Attenuation of monocyte chemotaxis--a novel anti-inflammatory mechanism of action for the cardio-protective hormone B-type natriuretic peptide

B-type natriuretic peptide (BNP) is a prognostic and diagnostic marker for heart failure (HF). An anti-inflammatory, cardio-protective role for BNP was proposed. In cardiovascular diseases including pressure overload-induced HF, perivascular inflammation and cardiac fibrosis are, in part, mediated by monocyte chemoattractant protein (MCP)1-driven monocyte migration. We aimed to determine the role of BNP in monocyte motility to MCP1. A functional BNP receptor, natriuretic peptide receptor-A (NPRA) was identified in human monocytes. BNP treatment inhibited MCP1-induced THP1 (monocytic leukemia cells) and primary monocyte chemotaxis (70 and 50 %, respectively). BNP did not interfere with MCP1 receptor expression or with calcium. BNP inhibited activation of the cytoskeletal protein RhoA in MCP1-stimulated THP1 (70 %). Finally, BNP failed to inhibit MCP1-directed motility of monocytes from patients with hypertension (n = 10) and HF (n = 6) suggesting attenuation of this anti-inflammatory mechanism in chronic heart disease. We provide novel evidence for a direct role of BNP/NPRA in opposing human monocyte migration and support a role for BNP as a cardio-protective hormone up-regulated as part of an adaptive compensatory response to combat excess inflammation.

Mechanical stretch up-regulates the B-type natriuretic peptide system in human cardiac fibroblasts:a possible defense against transforming growth factor-β mediated fibrosis

BACKGROUND: Mechanical overload of the heart is associated with excessive deposition of extracellular matrix proteins and the development of cardiac fibrosis. This can result in reduced ventricular compliance, diastolic dysfunction, and heart failure. Extracellular matrix synthesis is regulated primarily by cardiac fibroblasts, more specifically, the active myofibroblast. The influence of mechanical stretch on human cardiac fibroblasts' response to pro-fibrotic stimuli, such as transforming growth factor beta (TGFβ), is unknown as is the impact of stretch on B-type natriuretic peptide (BNP) and natriuretic peptide receptor A (NPRA) expression. BNP, acting via NPRA, has been shown to play a role in modulation of cardiac fibrosis.

METHODS AND RESULTS: The effect of cyclical mechanical stretch on TGFβ induction of myofibroblast differentiation in primary human cardiac fibroblasts and whether differences in response to stretch were associated with changes in the natriuretic peptide system were investigated. Cyclical mechanical stretch attenuated the effectiveness of TGFβ in inducing myofibroblast differentiation. This finding was associated with a novel observation that mechanical stretch can increase BNP and NPRA expression in human cardiac fibroblasts, which could have important implications in modulating myocardial fibrosis. Exogenous BNP treatment further reduced the potency of TGFβ on mechanically stretched fibroblasts.

CONCLUSION: We postulate that stretch induced up-regulation of the natriuretic peptide system may contribute to the observed reduction in myofibroblast differentiation.

Long-term statin therapy in patients with systolic heart failure and normal cholesterol:effects on elevated serum markers of collagen turnover, inflammation, and B-type natriuretic peptide

BACKGROUND: The role of statin therapy in heart failure (HF) is unclear. The amino-terminal propeptide of procollagen type III (PIIINP) predicts outcome in HF, and yet there are conflicting reports of statin therapy effects on PIIINP.

OBJECTIVES: This study determined whether there was an increase in serum markers of inflammation, fibrosis (including PIIINP), and B-type natriuretic peptide (BNP) in patients with systolic HF and normal total cholesterol and determined the effects of long-term treatment with atorvastatin on these markers.

METHODS: Fifty-six white patients with systolic HF and normal cholesterol levels (age 72 [13] years; 68% male; body mass index 27.0 [7.3] kg/m(2); ejection fraction 35 [13]%; 46% with history of smoking) were randomly allocated to atorvastatin treatment for 6 months, titrated to 40 mg/d (A group) or not (C group). Age- and/or sex-matched subjects without HF (N group) were also recruited. Biomarkers were measured at baseline (all groups) and 6 months (A and C groups).

RESULTS: Serum markers of collagen turnover, inflammation, and BNP were significantly elevated in HF patients compared with normal participants (all P < 0.05). There were correlations between these markers in HF patients but not in normal subjects. Atorvastatin treatment for 6 months caused a significant reduction in the following biomarkers compared with baseline: BNP, from median (interquartile range) 268 (190-441) pg/mL to 185 (144-344) pg/mL; high-sensitivity C-reactive protein (hs-CRP), from 5.26 (1.95 -9.29) mg/L to 3.70 (2.34-6.81) mg/L; and PIIINP, from 4.65 (1.86) to 4.09 (1.25) pg/mL (all P < 0.05 baseline vs 6 months). Between-group differences were significant for PIIINP only (P = 0.027). There was a positive interaction between atorvastatin effects and baseline hs-CRP and PIIINP (P < 0.01).

CONCLUSIONS: Long-term statin therapy reduced PIIINP in this small, selected HF population with elevated baseline levels. Further evaluation of statin therapy in the management of HF patients with elevated PIIINP is warranted.

Hazardous alcohol consumption is associated with increased levels of B-type natriuretic peptide: evidence from two population-based studies

Russia has very high mortality from cardiovascular disease (CVD), with evidence that heavy drinking may play a role. To throw further light on this association we have studied the association of alcohol with predictors of CVD risk including B-type natriuretic peptide (BNP). Levels of BNP increase primarily in response to abnormal cardiac chamber wall stretch which can occur both as a result of atherosclerosis as well as due to other types of damage to the myocardium. No previous population-based studies have investigated the association with alcohol. We analysed cross-sectional data on drinking behaviour in 993 men aged 25-60 years from the Izhevsk Family Study 2 (IFS2), conducted in the Russian city of Izhevsk in 2008-2009. Relative to non-drinkers, men who drank hazardously had an odds ratio (OR) of being in the top 20 % of the BNP distribution of 4.66 (95 % CI 2.13, 10.19) adjusted for age, obesity, waist-hip ratio, and smoking. Further adjustment for class of hypertension resulted in only slight attenuation of the effect, suggesting that this effect was not secondary to the influence of alcohol on blood pressure. In contrast hazardous drinking was associated with markedly raised ApoA1 and HDL cholesterol levels, but had little impact on levels of ApoB and LDL cholesterol. Similar but less pronounced associations were found in the Belfast (UK) component of the PRIME study conducted in 1991. These findings suggest that the association of heavy drinking with increased risk of cardiovascular disease may be partly due to alcohol-induced non-atherosclerotic damage to the myocardium.

Associates of an elevated natriuretic peptide level in stable heart failure patients:implications for targeted management

BACKGROUND: Persistently elevated natriuretic peptide (NP) levels in heart failure (HF) patients are associated with impaired prognosis. Recent work suggests that NP-guided therapy can improve outcome, but the mechanisms behind an elevated BNP remain unclear. Among the potential stimuli for NP in clinically stable patients are persistent occult fluid overload, wall stress, inflammation, fibrosis, and ischemia. The purpose of this study was to identify associates of B-type natriuretic peptide (BNP) in a stable HF population.

METHODS: In a prospective observational study of 179 stable HF patients, the association between BNP and markers of collagen metabolism, inflammation, and Doppler-echocardiographic parameters including left ventricular ejection fraction (LVEF), left atrial volume index (LAVI), and E/e prime (E/e') was measured.

RESULTS: Univariable associates of elevated BNP were age, LVEF, LAVI, E/e', creatinine, and markers of collagen turnover. In a multiple linear regression model, age, creatinine, and LVEF remained significant associates of BNP. E/e' and markers of collagen turnover had a persistent impact on BNP independent of these covariates.

CONCLUSION: Multiple variables are associated with persistently elevated BNP levels in stable HF patients. Clarification of the relative importance of NP stimuli may help refine NP-guided therapy, potentially improving outcome for this at-risk population.

Natriuretic peptide-based screening and collaborative care for heart failure:the STOP-HF randomized trial

IMPORTANCE: Prevention strategies for heart failure are needed.

OBJECTIVE: To determine the efficacy of a screening program using brain-type natriuretic peptide (BNP) and collaborative care in an at-risk population in reducing newly diagnosed heart failure and prevalence of significant left ventricular (LV) systolic and/or diastolic dysfunction.

DESIGN, SETTING, AND PARTICIPANTS: The St Vincent's Screening to Prevent Heart Failure Study, a parallel-group randomized trial involving 1374 participants with cardiovascular risk factors (mean age, 64.8 [SD, 10.2] years) recruited from 39 primary care practices in Ireland between January 2005 and December 2009 and followed up until December 2011 (mean follow-up, 4.2 [SD, 1.2] years).

INTERVENTION: Patients were randomly assigned to receive usual primary care (control condition; n=677) or screening with BNP testing (n=697). Intervention-group participants with BNP levels of 50 pg/mL or higher underwent echocardiography and collaborative care between their primary care physician and specialist cardiovascular service.

MAIN OUTCOMES AND MEASURES: The primary end point was prevalence of asymptomatic LV dysfunction with or without newly diagnosed heart failure. Secondary end points included emergency hospitalization for arrhythmia, transient ischemic attack, stroke, myocardial infarction, peripheral or pulmonary thrombosis/embolus, or heart failure.

RESULTS: A total of 263 patients (41.6%) in the intervention group had at least 1 BNP reading of 50 pg/mL or higher. The intervention group underwent more cardiovascular investigations (control, 496 per 1000 patient-years vs intervention, 850 per 1000 patient-years; incidence rate ratio, 1.71; 95% CI, 1.61-1.83; P<.001) and received more renin-angiotensin-aldosterone system-based therapy at follow-up (control, 49.6%; intervention, 56.5%; P=.01). The primary end point of LV dysfunction with or without heart failure was met in 59 (8.7%) of 677 in the control group and 37 (5.3%) of 697 in the intervention group (odds ratio [OR], 0.55; 95% CI, 0.37-0.82; P = .003). Asymptomatic LV dysfunction was found in 45 (6.6%) of 677 control-group patients and 30 (4.3%) of 697 intervention-group patients (OR, 0.57; 95% CI, 0.37-0.88; P = .01). Heart failure occurred in 14 (2.1%) of 677 control-group patients and 7 (1.0%) of 697 intervention-group patients (OR, 0.48; 95% CI, 0.20-1.20; P = .12). The incidence rates of emergency hospitalization for major cardiovascular events were 40.4 per 1000 patient-years in the control group vs 22.3 per 1000 patient-years in the intervention group (incidence rate ratio, 0.60; 95% CI, 0.45-0.81; P = .002).

CONCLUSION AND RELEVANCE: Among patients at risk of heart failure, BNP-based screening and collaborative care reduced the combined rates of LV systolic dysfunction, diastolic dysfunction, and heart failure.

TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00921960.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

The structure of helokinestatin-5 and its biosynthetic precursor from Gila monster (Heloderma suspectum) venom: Evidence for helokinestatin antagonism of bradykinin-induced relaxation of rat tail artery smooth muscle

Here we report the primary structure of a novel peptide, named helokinestatin-5 (VPPPLQMPLIPR), from the venom of the Gila monster (Heloderma suspectum). Helokinestatin-5 differs in structure from helokinestatin-3 by deletion of a single prolyl residue in the N-terminally located polyproline region. Two different biosynthetic precursors were consistently cloned from a venom-derived cDNA library. The first encoded helokinestatins 1–4 and a single copy of C-type natriuretic peptide, as previously described, whereas the second was virtually identical, lacking only a single prolyl codon as found in the mature attenuated helokinestatin-5 peptide. Helokinestatins 1–3 and 5 were synthesized by solid-phase fmoc chemistry and each synthetic replicate was found to antagonize the relaxation effect induced by bradykinin on rat tail artery smooth muscle. Helokinestatins thus represent a novel family of vasoactive peptides from the venom of helodermatid lizards

Molecular cloning and deduced organization of the pHpG/CNP precursor from the venom of the African forest viper, Atheris squamigera

The discovery that the hypotensive sequela of envenomation by the South American viper, Bothrops jararaca, was mediated by peptides, represented a milestone in drug discovery research that led to the introduction of ACE inhibitors. These bradykinin-potentiating peptides (BPPs) have been found in the venoms of many species of viper and molecular cloning of biosynthetic precursors has revealed that each encodes several different BPPs in tandem with a single copy of a C-type natriuretic peptide (CNP) located at the C-terminus. Venoms of the African forest vipers (Atheris) have been poorly studied possibly because they do not represent a major danger to humans. However, initial studies have indicated that they contain some of the “classical” protein toxins of viper venoms and a novel class of peptide, the polyglycine/polyhistidine (pGpH) peptides. These peptides occur in several molecular forms with different numbers of repetitive glycine and histidine repeats. We have cloned the biosynthetic precursor of A. squamigera pGpH peptides from a venom-derived cDNA library and have confirmed that a single copy of CNP is located at the C-terminus and additionally that, like BPPs in other vipers, pGpH peptides are encoded in tandem within this single precursor. Solid phase peptide synthesis of pGpH peptides has proven to be extremely difficult but is progressing and acquisition of synthetic replicates of each peptide is a necessary prerequisite for systematic pharmacological characterisation as establishment of a biological function for these peptides remains elusive. pGpH peptides may prove to play a role as fundamental as that of the BPPs.

Helokinestatins: A novel family of bioactive peptides with drug-lead potential fromthe venomof the Gila Monster (Heloderma suspectum)

Venom of the Gila Monster (Heloderma suspectum) has proven to be an unlikely source of lead compounds (exendins) for the development of new injectable peptide therapeutics for the treatment of Type 2 diabetes. However, no systematic searches for new classes of bioactive peptides in lizard venom have appeared until recently. Here we describe the discovery of a new class of peptides – the helokinestatins – from H. suspectum venom, their structural characterisation and that of their biosynthetic precursors from cloned cDNA. In addition, we have subjected members of the family to preliminary pharmacological characterisation. Helokinestatins 1–6 are a family of proline-rich peptides containing 10–15 amino acid residues terminating in a common -Pro-Arg.OH motif. They are encoded in tandem within two virtually identical biosynthetic precursors of 177 and 178 amino acid residues, differing by only a single Pro residue. Each precursor also encodes a single copy of a C-type natriuretic peptide located at the C-terminus. Synthetic replicates of all helokinestatins were shown to be devoid of any direct action on the smooth muscle of rat tail artery but were found to be potent inhibitors of bradykinin-induced relaxation in this preparation in a manner that is suggestive of a non-competitive mechanism. Helokinestatin-3 (VPPPPLQMPLIPR) and helokinestatin-5 (VPPPLQMPLIPR) were found to be most potent in this respect causing almost complete inhibition of bradykinin-induced relaxation. Helokinestatins and BPPs may have a shared evolutionary history but the former do not inhibit ACE. The bradykinin inhibitory potential of helokinestatins may be exploited in the local control of chronic inflammation.

Influence of diabetes on natriuretic peptide thresholds in screening for Stage B heart failure

CONTEXT: Natriuretic peptide (NP) has been shown to be an effective screening tool to identify patients with Stage B heart failure and to have clinical value in preventing heart failure progression. The impact of associated metabolic confounders on the screening utility of NP needs clarification.

OBJECTIVE: To assess the impact of diabetes mellitus (DM) on NP screening for asymptomatic Stage B heart failure.

MATERIALS AND METHODS: The study population consisted of 1368 asymptomatic patients with cardiovascular risk factors recruited from general practice as part of the STOP-HF trial. B-type NP (BNP) was quantified at point-of-care.

RESULTS: BNP was found to be as accurate for detecting Stage B heart failure in DM patients compared to non-DM patients (AUC 0.75 [0.71,0.78] and 0.77 [0.72,0.82], respectively). However, different BNP thresholds are required to achieve the same level of diagnostic sensitivity in DM compared with non-DM patients. To achieve 80% sensitivity a difference of 5-ng/L lower is required for patients with DM.

CONCLUSION: Although a significantly different BNP threshold is detected for patients with DM, the BNP concentration difference is small and unlikely to warrant a clinically different diagnostic threshold.

Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure

We examined the association of common variants at the NPPA-NPPB locus with circulating concentrations of the natriuretic peptides, which have blood pressure-lowering properties. We genotyped SNPs at the NPPA-NPPB locus in 14,743 individuals of European ancestry, and identified associations of plasma atrial natriuretic peptide with rs5068 (P = 8 × 10 -70), rs198358 (P = 8 × 10 -30) and rs632793 (P = 2 × 10 -10), and of plasma B-type natriuretic peptide with rs5068 (P = 3 × 10 -12), rs198358 (P = 1 × 10 -25) and rs632793 (P = 2 × 10 -68). In 29,717 individuals, the alleles of rs5068 and rs198358 that showed association with increased circulating natriuretic peptide concentrations were also found to be associated with lower systolic (P = 2 × 10 -6 and 6 × 10 -5, respectively) and diastolic blood pressure (P = 1 × 10 -6 and 5 × 10 -5), as well as reduced odds of hypertension (OR = 0.85, 95% CI = 0.79-0.92, P = 4 × 10 -5; OR = 0.90, 95% CI = 0.85-0.95, P = 2 × 10 -4, respectively). Common genetic variants at the NPPA-NPPB locus found to be associated with circulating natriuretic peptide concentrations contribute to interindividual variation in blood pressure and hypertension.

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)(5)-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides. (C) 2011 Published by Elsevier Inc.