Plasma levels of soluble VEGF receptor isoforms, circulating pterins and VEGF system SNPs as prognostic biomarkers in patients with acute coronary syndromes.

BACKGROUND: Development of collateral circulation in coronary artery disease is cardio-protective. A key process in forming new blood vessels is attraction to occluded arteries of monocytes with their subsequent activation as macrophages. In patients from a prospectively recruited post-acute coronary syndromes cohort we investigated the prognostic performance of three products of activated macrophages, soluble vascular endothelial growth factor (VEGF) receptors (sFlt-1 and sKDR) and pterins, alongside genetic variants in VEGF receptor genes, VEGFR-1 and VEGFR-2. METHODS: Baseline levels of sFlt-1 (VEGFR1), sKDR (VEGFR2) and pterins were measured in plasma samples from subgroups (n = 513; 211; 144, respectively) of the Coronary Disease Cohort Study (CDCS, n = 2067). DNA samples from the cohort were genotyped for polymorphisms from the VEGFR-1 gene SNPs (rs748252 n = 2027, rs9513070 n = 2048) and VEGFR-2 gene SNPs (rs2071559 n = 2050, rs2305948 n = 2066, rs1870377 n = 2042). RESULTS: At baseline, levels of sFlt-1 were significantly correlated with age, alcohol consumption, NTproBNP, BNP and other covariates relevant to cardiovascular pathophysiology. Total neopterin levels were associated with alcohol consumption at baseline. 7,8 dihydroneopterin was associated with BMI. The A allele of VEGFR-2 variant rs1870377 was associated with higher plasma sFlt-1 and lower levels of sKDR at baseline. Baseline plasma sFlt-1 was univariately associated with all cause mortality with (p < 0.001) and in a Cox's proportional hazards regression model sFlt-1 and pterins were both associated with mortality independent of established predictors (p < 0.027). CONCLUSIONS: sFlt-1 and pterins may have potential as prognostic biomarkers in acute coronary syndromes patients. Genetic markers from VEGF system genes warrant further investigation as markers of levels of VEGF system components in these patients. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry. ACTRN12605000431628 . 16 September 2005, Retrospectively registered.

Development of a BNP1-32 Immunoassay That Does Not Cross-React with proBNP.

BACKGROUND: Plasma B-type natriuretic peptide (BNP) concentration reflects cardiac dysfunction and assists in determining the diagnosis and prognosis of heart failure (HF). Current BNP assays overestimate circulating bioactive BNP1-32 concentrations as they also detect less active BNP metabolites and proBNP. A specific BNP1-32 assay with negligible cross-reactivity to proBNP and/or BNP metabolites may be advantageous. METHODS: We developed a Luminex-based specific BNP1-32 immunoassay and compared results obtained from 3 other BNP assays (a Luminex-based total-BNP assay, our BNP RIA, and the commercially available Abbott Architect BNP assay) in plasma from 42 patients with HF and 22 healthy controls. RESULTS: The BNP1-32 assay showed 57% cross-reactivity with BNP2-32, but ≤0.1% cross-reactivity to BNP3-32, other BNP metabolites, and proBNP; its detection limit was 0.35 ng/L; and intra- and interassay CVs were <15%. BNP immunoreactivity increased with HF severity (median concentrations being 0.3, 0.8, 26.2, and 17.3 ng/L in healthy controls and 40.7, 139, 465, and 1778 ng/L in HF patients for the BNP1-32, total-BNP, BNP RIA, and Abbott BNP assays respectively). The fold increase between HF cases with the New York Heart Association (NYHA) class IV was significantly greater with the BNP1-32 assay than the Abbott BNP (P = 0.026) and the BNP RIA (P < 0.0001) but not the total-BNP assay. CONCLUSIONS: We have developed the first assay that measures BNP1-32 in plasma without interference by proBNP. Analysis of larger patient cohorts is now required to compare the performance of this assay with current less specific assays for the diagnosis or prognosis of HF.

(Pro)renin Receptor Blockade Ameliorates Cardiac Injury and Remodeling and Improves Function After Myocardial Infarction.

BACKGROUND: The (pro)renin receptor [(P)RR] is implicated in the pathogenesis of cardiovascular disease. We investigated the effects of (P)RR blockade after myocardial infarction (MI) in a mouse coronary-ligation model. METHODS AND RESULTS: Mice underwent sham control surgeries (n = 8) or induction of MI followed by 28 days' treatment with a vehicle control (n = 8) or (P)RR antagonist (n = 8). Compared with sham control subjects, MI + vehicle mice demonstrated reduced left ventricular (LV) ejection fraction (LVEF: P < .001) and fractional shortening (P < .001), and increased LV end-systolic and -diastolic volumes (LVESV: P < .001; LVEDV: P < .001) 28 days after MI. In addition, MI decreased LV posterior wall and septal diameters (both P < .001), increased heart weight-body weight ratios (P < .05), LV collagen deposition, and cardiomyocyte diameter (both P < .001), and up-regulated collagen 1 (P < .01) and ß-myosin heavy chain (ß-MHC: P < .05) mRNA. Compared with MI + vehicle mice, (P)RR antagonism after MI reduced infarct size (P < .01), improved LVEF (P < .001), fractional shortening (P < .001), and stroke volume (P < .05), and decreased LVESV (P < .001) and LVEDV (P < .001). (P)RR antagonism also reversed MI-induced transmural thinning (P < .001) and reduced LV fibrosis (P < .01), cardiomyocyte size (P < .001), and ventricular collagen 1 (P < .01), ß-MHC (P = .06), transforming growth factor ß1 (P < .01), and angiotensin-converting enzyme (P < .05) expression. CONCLUSIONS: The present study found that (P)RR blockade after MI in mice ameliorates infarct size, cardiac fibrosis/hypertrophy, and cardiac dysfunction and identifies the receptor as a potential therapeutic target in this setting.

First identification of circulating prepro-A-type natriuretic peptide (preproANP) signal peptide fragments in humans: initial assessment as cardiovascular biomarkers.

BACKGROUND: New biomarkers are needed to assist clinical decision making in cardiovascular disease. We have recently shown that signal peptides may represent a novel biomarker target in cardiovascular diseases. METHODS: We developed a novel immunoassay for the signal peptide of preproANP (ANPsp) and used it to document cardiac tissue levels of ANPsp in explant human hearts (n = 9), circulating venous concentrations of ANPsp in healthy volunteers (n = 65), temporal ANPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4 h after chest pain onset (n = 23), and regional plasma ANPsp concentrations in patients undergoing clinically indicated catheterization (n = 10). We analyzed the structure and sequence of circulating ANPsp by tandem mass spectrometry (MS/MS). RESULTS: ANPsp levels in human heart tissue were 50-1000 times lower than those of ANP/NT-proANP. ANPsp was detectable in control human plasma at concentrations comparable with ANP itself (approximately 20 ng/L). In STEMI patients, plasma concentrations of ANPsp rose to peak values at 5 h after symptom onset, significantly earlier than myoglobin, creatine kinase-MB, and troponin (P < 0.001). There were significant arteriovenous increases in ANPsp concentrations (P < 0.05) across the heart and kidney; arterial and coronary sinus concentrations of ANPsp both negatively correlated with systolic and mean arterial blood pressures (both P < 0.01). MS/MS verified circulating ANPsp to be preproANP(16-25) and preproANP(18-25). CONCLUSIONS: ANPsp is a novel circulating natriuretic peptide with potential to act as a cardiovascular biomarker. The rapid increase of plasma ANPsp in STEMI and its significant relationship with blood pressure encourage further study of its potential clinical utility.

KCNE5 polymorphism rs697829 is associated with QT interval and survival in acute coronary syndromes patients.

INTRODUCTION: The KCNE family is a group of small transmembrane channel proteins involved in potassium ion (K(+)) conductance. The X-linked KCNE5 gene encodes a regulator of the K(+) current mediated by the potassium channel KCNQ1. Polymorphisms in KCNE5 have been associated with altered cardiac electrophysiological properties in human studies. We investigated associations of the common rs697829 polymorphism from KCNE5 with baseline characteristics, baseline electrocardiographic (ECG) measurements, and patient survival in a cohort of post-acute coronary syndromes (ACS) patients (the Coronary Disease Cohort Study cohort). METHODS AND RESULTS: DNA samples (n = 1,740) were genotyped for rs697829 using a TaqMan assay. Baseline ECG data revealed corrected QT (QTc) interval was associated with rs697829 in male, but not female, patients, being extended in the G genotype group (A 416 ± 1.71; G 431 ± 4.25 ms, P = 0.002). Covariate-adjusted survival was poorest in G genotype patients in Cox proportional hazard modeling of mortality data of males (P(overall) = 0.020). Male patients with G genotype had a hazard ratio of 1.44 (1.11-2.33) for death when compared to the A genotype male patients (P = 0.048) after adjustment for age, baseline log-transformed N-terminal pro-B-type natriuretic peptide (NTproBNP), ß-blocker and insulin treatment, QTc interval, history of myocardial infarction, and physical activity score. CONCLUSION: This study suggests an association between rs697829, a common single nucleotide polymorphism (SNP) from KCNE5, and ECG measurements and survival in postacute ACS patients. Prolonged subclinical QT interval may be a marker of adverse outcome in this group of patients.

Association of genetic variation in the natriuretic peptide system with cardiovascular outcomes.

Polymorphisms within individual natriuretic peptide genes have been associated with risk factors for cardiovascular disease, but their association with clinical outcomes was previously unknown. This study aimed to investigate the association between genetic variants in key genes of the natriuretic peptide system with cardiovascular outcomes in patients with coronary artery disease. Coronary disease patients (n=1810) were genotyped for polymorphisms within NPPA, NPPB, NPPC, NPR1 and NPR2. Clinical history, natriuretic peptide concentrations, echocardiography, all-cause mortality and cardiovascular hospital readmissions were recorded over a median 2.8 years. Minor alleles of NPPA rs5068, rs5065 and rs198358 were associated with less history of hypertension; minor alleles of NPPA rs5068 and rs198358 was also associated with higher circulating natriuretic peptide levels (p=0.003 to p=0.04). Minor alleles of NPPB rs198388, rs198389, and rs632793 were associated with higher circulating BNP and NT-proBNP (p=0.001 to p=0.03), and reduced E/E(1) (p=0.011), or LVESVI (p=0.001) and LVEDVI (p=0.004). Within NPPC, both rs11079028 and rs479651 were associated with higher NT-proBNP and CNP (p=0.01 to p=0.03), and rs479651 was associated with lower LVESVI (p=0.008) and LVEDVI (p=0.018). NPR2 rs10758325 was associated with smaller LVMI (p<0.02). A reduced rate of cardiovascular readmission was observed for minor alleles of NPPA rs5065 (p<0.0001), NPPB rs632793 (p<0.0001), rs198388 (p<0.0001), rs198389 (p<0.0001), and NPR2 rs10758325 (p<0.0001). There were no associations with all-cause mortality. In established cardiovascular disease, natriuretic peptide system polymorphisms were associated with natriuretic peptide levels, hypertension, echocardiographic indices and the incidence of hospital readmission for cardiovascular events.

B-type natriuretic peptide signal peptide circulates in human blood: evaluation as a potential biomarker of cardiac ischemia.

BACKGROUND: The diagnosis of cardiac necrosis such as myocardial infarction can be difficult and relies on the use of circulating protein markers like troponin. However, there is a clear need to identify circulating, specific biomarkers that can detect cardiac ischemia without necrosis. METHODS AND RESULTS: Using specific immunoassay and tandem mass spectrometry, we show that a fragment derived from the signal peptide of B-type natriuretic peptide (BNPsp) not only is detectable in cytosolic extracts of explant human heart tissue but also is secreted from the heart into the circulation of healthy individuals. Furthermore, plasma levels of BNPsp in patients with documented acute ST-elevation myocardial infarction (n=25) rise to peak values ( approximately 3 times higher than the 99th percentile of the normal range) significantly earlier than the currently used biomarkers myoglobin, creatine kinase-MB, and troponin. Preliminary receiver-operating characteristic curve analysis comparing BNPsp concentrations in ST-elevation myocardial infarction patients and other patient groups was positive (area under the curve=0.97; P<0.001), suggesting that further, more rigorous studies in heterogeneous chest pain patient cohorts are warranted. CONCLUSIONS: Our results demonstrate for the first time that BNPsp exists as a distinct entity in the human circulation and could serve as a new class of circulating biomarker with the potential to accelerate the clinical diagnosis of cardiac ischemia and myocardial infarction. Clinical Trial Registration- URL: http://www.anzctr.org.au. Unique identifier: ACTRN12609000040268.

CYP1A1 MSPI (T6235C) gene polymorphism is associated with mortality in acute coronary syndrome patients.

1. The CYP1A1 T6235C polymorphism (rs4646903) gene polymorphism has been linked to the development of coronary heart disease and cigarette smoking-related lung cancer. The present study investigated associations between survival in acute coronary syndromes (ACS), smoking and the CYP1A1 T6235C polymorphism. 2. Patients with ACS (n = 1251) were genotyped for the CYP1A1 T6235C polymorphism. Patients had a mean age of 67.0 years, 69.8% were male and follow up occurred over a median of 1.9 years. 3. Overall genotype frequencies were CC 2.2%, TC 21.7% and TT 76.1%. The CC genotype was associated with baseline characteristics of a higher incidence of Type 2 diabetes (P = 0.017), elevated body mass index (P = 0.001) and younger age (P = 0.045). Patients with the CC genotype had significantly worse survival than TT/TC patients (P = 0.014), independent of ethnicity and established clinical risk factors. When survival was stratified by smoking history, the T6235C genotype was particularly associated with mortality in past or current smokers (mortality 23.5 vs 9.4% in CC and TT/TC patients, respectively; P = 0.019) compared with those who had never smoked (mortality 11.1 vs 11.5% in CC and TT/TC patients, respectively; P = 0.853). 4. The results indicate that the homozygous CYP1A1 6235C genotype is associated with greater mortality following the onset of ACS, independent of ethnicity and clinical risk factors, but related to smoking history.

Ouabain, a circulating hormone secreted by the adrenals, is pivotal in cardiovascular disease. Fact or fantasy?

Substantial evidence points to the presence in human plasma of an inhibitor of the sodium/potassium pump which plays a central role in the pathophysiology of circulatory disorders, including essential hypertension. Studies from the 1980/90s claimed that this inhibitor was identical or very similar in structure to plant-derived ouabain and was synthesized by the adrenal cortex. However, the physical evidence in studies reporting isolation and identification of ouabain in human plasma appears insecure on closer examination. Additionally, reported circulating levels of immunoreactive ouabain in humans vary greatly, the ability of the human adrenal glands to secrete ouabain is questionable and the original commercial assay for measuring immunoreactive ouabain is no longer available. We submit that the position of ouabain as an endogenous, adrenally produced regulator of the sodium pump is of such importance that the current evidence needs either to put on a more secure footing or to lose its current status.

The common G-866A polymorphism of the UCP2 gene and survival in diabetic patients following myocardial infarction.

BACKGROUND: A variant in the promoter of the human uncoupling protein 2 (UCP2) gene, the G-866A polymorphism, has been associated with future risk of coronary heart disease events, in those devoid of traditional risk factors and in those suffering from diabetes. We thus examined the impact of the G-866A polymorphism on 5-year survival in a cohort of 901 post-myocardial infarction patients, and the impact of type-2 diabetes on this relationship. The association of UCP2 with baseline biochemical and hormonal measurements, including levels of the inflammatory marker myeloperoxidase, was also examined. METHODS: UCP2 G-866A genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol. Myeloperoxidase levels were measured in plasma samples taken from 419 cohort patients 24-96 hours after admission. RESULTS: Genotypes were obtained for 901 patients with genotype frequencies AA 15.5%, GA 45.5%, and GG 39.0%. Genotype was not associated with survival in the overall cohort (mortality: AA 15.6%, GA 16.8%, GG 19.4%, p = 0.541). However, amongst diabetics, AA and GA genotype groups had significantly worse survival than GG diabetic patients (p < 0.05) with an attributable risk of 23.3% and 18.7% for those of AA and GA genotype respectively. Multivariate analysis using a Cox proportional hazards model confirmed that the interaction of diabetes with genotype was significantly predictive of survival (p = 0.031). In the cohort's diabetic subgroup AA/GA patients had higher myeloperoxidase levels than their GG counterparts (GA/AA, n = 51, 63.9 +/- 5.23; GG, n = 34, 49.1 +/- 3.72 ng/ml, p = 0.041). Further analysis showed that this phenomenon was confined to male patients (GA/AA, n = 36, 64.3 +/- 6.23; GG, n = 29, 44.9 +/- 3.72 ng/ml, p = 0.015). CONCLUSION: Diabetic patients in this post-myocardial infarction cohort with UCP2 -866 AA/GA genotype have poorer survival and higher myeloperoxidase levels than their GG counterparts.

Plasma aldosterone levels during hospitalization are predictive of survival post-myocardial infarction.

AIMS: Plasma aldosterone levels have been shown to be associated with adverse clinical outcomes after ST-elevation myocardial infarction (STEMI). We investigated whether aldosterone levels in patients presenting with STEMI or non-STEMI, are predictive of mortality during prolonged follow-up. METHODS AND RESULTS: Aldosterone levels were assayed in plasma taken from 583 patients within 24-96 h following acute myocardial infarction (MI). The median plasma aldosterone level was 108 pmol/L and all values were below the upper limit of the normal range (800 pmol/L) except for five patients (0.9%). Aldosterone tertile was significantly associated with increasing plasma levels of NTproBNP (N-terminal pro-B-type natriuretic peptide), BNP (B-type natriuretic peptide), epinephrine, and endothelin-1 (P or=25.3% mortality, P >or= 0.026). CONCLUSION: Plasma aldosterone levels post-MI are independent predictors of survival and hospitalization for heart failure over a 5-year-follow-up period.

Angiotensin-converting enzyme 2 A1075G polymorphism is associated with survival in an acute coronary syndromes cohort.

BACKGROUND: Polymorphisms of the angiotensin-converting enzyme 2 (ACE2) gene, which is located on the X chromosome, have been associated with hypertension and left ventricular hypertrophy in previous studies. We tested the hypothesis that the rare allele of an ACE2 gene polymorphism was associated with risk factors for and adverse outcome after acute coronary syndrome (ACS) events. METHODS: Patients (n = 1,042) were recruited after admission for an ACS event and were genotyped for the A1075G polymorphism of the angiotensin-converting enzyme 2 gene. This genetic marker was tested for association with baseline measurements, echocardiographic measurements, and clinical outcome, over a median 2.19 years follow-up. As the ACE2 gene is X-linked, analyses were performed separately for males and females. Patients were predominantly of European ethnicity (90.1%). RESULTS: The A1075 allele was significantly associated with covariate-adjusted mortality in male patients (hazard ratio 1.95, 95% CI 1.10-3.46, P = .047) but not unadjusted (hazard ratio 1.14, 95% CI 0.736-1.76, P = .56). The G1075 (P < .035) allele was more frequent in patients of Maori compared to European ancestry. E/E', an echocardiographic index of left ventricular diastolic function and filling pressure, was higher in males in the A1075 group (G allele group 10.5 [95% CI 10.0-11.0], A allele group 11.4 [95% CI 10.8-12.1], P = .024). A1075 genotype was significantly associated with male survival in the absence of (mortality: A 12.8%, n = 39; G 29.2%, n = 48; P = .037) but not in the presence of beta-blocker treatment (mortality: A 13.5% n = 273; G 8.2% n = 304, P = nonsignificant). CONCLUSIONS: The A1075 allele was associated with covariate-adjusted mortality in male patients.

An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency.

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.

Characterisation of proghrelin peptides in mammalian tissue and plasma.

Ghrelin is a 28 amino acid stomach peptide, derived from proghrelin(1-94), that stimulates GH release, appetite and adipose deposition. Recently, a peptide derived from proghrelin(53-75) -- also known as obestatin -- has been reported to be a physiological antagonist of ghrelin in the rat. Using four specific RIAs, we provide the first characterisation of proghrelin(1-94) peptides in human plasma, their modulation by metabolic manipulation and their distribution in mammalian tissues. ghrelin(1-28) immunoreactivity (IR) in human plasma and rat plasma/stomach consisted of major des-octanoyl and minor octanoylated forms, as determined by HPLC/RIA. Human plasma ghrelin(1-28) IR was significantly suppressed by food intake, oral glucose and 1 mg s.c. glucagon administration. ghrelin(1-28) IR and proghrelin(29-94) IR peptide distributions in the rat indicated that the stomach and gastrointestinal tract contain the highest amounts of the peptides. Human and rat plasma and rat stomach extracts contained a major IR peak of proghrelin(29-94)-like peptide as determined by HPLC/RIA, whereas no obestatin IR was observed. Human plasma proghrelin(29-94)-like IR positively correlated with ghrelin(1-28) IR, was significantly suppressed by food intake and oral glucose and shared with ghrelin(1-28) IR a negative correlation with body mass index. We found no evidence for the existence of obestatin as a unique, endogenous peptide. Rather, our data suggest that circulating and stored peptides derived from the carboxyl terminal of proghrelin (C-ghrelin) are consistent in length with proghrelin(29-94) and respond to metabolic manipulation, at least in man, in similar fashion to ghrelin(1-28).

Evaluation of AMPD1 C34T genotype as a predictor of mortality in heart failure and post-myocardial infarction patients.

BACKGROUND: The AMPD1 gene C34T polymorphism has previously been associated with prolonged survival in small cohorts of heart failure (HF) and coronary artery disease patients. This study aimed to corroborate the association of the AMPD1 C34T polymorphism with survival in larger myocardial infarction (MI) and HF cohorts. METHODS: Genotypes were obtained for 935 post-MI (PMI) and 433 patients with established HF, with median follow-up times of 5.4 and 3.1 years, respectively. At admission, cardiac function was assessed by nuclear ventriculography (PMI) and echocardiography (HF) and plasma cardiac neurohormones were assayed. RESULTS: Differences in mortality by AMPD1 genotype did not achieve significance, either for the overall HF (P = .07) or the overall PMI group (P = .28), but AMPD1 genotype predicted mortality in patients of both cohorts with a history of MI (HxMI). In contrast to previous studies, the mutant T allele was associated with poorer outcome. Mortality in HF HxMI patients was significantly different between genotype groups (n = 144, mortality CC 56.5%, CT/TT 77.8%, P = .027), but not in patients without HxMI. In PMI patients, the association of genotype with survival in the HxMI subgroup trended toward significance (n = 147, mortality CC 29.8%, CT/TT 45.5%, P = .093). Multivariate analysis of combined PMI and HF cohorts showed that HxMI patients with CT/TT genotype were at greater risk than all other groups (P < .001). CONCLUSION: This study suggests that AMPD1 C34T genotype is not a predictor of survival in heart disease patients, except possibly those with HxMI.

Plasma cardiotrophin-1 is elevated in human hypertension and stimulated by ventricular stretch.

OBJECTIVE: Cardiotrophin-1 (CT-1) is an interleukin-6-related cytokine with known hypertrophic and protective actions upon cardiac myocytes. We provide here the first report of cardiac tissue and plasma levels of CT-1 in human and experimental hypertension, demonstrate cardiac CT-1 secretion stimulated by ventricular stretch, and characterise molecular forms of CT-1 in tissue and plasma. METHODS: CT-1 levels in human and rat plasma and in rat cardiac tissue extracts were determined by specific radioimmunoassay (RIA). Cardiac CT-1 secretion during ventricular stretch was studied in isolated, perfused hearts. Molecular forms of CT-1 were identified using RIA coupled with high performance liquid chromatography (HPLC). Results are given as mean+/-SEM. RESULTS: Plasma levels of CT-1 in patients with untreated hypertension (UTH, 606+/-18 pmol/L, n=24) were significantly higher than those in age-and BMI-matched normotensive volunteers (NT, 546+/-12 pmol/L, n=31, P<0.01 vs. UTH). CT-1 levels in matched patients with treated hypertension (THT, 618+/-10 pmol/L, n=35) were similar to those in UTH patients, but higher than in NT controls (P<0.01). Plasma CT-1 demonstrated a weak but significant correlation with systolic blood pressure in all patients (r=0.241, P<0.05, n=90). In contrast, CT-1 levels in male, 40-week-old, NT-WKY rats (1295+/-98 pmol/L) were significantly higher than those in matched UTH-SHR (937+/-31 pmol/L, P<0.01). In both WKY and SHR rats, atrial tissue concentrations of CT-1 were 8-fold higher than ventricular levels. Left ventricular tissue CT-1 protein concentrations were significantly higher in 40-week-old SHR compared with age-matched WKY (SHR 12.6+/-0.5 fmol/g vs. WKY 9.5+/-0.8 fmol/g, P<0.01). Ventricular stretch of Langendorff perfused, isolated WKY/SHR hearts resulted in significant, acute release of CT-1 and BNP. HPLC coupled with specific RIA revealed CT-1 in human/rat plasma, isolated rat heart perfusate, and rat heart tissue extracts to consist of complex, high molecular weight forms. CONCLUSIONS: This is the first report to show increased levels of plasma CT-1 in hypertensive disease. CT-1 is a unique cardiac cytokine whose release is stimulated by ventricular stretch. The atrium contains the highest levels of the protein. The stored and circulating molecular form of CT-1 is complex, which may modulate its in vivo role in cardiovascular disease.

Angiotensin-converting enzyme gene polymorphism interacts with left ventricular ejection fraction and brain natriuretic peptide levels to predict mortality after myocardial infarction.

OBJECTIVES: The goal of this study was the exploration of the associations between the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and post-myocardial infarction (MI) outcomes, especially any interaction with the accepted clinical prognostic markers brain natriuretic peptide (BNP) and left ventricular ejection fraction (LVEF). BACKGROUND: The ACE gene I/D polymorphism has been implicated in the development of MI, hypertension, and left ventricular hypertrophy. We examined the association of ACE I/D and prognosis after acute MI. METHODS: Patients incurring acute MI were genotyped for the ACE I/D polymorphism. Clinical data included assays of neurohormones, radionuclide ventriculography, and mortality over a mean 2.6 years of follow-up. RESULTS: Patients (n = 978) had a mean age of 62.1 years, and 78% were male. Overall genotype frequencies were II 23.2%, ID 49.5%, and DD 27.3%. Chi-square analysis revealed an association between the ACE D allele and death after MI (88 of 103 who died were DD or ID; p < 0.05), with an odds ratio for mortality of 8.03 (95% confidence interval, 2.16 to 29.88). Patients with the DD genotype had higher (p < 0.05) plasma BNP, N-terminal BNP (N-BNP), and endothelin-1 levels within 96 h after MI than grouped ID/II patients. Multivariate analysis indicated ACE genotype, age, and previous MI were independent predictors of death (p < 0.05). Patients with an ACE D allele in combination with either a lower than median LVEF or greater than median BNP had a higher mortality (p < 0.001 and p < 0.025, respectively) than the risk associated with the D allele itself. CONCLUSIONS: Angiotensin-converting enzyme genotyping may provide additional prognostic information in patients after MI in combination with the proven utility of LVEF, plasma BNP, and N-BNP measurements.

B-type Natriuretic Peptide circulating forms: Analytical and bioactivity issues.

B-type Natriuretic Peptide (BNP), A-type and C-type Natriuretic Peptides (ANP and CNP) comprise a family of peptides that retain a common ring structure and conserved amino acid sequences. All are present in the heart, but only BNP and ANP are regarded as primarily cardiac secretory products. BNP and ANP, acting through a guanylyl cyclase receptor, increase sodium and water excretion by the kidney, induce vasodilation, reduce blood pressure, counteract the bioactivity of the renin-angiotensin-aldosterone and sympathetic nervous systems and possess anti-hypertrophic and anti-fibrotic properties. BNP is synthesised in cardiomyocytes first as the precursor peptide preproBNP. Removal of the signal peptide from preproBNP produces proBNP which is cleaved to produce the biologically active carboxy-terminal BNP peptide and the inactive N-terminal fragment, NT-proBNP. BNP, NT-proBNP, proBNP and the C-terminal portion of the BNP signal peptide have been detected in human plasma as well as multiple sub-forms including truncated forms of BNP and NT-proBNP, as well as variable glycosylation of NT-proBNP and proBNP. The origin of these circulating forms, their potential bioactivity and their detection by current analytical methods are presented in this review.

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure.

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26+/-0.02 pmol/g wet weight versus heart failure 0.16+/-0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.