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1
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
635
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:[email protected] OR Dr. Jean-MarieLaunay,
Department of Biochemistry, LariboisièreHospital, 2, Rue A. Paré,
75475 Paris, Cedex 10,France. E-mail:
[email protected].
mailto:[email protected]:[email protected]
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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