of a scuba dive, focusing on the acute changes in the plasma concentrations of the different peptide fragments from the B-type natriuretic peptide (BNP) precursor.

METHODS

We studied 12 healthy scuba divers (mean age ± standard deviation: 44 ± 7 years; range: 34-55 years; BMI: 24.8 ± 2.8 kg/m(2)), who performed a 15-m depth dive in salt water, with a bottom time of 30 min. Blood samples for BNPs (pro-B-type natriuretic peptide [pro-BNP], BNP and aminoterminal pro-BNP) and catecholamines were measured in plasma before immersion, and after the dive. A continuous electrocardiographic recording was obtained during the entire protocol.

RESULTS

BNP, aminoterminal pro-BNP and pro-BNP plasma concentrations slightly, but significantly, increased after the scuba dive (18 ± 15 to 21 ± 11 ng/l, p = 0.020; 32 ± 19 to 38 ± 21 ng/l, p = 0.008; and 7.8 ± 1.6 to 10.3 ± 3.6 ng/l, p = 0.028, respectively) in parallel with norepinephrine concentration (743 ± 323 to 1163 ± 656 ng/l, p = 0.014), with no variations in total plasma proteins, hematocrit or osmolality. A persistent sinus tachycardia was observed during all phases of the dive.

CONCLUSIONS

A 15-m depth scuba dive induces an acute slight release of the different peptide fragments from the BNP precursor, likely through the stimulation of a constitutive secretory pathway promoted by adrenergic activation and cardiac chamber dilation.

BACKGROUND

Plasma measurement of cardiac natriuretic peptides and their biosynthetic precursors is helpful in chronic heart failure patients. In contrast, information on circulating B-type natriuretic peptide (BNP) and its molecular precursor (proBNP) in patients with cardiogenic shock is scarce. We therefore examined proBNP-derived peptides in plasma from patients with myocardial infarction complicated by cardiogenic shock.

RESULTS

Patients were referred for early, invasive therapy because of myocardial infarction complicated by cardiogenic shock (n = 13). Plasma proBNP was measured with an automated assay (NT-proBNP) and an in-house radioimmunoassay (proBNP); BNP concentrations were quantitated with an immunoradiometric assay. The median NT-proBNP concentration was 8.2-fold higher than the corresponding BNP concentration (873 pmol/L [range 41-12,486] versus 107 pmol/L [1-1041], P < .001). Moreover, the NT-proBNP concentration was 3.3-fold higher compared with proBNP (268 pmol/L [19-12,220], P < .01). Despite the molar differences, there was a strong correlation between NT-proBNP and proBNP (r = 0.84, P < .0001) and BNP (r = 0.82, P < .0001) concentrations. Gel filtration chromatography suggested that the proBNP immunoreactivity reflect a molecular form larger than the N-terminal 1-76 fragment.

CONCLUSIONS

The study reveals the plasma profile of proBNP-derived peptides during myocardial infarction complicated by cardiogenic shock. Peripheral concentrations of NT-proBNP, proBNP, and BNP were highly correlated despite marked differences between assays. The results also suggest an increase in cardiac proBNP processing after myocardial infarction and cardiogenic shock.

BACKGROUND

ProBNP, the precursor peptide to BNP and NT-proBNP (NP), circulates in patients with chronic heart failure (HF) and appears to be the predominant form of NP. This heterogeneity may confound interpretation of NP concentrations. The aim of this study was to evaluate the response to therapy and prognostic influence of proBNP in a cohort of patients admitted to hospital for decompensated HF.

METHODS

We prospectively evaluated 40 Class III-IV patients who received clinically-indicated nesiritide infusions as part of their care. Blood was drawn before, during, and post-infusion, and assayed for proBNP, BNP, and NT-proBNP.

RESULTS

All biomarkers were increased at baseline consistent with HF. ProBNP and NT-proBNP demonstrated significant reductions in response to therapy (42% and 18% post-infusion, respectively). In the patients who experienced post-hospital mortality (40% at 6 months), baseline proBNP and BNP concentrations were significantly lower than in survivors. This paradoxical finding may be explained by the end-stage nature of this patient cohort possibly experiencing exhaustion of their NP systems.

CONCLUSIONS

Circulating concentrations of proBNP are increased in decompensated HF and similar to NT-proBNP are reduced in response to acute therapy. Paradoxically and similar to BNP, baseline proBNP concentrations were lower in post-hospital non-survivors. While hypothesis generating, the results of this study support a role for proBNP in monitoring therapy and predicting short-term outcome. These findings need to be confirmed in a patient cohort without nesiritide therapy and more moderate HF.

BACKGROUND

Protease neprilysin is known to be responsible for the degradation of natriuretic peptides. A recent heart failure (HF) drug, LCZ696 (Entresto(TM)), that combines a neprilysin inhibitor and an angiotensin II receptor inhibitor was suggested to augment circulating B-type natriuretic peptide (BNP) concentrations, making the results of BNP measurements diagnostically ambiguous. Because the main form of measured BNP in HF patients is represented by its uncleaved precursor, proBNP, it is important to know the susceptibility of proBNP to cleavage by neprilysin.

METHODS

BNP 1-32 and nonglycosylated and glycosylated forms of proBNP 1-108 were incubated with neprilysin for different time periods. BNP immunoreactivity was analyzed using 2 sandwich immunoassays: one utilizing monoclonal antibody (mAb) KY-BNP-II (epitope 14-21) as capture with mAb 50E1 (epitope 26-32) for detection and a single-epitope sandwich BNP (SES-BNP) immunoassay specific to the epitope 11-17. Mass-spectrometry was applied to determine the sites of BNP cleavage.

RESULTS

In contrast to BNP, both forms of proBNP were resistant to degradation by neprilysin. The SES-BNP assay was much less susceptible to the BNP cleavage by neprilysin compared with the immunoassay utilizing antibodies specific to the region 14-21, comprising the site Arg17-Ile18, known as the site of BNP cleavage by neprilysin.

CONCLUSIONS

These findings suggest that modulation of neprilysin activity by specific inhibitors may not greatly influence the circulating concentrations of immunoreactive BNP, mostly represented in HF by proBNP, which is not susceptible to neprilysin. The different susceptibility of the BNP regions to neprilysin-dependent degradation highlights the importance of the choice of epitopes for reliable BNP immunodetection.

BACKGROUND

In a large collaborative study (n>> 50,000), common variants in the natriuretic peptide (NP) genes were found to be associated with circulating NP levels and also with blood pressure (BP) levels based on office BP measurements (OBPMs). It is unknown if determining an individual's BP by 24-h ambulatory BP measurements (ABPMs) will influence the effect of NP gene variations on BP levels.

METHODS

We used rs632793 at the NPPB (NP precursor B) locus to investigate the relationship between genetically determined serum N-terminal pro-brain NP (NT-proBNP) concentrations and BP levels determined by both 24-h ABPMs and OBPMs in a population consisting of 1,397 generally healthy individuals taking no BP-lowering drugs.

RESULTS

rs632793 was significantly correlated with serum Nt-proBNP levels (r = 0.10, P = 0.0003), and participants with the A:A genotype had lower serum Nt-proBNP levels than participants with the G:G genotype (geometric mean (95% confidence interval (CI)): 34.8 (31.5-38.4) pg/ml vs. 48.1 (41.9-55.3) pg/ml, P = 0.0002), but higher 24-h ambulatory BP levels (mean difference (95% CI): 2.0 (0.1-4.1) mm Hg, P = 0.043, for systolic BP and 1.7 (0.4-3.1) mm Hg, P = 0.011, for diastolic BP). Office BP decreased across the genotypes from A:A to G:G, but the differences did not reach statistical significance (P ≥ 0.12).

CONCLUSIONS

This study suggests that 24-h ABPMs is a better method than OBPMs to detect significant differences in BP levels related to genetic variance and provides further evidence that the NP system plays an important role in BP regulation.

Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by approximately 11 wk, cardiac dysfunction at approximately 17 wk, and death from cardiac dysfunction at approximately 21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1beta (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, beta(1)-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.

Using the "Bi-Digital O-Ring Test Imaging Technique", the author has been able to accurately localize meridians and acupuncture points that correspond to specific internal organs and has found that most general patterns of meridians and the number of acupuncture points on each of the meridians of specific internal organs of the 12 main internal organs described in the literature of ancient Chinese medicine, are more or less correct, with the exception of some variations and inaccuracies. Each meridian of specific internal organs was found to be connected to the organ representation area in the cerebral cortex of specific internal organs. The acupuncture point has an area and occupies 3-dimensional space. It has a circular or slightly oval boundary with diameter in the range of 3 mm to 2.7 cm, although 6-12 mm are the most common diameters in human adults, with the exception of the area outside the corners of the nailbeds of the fingers and toes. Using the "Bi-Digital O-Ring Test Molecular Identification Method", the author also found that within the boundary of most acupuncture points and meridian lines (including Heart, Stomach, and Triple Burner) were high concentrations of neurotransmitters and hormones, including Acetylcholine, Methionine-Enkephalin, Beta-Endorphin, ACTH, Secretin, Cholecystokinin, Norepinephrine, Serotonin, and GABA. On all these meridian lines, in addition to the above neurotransmitters and hormones, Dopamine, Dynorphin 1-13, Prostaglandin E1 (PGE1) and VIP were found, but the latter do not usually exist within the boundary of the acupuncture point with the exception of the center midline of the acupuncture point where the meridian line is situated. Serotonin, Norepinephrine, and Cholecystokinin appeared in either one of the above 2 patterns, depending on the individual. Usually, no significant amounts of these neurotransmitters and hormones were found at the surrounding area outside of meridian and acupuncture points. However, the essential amino acid L-Tryptophan (which is a precursor of Serotonin), was usually found outside of the boundary of the acupuncture point and the meridian but not within the boundary of the acupuncture point and the meridian. Wherever Serotonin appeared, L-Tryptophan disappeared significantly and when the Serotonin disappeared, L-Tryptophan reappeared. In addition to the above common neurotransmitters and hormones, the Heart meridian had additional Atrial Natriuretic Peptide in both the meridian and its acupuncture points. Similarly, the Stomach meridian had additional Gastrin in both the meridian and its acupuncture points. Likewise,the Triple Burner meridian had additional Testosterone (in the male) and Estrogen (especially Estriol and Estradiol in the female.

A novel fluorogenic peptide, dansyl-Gly-(p-NO2) Phe-beta Ala (DGNPA), was synthesized as a selective substrate for neutral endopeptidase 24.11, an enzyme involved in enkephalin and atrial natriuretic peptide degradation and a marker of differentiation (CD10) on the surface of lymphohematopoietic cells. Cleavage of the substrate Gly-(p-NO2)Phe amide bond leads to an increase in fluorescence related to the disappearance of the intramolecular quenching of the dansyl fluorescence by the nitrophenyl residue. This new fluorogenic substrate is an improvement over the commercially available dansyl-D-Ala-Gly-(p-NO2)Phe-Gly, as the Gly4 residue of the latter has been replaced by a beta-alanine, therefore eliminating a residual sensitivity of the peptide toward angiotensin converting enzyme. Moreover, deletion of the D-Ala2 residue was shown to increase the quenching efficiency, thus raising the sensitivity of the assay, which was further improved by stopping the reaction with dioxane. The present substrate has improved affinity (Km = 37 microM, V = 0.72 mumol min-1 mg protein-1), selectivity, and sensitivity over its precursor and was used in automated assays using 96-well microplates and a fluorescence plate reader.

A cDNA clone, 1.8 kb long, was isolated from a venom gland cDNA library of Agkistrodon blomhoffi that encodes a large plurifunctional precursor composed of 263 amino-acid residues. Nucleotide sequence analysis of this clone revealed that sequences which code for blomhotin and a novel peptide Leu3-blomhotin are located in the N-terminal region of the precursor polypeptide, followed by four tandemly aligned sequences which code for three types of bradykinin-potentiating peptide. In the C-terminal region, the sequence for the C-type natriuretic peptide was located along with a preceding processing signal. The deduced amino-acid sequences for the four bradykinin-potentiating peptides coincided exactly with previously known sequences for potentiator B, potentiator C and potentiator E. The actual Leu3-blomhotin peptide was subsequently isolated from the venom of A. blomhoffi and characterized. Leu3-blomhotin possesses contractile activity in isolated rat stomach fundus smooth muscle in the same manner as blomhotin. Furthermore, it was shown that blomhotin and Leu3-blomhotin retained activity to inhibit the angiotensin-converting enzyme.