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Elevated Plasma B-Type NatriureticPeptide Concentrations DirectlyInhibit Circulating Neprilysin Activityin Heart Failure

Nicolas Vodovar, PHD,*y Marie-France Séronde, MD, PHD,*z Said Laribi, MD, PHD,*x Etienne Gayat, MD, PHD,*k{Johan Lassus, MD, PHD,# James L. Januzzi, JR, MD,** Riadh Boukef, MD,yy Semir Nouira, MD,yyPhilippe Manivet, PHARMD, PHD,*yzzxx Jane-Lise Samuel, MD, PHD,*y{ Damien Logeart, MD, PHD,*ykk{{Alain Cohen-Solal, MD, PHD,*ykk{{ A. Mark Richards, MD,***yyy Jean-Marie Launay, PHARMD, PHD,*yzzxxAlexandre Mebazaa, MD, PHD,*k{ {{ on behalf of the GREAT Network

ABSTRACT

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OBJECTIVES This study sought to hypothesize that elevated B-type natriuretic peptide (BNP) could act as an

endogenous neprilysin inhibitor.

BACKGROUND A hallmark of acute decompensated heart failure (ADHF) is the overproduction of natriuretic peptides

(NPs) by stretched cardiomyocytes. Various strategies have been developed to potentiate the beneficial effect of the

NPs, including the recent use of neprilysin angiotensin receptor inhibitors. Contrary to rodents, human BNP is poorly

sensitive to neprilysin degradation while retaining affinity to neprilysin.

METHODS We enrolled 638 patients presenting to the emergency department with acute dyspnea of which 468 had

ADHF and 169 had dyspnea of noncardiac origin. We also included 46 patients with stable chronic heart failure (HF) and

10 age-matched healthy subjects. Plasma samples were collected within 4 h after emergency department admission.

BNP, neprilysin concentration and activity, and the neprilysin substrate substance P concentration were measured.

RESULTS We found that when plasma BNP rose above 916 pg/ml, neprilysin activity was markedly reduced

(p< 0.0001) and stratified 95% of the population into 2 groups: BNP

ABBR EV I A T I ON S

AND ACRONYMS

ADHF = acute decompensated

heart failure

ANP = A-type natriuretic

peptide

ARNi = angiotensin receptor-

neprilysin inhibitor

BNP = B-type natriuretic

peptide

CHF = chronic heart failure

CV = coefficient of variation

HF = heart failure

IQR = interquartile range

irBNP = immunoreactive

B-type natriuretic peptide

NP = natriuretic peptide

NT-proBNP = N-terminal

pro–B-type natriuretic peptide

ROC = receiver-operating

characteristic

grant agree

Myers Squi

Dr. Januzz

Roche, Crit

entific, Cor

Ipsen, Sori

Dr. Richard

received sp

authors hav

contributed

Manuscript

Vodovar et al. J A C C : H E A R T F A I L U R E V O L . 3 , N O . 8 , 2 0 1 5

Inhibition of Neprilysin Activity by BNP in HF A U G U S T 2 0 1 5 : 6 2 9 – 3 6630

O ne of the hallmarks of acute de-compensated heart failure (ADHF)is the rapid large-scale productionof natriuretic peptides (NPs), which occursin response to mechanical and neurohor-monal effects on cardiomyocytes. B-type NP(BNP) and its N-terminal pro-peptide equiva-lent (NT-proBNP) are established biomarkersfor the diagnosis and prognosis of ADHF asarticulated in international clinical practiceguidelines (1,2). It is worth noting thatimmunoreactive BNP (irBNP), as quantifiedby conventional routine BNP assays, corre-sponds to both bioactive BNP and its precur-sor (proBNP) (3). Furthermore, it has beenshown that proBNP was predominant inpatients with severe heart failure (HF) andhigh irBNP values (4–6).

SEE PAGE 637

Production of BNP by stretched car-

diomyocytes promotes natriuresis, diuresis, andvasorelaxation to mitigate cardiac overload (7).In addition, BNP also exhibits antifibrotic (8),antihypertrophic (9), anti-inflammatory (10), andangiogenic (11) actions, and acutely opposes the renin-angiotensin-aldosterone (12) and sympathetic nervoussystems. Various strategies have been attempted topotentiate the beneficial effects of NPs in both ADHFand stable chronic HF (CHF) patients (13). These haveincluded the infusion of either recombinant (e.g.,nesiritide [14] or ularitide [15]) or synthetic NPs (e.g.,vasonatrin [16] or CD-NP [17]) and inhibition of the NP-degrading neprilysin (also known as neutral endo-peptidase) in combination either with inhibition of theangiotensin-converting enzyme (18) or angiotensinreceptor blockade (ARNi, LCZ696 [19]). Importantly,although the former strategies aim to simply leveragethe biological effect of the NPs, the latter approach(i.e., combined agents) may have broader effectsbecause of the broader biological benefits of neprilysininhibition plus the presence of vasodilator therapy.

ment No. 305507 (HOMAGE). Dr. Gayat has received travel fees fro

bb. Dr. Lassus has received honoraria from Roche Diagnostics, Nov

i has received research grants from Siemens, Singulex, and The

ical Diagnostics, Sphingotc, Amgen, and Novartis. Dr. Logeart h

dis, Roche Diagnostics, Novartis, Pfizer, and Servier. Dr. Solal has

n, Abbott, Novartis, Thermo Fisher, Alere, Pfizer, Vifor, Amgen

s has received research grants, speaker fees, and travel support

eaker honoraria from Cardiorentis, The Medicines Company, Crit

e reported that they have no relationships relevant to the content

equally to this work.

received December 3, 2014; revised manuscript received Februa

Neprilysin is a membrane-bound and circulatingprotease that has been involved in the catabolism ofnumerous vasoactive peptides, including A-type NP(ANP), bradykinins, angiotensin I, substance P, andendothelin-1 (20,21). In many animals, neprilysin isalso responsible for the degradation of the BNP (22).In particular, mouse BNP is successively cleaved bymeprinA and neprilysin (22). MeprinA cleaves BNP1-32into still-bioactive BNP7-32, which makes mouse BNPamenable to neprilysin cleavage within the ringstructure. By contrast, human BNP is poorly sensitiveto neprilysin degradation in vitro (23–25) possibly as itis not cleaved by meprinA (26). However, a shorterversion of human BNP, that is, BNP9-32, is also poorlysensitive to degradation by neprilysin in vitro (27),suggesting that human BNP is susceptible to neithermeprinA nor neprilysin degradation. Instead, humanBNP appears to be degraded by leupeptin-sensitiveproteases (28,29), possibly from the kallikrein family(28). Nevertheless, human BNP retains affinity to bindneprilysin (23,27) and has been shown to interferewith ANP cleavage in vitro (27). Because proBNP isalso detected by proBNP assays and differs from BNPby a longer N-terminal extremity, proBNP may alsointeract with neprilysin. We therefore hypothesizedthat human irBNP—that is, BNP and proBNP—besidesbeing effectors of the cardiac response to car-diomyocytes stretching in ADHF, could also act as anendogenous neprilysin inhibitor in vivo.

METHODS

This study was approved by local ethic committeesand written consent was obtained from patients ornext of kin. This study using human samples was per-formed according to the current revision of the Hel-sinki Declaration and registered at Clinical Trials.govunder the NCT01374880 identifier.

STUDY POPULATION. The studied population (n¼684)has been previously described (30). In brief, thecohort consisted of 638 patients presenting to theemergency department with shortness of breath as

m Servier; and has received lecture fees from Bristol-

artis, Vifor Pharma, Bayer, Orion Pharma, and Pfizer.

rmo Fisher; and has received consulting fees from

as received honorarium from Biotronic, Boston Sci-

received speaker and consulting fees from Actelion,

, Servier, Bayer, Sanofi, and Boehringer Ingelheim.

from Roche Diagnostics and Alere. Dr. Mebazaa has

ical Diagnostics, Vifor, Orion, and Novartis. All other

s of this paper to disclose. Drs. Vodovar and Séronde

ry 23, 2015, accepted March 6, 2015.

http://Clinical%20Trials.govhttps://clinicaltrials.gov/ct2/show/NCT01374880?term=NCT01374880%26rank=1

FIGURE 1 irBNP, Neprilysin Concentration, and Activity in the Study Population

(A) Comparison on plasma immunoreactive B-type natriuretic peptide (irBNP) concentra-

tions in acute decompensated heart failure (ADHF) (n ¼ 468), non-ADHF (n ¼ 169), andchronic heart failure (CHF) (n ¼ 46) patients. (B) Comparison of plasma neprilysin con-centrations in ADHF, non-ADHF, and CHF patients. (C) Comparison of circulating neprilysin

activity in ADHF, non-ADHF, and CHF patients. (D) Distribution of circulating neprilysin

activity according to the plasma neprilysin concentrations (ADHF ¼ open purple circles,non-ADHF ¼ open green circles, and CHF ¼ solid gray circles).

J A C C : H E A R T F A I L U R E V O L . 3 , N O . 8 , 2 0 1 5 Vodovar et al.A U G U S T 2 0 1 5 : 6 2 9 – 3 6 Inhibition of Neprilysin Activity by BNP in HF

631

their primary complaint: 469 were diagnosed withADHF and 169 suffered from noncardiac dyspnea(non-ADHF). The diagnosis of cardiac or noncardiacorigin of the dyspnea was performed by the emer-gency physicians according to clinical practiceguidelines (1). All plasma samples were collectedwithin 4 h after admission to the emergency depart-ment; 30-day and 1-year mortality was available for507 of these patients. Plasma was also obtained from46 patients with stable CHF in the cardiology outpa-tient center of Lariboisière Hospital (Paris). Lastly,plasma samples were obtained from 10 age-matchedhealthy controls with no recorded history of cardiacor neurological disease.

BIOMARKER QUANTIFICATION. Venous blood sam-ples were collected in tubes containing EDTA (eth-ylenediaminetetraacetic acid). Blood samples wereimmediately centrifuged at 3,500 rpm for 15 minat 4�C. BNP plasma levels were immediately mea-sured on an Abbott Architect system (Abbott Labo-ratories, Abbott Park, Illinois); the remaining of theplasma samples was immediately stored at �80�Cuntil further use. Neprilysin (EC 3.4.24.11) concen-tration was measured using the SEB785Hu ELISAkit from Uscn Life Science (Wuhan, China). Theanalytical parameters for the assay were intra-assaycoefficient of variation (CV): 4.6%, interassay CV:7.3%, limit of detection: 5 pg/ml, and linearity: 12 to350 pg/ml. Neprilysin activity was determined byfluorometry as previously described (31). The an-alytical parameters for the assay were intra-assayCV: 5.9%, interassay CV: 8.7%, limit of detection:0.012 nmol/ml/min, and linearity: 0.025 to 0.700nmol/ml/min. Substance P plasma concentrationswere determined by a radioimmunoassay (PhoenixPharmaceuticals, Belmont, California) according tothe manufacturer’s instructions.

IN VITRO ADDITION OF HUMAN RECOMBINANT BNP

TO PLASMA SAMPLES. Human recombinant BNP(MolBio, Lohmar, Germany) or human syntheticnonglycosylated proBNP (Roche, Basel, Switzerland)were reconstituted in 0.1% Bovine Serum Albumin(Pierce, Life Technologies, Carlsbad, California) insaline to a 100 mg/ml concentration. BNP and proBNPwere serially diluted into saline before being added tothe plasma of age-matched healthy controls at finalconcentrations of 300 and 1,600 pg/ml. The volume ofrecombinant BNP solution added to the samples was10% of the final volume. Plasma samples were incu-bated for 2 h at 37�C before neprilysin concentrationand activity measurements.

STATISTICAL ANALYSES. All statistical analyseswere performed using the R-statistical software

(R Foundation for Statistical Computing, Vienna,Austria). Data are expressed as median [interquartilerange (IQR)]. Variables were tested with Wilcoxonrank sum test or Kruskal-Wallis test, as appropriate; pvalues for multiple comparisons were adjusted usingthe Holm’s method. Paired data were analyzed usingpaired Wilcoxon rank sum test. Relationships be-tween variables were assessed using Spearman cor-relation coefficient. A 2-sided p value

FIGURE 2 Relationship Between irBNP and Neprilysin Activity

(A) Distribution of neprilysin circulating activity according to irBNP plasma concentration in

ADHF (n ¼ 468, open purple circles), non-ADHF (n ¼ 169, open green circles), and CHF(n ¼ 46, solid gray circles) patients. The vertical dashed line indicates the threshold forBNP at 916 pg/ml; the horizontal dashed line indicates the threshold for circulating

neprilysin activity at 0.21 nmol/ml/min. The large majority (61.8% þ 33.5% ¼ 95.3%) wasdistributed in the top-left and bottom-right quadrants. (B) Comparison of circulating

neprilysin activity in patients across the 916 pg/ml threshold (irBNP ADHF (314 pg/ml [IQR: 257 to 377 pg/ml]) > non-ADHF (256 pg/ml [IQR: 58 to 339 pg/ml]) (Figure 1B).Conversely, circulating neprilysin activity was thehighest in non-ADHF and the lowest in ADHF pa-tients: non-ADHF (0.37 nmol/ml/min [IQR: 0.3 to0.5 nmol/ml/min]) > CHF (0.29 nmol/ml/min [IQR:0.22 to 0.35]) > ADHF (0.22 nmol/ml/min [IQR: 0.13 to0.34 nmol/ml/min]) (Figure 1C). There was no corre-lation between circulating neprilysin activity andconcentration (r ¼ �0.04; p ¼ 0.27) (Figure 1D).

RELATION BETWEEN PLASMA IRBNP LEVELS AND

CIRCULATING NEPRILYSIN ACTIVITY. We nextexamined the distribution of circulating neprilysinactivity with respect to the plasma concentrations ofirBNP. Figure 2A shows an inverse relationshipbetween plasma irBNP and circulating neprilysinactivity. Notably, patients with irBNP $916 pg/mlexhibited an almost 3-fold reduction in circulatingneprilysin activity compared with those withirBNP

FIGURE 3 Inhibitory Effect of Exogenous BNP and proBNP on

Neprilysin Activity In Vitro

(A) Kinetics of neprilysin activity measured in the plasma of 10 age-matched healthy

controls spiked with either 300 pg/ml (green) or 1,600 pg/ml (purple) human recombi-

nant BNP. (B) Kinetics of neprilysin concentration measured in the plasma of 10 age-

matched healthy controls spiked with either 300 pg/ml (green) or 1,600 pg/ml (purple)

human recombinant BNP. (C) Kinetics of neprilysin activity measured in the plasma of 10

age-matched healthy controls spiked with either 300 pg/ml (green) or 1,600 pg/ml

(purple) human synthetic proBNP. (D) Kinetics of neprilysin concentration measured in the

plasma of 10 age-matched healthy controls spiked with either 300 pg/ml (green) or

1,600 pg/ml (purple) human synthetic proBNP. BNP ¼ B-type natriuretic peptide;proBNP ¼ pro–B-type natriuretic peptide.

J A C C : H E A R T F A I L U R E V O L . 3 , N O . 8 , 2 0 1 5 Vodovar et al.A U G U S T 2 0 1 5 : 6 2 9 – 3 6 Inhibition of Neprilysin Activity by BNP in HF

633

NEPRILYSIN DIRECT INHIBITION BY BNP AND

proBNP. To test a potential direct inhibitory effect ofirBNP, that is, BNP and proBNP, on neprilysin activ-ity, the plasma of 10 age-matched healthy controls(irBNP: 19 pg/ml [IQR: 14 to 28 pg/ml]) was spikedwith human recombinant BNP or human syntheticproBNP. Both recombinant peptides were addedindependently to final concentrations of 300 pg/ml or1,600 pg/ml to achieve levels < and $916 pg/ml,respectively.

Before addition of recombinant BNP, neprilysinactivity was similar in both groups (300 pg/ml:0.38 nmol/ml/min [IQR: 0.37 to 0.46 pg/ml],1,600 pg/ml: 0.39 nmol/ml/min [IQR: 0.37 to0.46 nmol/ml/min]; p ¼ 0.68). After incubation for2 h at 37�C, neprilysin activity was markedly reducedin the sample that contained 1,600 pg/ml BNP(0.06 nmol/ml/min [IQR: 0.05 to 0.08 nmol/ml/min])compared with the samples containing 300 pg/mlBNP (0.32 nmol/ml/min [IQR: 0.23 to0.39nmol/ml/min];p < 0.0001) (Figure 3A). Neprilysin concentrationremained unchanged throughout the experiment(Figure 3B).

Before the addition of synthetic proBNP, neprily-sin activity was similar in both groups (300 pg/ml:0.39 nmol/ml/min [IQR: 0.35 to 0.45 nmol/ml/min],1,600 pg/ml: 0.39 nmol/ml/min [IQR: 0.35 to0.45 nmol/ml/min]; p ¼ 1). After incubation for 2 h at37�C, neprilysin activity was markedly reduced inthe sample that contained 1,600 pg/ml proBNP(0.05 nmol/ml/min [IQR: 0.04 to 0.07 nmol/ml/min])compared with the samples containing 300 pg/mlproBNP (0.40 nmol/ml/min [IQR: 0.37 to 0.41nmol/ml/min]; p ¼ 0.002) (Figure 3C). Again, neprilysinconcentration remained unchanged throughout theexperiment (Figure 3D).CIRCULATING NEPRILYSIN ACTIVITY AND PLASMA

SUBSTANCE P CONCENTRATION. We next investi-gated the relationship between circulating neprily-sin activity and the plasma concentration of thevasoactive peptide substance P, a known substrateto neprilysin. Figure 4A shows a strong negativecorrelation between circulating neprilysin ac-tivity and plasma concentrations of substance P(r ¼ �0.80; p < 0.0001): the lower the circulatingneprilysin activity, the higher the plasma concen-tration of substance P. Figure 4B further shows thatirBNP at 916 pg/ml could stratify more than 91% ofthe patients into 2 subpopulations across athreshold of substance P at 54 pg/ml: patients withirBNP $916 pg/ml had higher substance P concen-trations (59 pg/ml [IQR: 57 to 64 pg/ml]) thanthose with irBNP

FIGURE 4 Relationship Between Neprilysin Activity, Substance P,

and irBNP Concentrations

(A) Distribution of plasma substance P according to circulating neprilysin activity con-

centration in ADHF (n ¼ 468, open purple circles), non-ADHF (n ¼ 169, open greencircles), and CHF (n ¼ 46, solid gray circles) patients. (B) Distribution of substance Pplasma concentration according to irBNP plasma level in ADHF, non-ADHF, and CHF

patients. The vertical dashed line indicates the threshold for irBNP at 916 pg/ml; the

horizontal dashed line indicates the threshold for substance P at 54 pg/ml. (C) Com-

parison of substance P plasma concentration in patients across the 916 pg/ml threshold

(irBNP

FIGURE 5 Schematic Representation of the Molecular Switch Induced by the irBNP-Mediated Neprilysin Inhibition

(Left) Patients with moderate irBNP levels and high NEP activity accumulate inactive vasoactive peptides and AngI1-7. (Right) Patients with high

irBNP levels and low NEP activity accumulate active vasoactive peptides and AngII. AngI ¼ angiotensin I; AngII ¼ angiotensin II; ANP ¼ A-typenatriuretic peptide; BK ¼ bradykinins; CNP ¼ C-type natriuretic peptide; ET ¼ endothelin; NEP ¼ neprilysin; SP ¼ substance P; URO ¼urodilatin; other abbreviations as in Figures 1 and 3.

PERSPECTIVES

COMPETENCY IN MEDICAL KNOWLEDGE: Immunoreactive

BNP is a gold-standard biomarker in HF; a cutoff at 916 pg/ml

discriminates patients with high and low neprilysin activity,

hence potentially predicts responders to neprilysin inhibition.

TRANSLATIONAL OUTLOOK: We identified 916 pg/ml as a

biologically relevant BNP cutoff that discriminated most of the

HF patients with different outcomes and vasoactive profiles.

Further analysis is needed to confirm and precisely identify the

impact of BNP-mediated neprilysin inhibition, and to determine

whether patients across this cutoff need different management.

J A C C : H E A R T F A I L U R E V O L . 3 , N O . 8 , 2 0 1 5 Vodovar et al.A U G U S T 2 0 1 5 : 6 2 9 – 3 6 Inhibition of Neprilysin Activity by BNP in HF

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STUDY LIMITATIONS. We did not assess the plasmaconcentrations of other vasoactive peptides that areneprilysin substrates other than substance P, and theyshould be considered in further studies. Furthermore,the number of patients tested is limited. However, therelationships between plasma irBNP and circulatingneprilysin activity, and between neprilysin activityand plasma substance P concentrations were unam-biguous. Finally, we did not assess the effect of thetruncated forms of BNP on neprilysin activity. How-ever, because neprilysin-mediated BNP cleavage onlyoccurs when the C-terminus is truncated (27), it isanticipated that most of BNP truncated forms wouldact as neprilysin inhibitors.

CONCLUSIONS

We showed that elevated irBNP acts as an endogenousinhibitor to neprilysin, with clear ramifications withrespect to the pathophysiology of HF as well asproviding insights into recent important clinical trialsin HF. It is widely anticipated that neprilysin inhibitionwill become a primary treatment option for patientswith HF, representing the first potential change to theusual HF care algorithm in a decade (34). Our dataprovide useful insights into the mechanism of thebenefit of neprilysin inhibition, and inform importantdata regarding this novel class of HF therapeutics.

ACKNOWLEDGMENTS The authors thank MalhaSadoune for technical assistance, and BéatriceFoucher at the Centre for Biological Resources,Lariboisière Hospital.

REPRINT REQUESTS AND CORRESPONDENCE: Dr.Alexandre Mebazaa, Department of Anesthesiologyand Intensive Care, Lariboisière Hospital, 2,Rue A. Paré, 75475 Paris, Cedex 10, France. E-mail:alexandre.mebazaa@lrb.aphp.fr OR Dr. Jean-MarieLaunay, Department of Biochemistry, LariboisièreHospital, 2, Rue A. Paré, 75475 Paris, Cedex 10,France. E-mail: jean-marie.launay@lrb.aphp.fr.

mailto:alexandre.mebazaa@lrb.aphp.frmailto:jean-marie.launay@lrb.aphp.fr

Vodovar et al. J A C C : H E A R T F A I L U R E V O L . 3 , N O . 8 , 2 0 1 5

Inhibition of Neprilysin Activity by BNP in HF A U G U S T 2 0 1 5 : 6 2 9 – 3 6636

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KEY WORDS BNP, BNP-mediatedneprilysin inhibition, heart failure, neprilysin,substance P

APPENDIX For a supplemental figure, pleasesee the online version of this article.

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Elevated Plasma B-Type Natriuretic Peptide Concentrations Directly Inhibit Circulating Neprilysin Activity in Heart FailureMethodsStudy populationBiomarker quantificationIn vitro addition of human recombinant BNP to plasma samplesStatistical analyses

ResultsNeprilysin concentration and activity in the study populationRelation between plasma irBNP levels and circulating neprilysin activityNeprilysin direct inhibition by BNP and proBNPCirculating neprilysin activity and plasma substance P concentration

DiscussionStudy limitations

ConclusionsAcknowledgmentsReferences