Author + information
- Received October 29, 2014
- Accepted November 4, 2014
- Published online February 24, 2015.
- Antoni Bayés-Genís, MD, PhD∗,†∗ (, )
- Jaume Barallat, MD†,‡,
- Amparo Galán, MD, PhD†,‡,
- Marta de Antonio, MD, PhD∗,†,
- Mar Domingo, MD, PhD∗,
- Elisabet Zamora, MD, PhD∗,†,
- Agustín Urrutia, MD, PhD∗,† and
- Josep Lupón, MD, PhD∗,†
- ∗Heart Failure Clinic, Cardiology Service, Hospital Universitari Germans Trias i Pujol Badalona, Barcelona, Spain
- †Department of Medicine, Universitat Autonoma de Barcelona, Barcelona, Spain
- ‡Biochemistry Service, Hospital Universitari Germans Trias i Pujol Badalona, Barcelona, Spain
- ↵∗Reprint requests and correspondence
: Dr. Antoni Bayes-Genis, Department of Medicine, Universitat Autonoma de Barcelona, Hospital Universitari Germans Trias i Pujol, Carretera del Canyet s/n, 08916 Badalona, Barcelona, Spain.
Background Neprilysin is a membrane-bound enzyme that breaks down natriuretic peptides. The PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) trial showed that patients with heart failure (HF) treated with an angiotensin receptor neprilysin inhibitor lived longer without being hospitalized for HF than those receiving standard care with enalapril.
Objectives This study sought to assess the presence of circulating soluble neprilysin in a real-life cohort of HF patients and correlate neprilysin levels with outcomes.
Methods Circulating soluble neprilysin was measured with a modified sandwich immunoassay in consecutive ambulatory patients with HF who were followed up for 4.1 years. Associations between neprilysin level and a composite endpoint that included cardiovascular death or HF hospitalization were explored.
Results Median neprilysin concentration in 1,069 patients was 0.642 ng/ml (median quartile 1 to 3: 0.385 to 1.219). Neprilysin weakly but significantly correlated with age (rho = 0.16; p < 0.001). In age-adjusted Cox regression analyses, neprilysin concentrations were significantly associated with the composite endpoint (hazard ratio [HR]: 1.17; 95% confidence interval [CI]: 1.06 to 1.29; p = 0.001) and cardiovascular death (HR: 1.19; 95% CI: 1.06 to 1.32; p = 0.002). In comprehensive multivariable analyses, soluble neprilysin remained significantly associated with both the composite endpoint (HR: 1.18; 95% CI: 1.07 to 1.31; p = 0.001) and cardiovascular death (HR: 1.18; 95% CI: 1.05 to 1.32; p = 0.006).
Conclusions Identification of circulating neprilysin in HF patients and the positive association of neprilysin with cardiovascular mortality and morbidity further support the importance of NEP inhibition for augmenting natriuretic peptides as a therapeutic target.
Neprilysin is known by a variety of other names, including neutral endopeptidase, CD10, enkephalinase, common acute lymphoblastic leukemia antigen (CALLA), and endopeptidase-24.11. Neprilysin catalyzes the degradation of several vasodilator peptides, including natriuretic peptides, angiotensin II, bradykinin, substance P, adrenomedullin, and endothelin-1 (1–3). Therefore, inhibiting neprilysin will augment the naturally occurring natriuretic peptides, which promote natriuresis, induce vasodilation, and reduce cardiac hypertrophy and fibrosis. Because neprilysin participates in the breakdown of angiotensin II, neprilysin inhibitors might increase circulating angiotensin II levels. This provides a rationale for agents that block both neprilysin and angiotensin II.
LCZ696, an angiotensin receptor neprilysin inhibitor (ARNI), contains the neprilysin inhibitor prodrug AHU377 and the angiotensin II receptor antagonist valsartan (4). It has been tested successfully in patients with hypertension (5) and with heart failure (HF) with preserved ejection fraction (6). Early closure of the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) trial in patients with HF with reduced ejection fraction (HFrEF) (7) was recently announced because patients who received LCZ696 lived longer without being hospitalized for HF than those who received standard care with the angiotensin-converting enzyme inhibitor (ACEI) enalapril.
Neprilysin is a membrane-bound enzyme with a large extracellular catalytic domain, a single transmembrane region, and a short (27 amino acids) cytoplasmic N-terminal domain (8,9). Previous studies showed that neprilysin, like many other membrane-bound metalloproteases, can be released from the cell surface, producing a non–membrane-associated form that retains catalytic activity (10,11). Previous reports have identified neprilysin in biological fluids (12,13), but no evidence exists of circulating neprilysin as a pathobiological surrogate in patients with HF. Accordingly, our aim was to assess for the first time the presence of circulating soluble neprilysin in a real-life cohort of HF patients and to correlate neprilysin levels with outcomes.
Ambulatory patients treated at a multidisciplinary HF clinic between May 22, 2006 and May 22, 2013 were consecutively included in the study. Referral inclusion criteria and blood sample collection have been described elsewhere (14). In summary, patients were referred to the HF clinic by cardiology or internal medicine departments and, to a lesser extent, from the emergency or other hospital departments. The principal referral criterion was HF according to the European Society of Cardiology guidelines irrespective of pathogenesis, at least 1 hospitalization for HF, or a reduced left ventricular ejection fraction (LVEF). Neprilysin and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) were analyzed from the same blood sample stored at −80°C, without previous freeze-thaw cycles. All samples were obtained between 9 am and noon.
All participants provided written informed consent, and the local ethics committee approved the study. All study procedures were in accord with the ethical standards outlined in the Helsinki Declaration of 1975, as revised in 1983.
Follow-up and outcomes
All patients were followed up at regular pre-defined intervals, with additional visits as required in case of decompensation. The regular visitation schedule included a minimum of quarterly visits with nurses, biannual visits with physicians, and elective visits with geriatricians, psychiatrists, and rehabilitation physicians (14,15). Patients who did not attend the regular visits were contacted by telephone.
The primary outcome was the composite of cardiovascular death or HF hospitalization. Secondary outcomes included cardiovascular and all-cause death. A death was considered of cardiovascular origin if it was caused by HF (decompensated HF or treatment-resistant HF, in the absence of another cause); sudden death (unexpected death, witnessed or not, of a previously stable patient with no evidence of worsening HF or any other cause of death); acute myocardial infarction (directly related in time with acute myocardial infarction, whether attributable to mechanic, hemodynamic, or arrhythmic complications); stroke (associated with recent acute neurological deficit); procedural (post-diagnostic or post-therapeutic procedural death); or other cardiovascular causes (e.g., rupture of an aneurysm, peripheral ischemia, or aortic dissection). HF hospitalizations were identified from clinic records, hospital wards, or electronic Catalan history record. Fatal events were identified from the clinical records of patients with HF, hospital wards, the emergency department, or general practitioners, or by contacting the patient’s relatives. Furthermore, data were verified from the databases of the Catalan and Spanish Health Systems. Adjudication of events was performed by 2 of the authors (M.D and J.L.).
Human neprilysin was measured with a modified sandwich immunoassay (human neprilysin/CD10 ELISA kit, Aviscera Bioscience, Santa Clara, California, code No. SK00724-01, lot No. 20111893). To improve the analytic sensitivity of the method and to obtain a lower limit of sample quantification, several modifications were made: 1) serum aliquots were diluted one-quarter in dilution buffer provided by the manufacturer (DB09) before incubation; 2) the kit was transferred to an automated robotic platform (Basic Radim Immunoassay Operator 2 [BRIO 2], Radim SpA, Pomezia, Italy) that performed all incubations at a constant temperature of 30°C, with 1,000 revolutions/min mixing; and 3) initial sample incubation was extended to 150 min to achieve a higher slope in the calibration curve and better assay sensitivity (Figure 1). The studied assay measures the 52 to 750 amino acid fraction of neprilysin as immunogen (extracellular soluble fraction). This assay displays 0% cross-reactivity with the 2 metallopeptidases most similar to this sequence, namely, endothelin-converting enzymes 1 and 2. It also does not display cross-reactivity with erythrocyte cell-surface antigen (KELL), another protein with strong homology with neprilysin (16). The modified protocol displayed an analytic linearity from 0.250 to 4 ng/ml. Samples with concentrations higher than 4 ng/ml were further diluted to a final measurement range from 0.250 to 64 ng/ml. At a positive control value of 1.4 ng/ml, intra-assay and interassay coefficients of variation were 3.7% and 8.9%, respectively. The intra-assay coefficient of variation at 0.642 ng/ml (median value) was 6.5%.
NT-proBNP levels were determined with an immunoelectrochemiluminescence assay on a Modular Analytics E170 analyzer (Roche Diagnostics, Indianapolis, Indiana). This assay has <0.001% cross-reactivity with bioactive B-type natriuretic protein (BNP), although as has been described for all the main BNP and NT-proBNP assays, it may display substantial cross-reactivity to proBNP peptides (17). In the constituent studies in this report, the assay had interrun coefficients of variation ranging from 0.9% to 5.5%.
Categorical variables were expressed as percentages. Continuous variables were expressed as the mean (SD) or median (quartile 1 to quartile 3 [Q1 to Q3]) according to normal or nonnormal distributions. Normal distribution was assessed with normal Q to Q plots. The correlation between neprilysin levels and age, LVEF, NT-proBNP, and estimated glomerular filtration rate (eGFR; Chronic Kidney Disease Epidemiology Collaboration formula) were analyzed with the Spearman rank correlation coefficient (rho). Differences in neprilysin concentrations among sex and pathogenic groups were assessed with the Mann-Whitney U test. Statistical differences (p value for trend) in neprilysin levels for New York Heart Association (NYHA) functional class groups were computed by Spearman test. Cox regression analyses that included age as a covariate and survival curves were plotted for the composite primary endpoint and for cardiovascular mortality relative to neprilysin median values. Multivariable survival analyses were also performed with Cox regression models. Neprilysin values were log-transformed, and 1 SD was used for hazard ratio (HR) calculation. In those patients with levels inferior to the lower range of detection (0.250 ng/ml), neprilysin levels were considered below median values in the dichotomous analysis and a concentration of 0.249 ng/ml was introduced in the analysis as a continuous variable. The following variables were incorporated into the model: age, sex, ischemic pathogenesis of HF, LVEF, NYHA functional class, presence of diabetes mellitus, hemoglobin (g/dl), serum sodium (mmol/l), eGFR, NT-proBNP, β-blocker treatment, ACEI or angiotensin II receptor blocker treatment, and neprilysin. In a subgroup of patients, other biomarkers such as ST2 (available in 814 patients) and high-sensitivity troponin T (hsTnT, available in 803 patients) were also included in a multivariable analysis.
Finally, we used different measurements of performance to test the potential incremental prognostic value of neprilysin (discrimination, calibration, and reclassification) as described previously (18). For net reclassification improvement calculation, we used risk categories based on tertiles of risk for the composite endpoint (<21%, 21% to 39%, and >39%) and cardiovascular death (<11%, 11% to 23%, and >23%).
Statistical analyses were performed with SPSS 15 (SPSS Inc., Chicago, Illinois) and the software R (version 2.11.1) statistical package (Foundation for Statistical Computing, Vienna, Austria). A 2-sided p < 0.05 was considered significant.
Circulating soluble neprilysin was measured in 1,069 patients with HF who were consecutively enrolled in the study from May 2006 to May 2013. Table 1 shows the baseline characteristics of the entire sample and the subgroup of patients who fulfilled the PARADIGM-HF study inclusion criteria (NYHA class II to IV, LVEF ≤35%, NT-proBNP ≥600 ng/l or NT-proBNP ≥400 ng/l with an admission for HF in the previous year, treated with ACEI or angiotensin II receptor blocker and with β-blockers unless contraindicated or not tolerated; PARADIGM-like cohort, n = 480). During a mean follow-up period of 4.1 ± 2.4 years, 449 patients died; 247 deaths were of cardiovascular causes, 169 of noncardiovascular causes (37.6%), and 33 of unknown causes (7.3%). Among known cardiovascular causes of death, refractory HF was the cause in 128 patients (51.8%), sudden death in 53 (21.4%), and acute myocardial infarction in 23 (9.3%). During the follow-up, 231 patients were admitted to the hospital for HF and 356 patients fulfilled the primary endpoint of cardiovascular death or HF hospitalization. Five patients were lost to follow-up and adequately censored.
The median soluble neprilysin level was 0.642 ng/ml (Q1 to Q3: 0.385 to 1.219 ng/ml). Neprilysin levels were below the analytic measurement range in 165 patients (14.6%). Neprilysin levels modestly but significantly correlated with age (rho = 0.16; p < 0.001). In contrast, no correlations were found between neprilysin and LVEF (rho = 0.02; p = 0.35), eGFR (rho = 0.05; p = 0.1), NT-proBNP (rho = −0.01; p = 0.68), NYHA functional class (p for trend 0.72), or blood pressure (rho = 0.01; p = 0.70), and neprilysin concentrations were not different in hypotensive patients (systolic blood pressure <100 mm Hg) versus nonhypotensive patients (p = 0.57). Neprilysin levels were not different between males and females (p = 0.28) but were significantly higher in nonischemic versus ischemic patients (0.690 ng/ml [0.450 to 1.401] vs. 0.611 ng/ml [0.328 to 1.046], respectively; p = 0.002).
Neprilysin and outcomes
Neprilysin values relative to the median were significantly associated with the composite primary endpoint of cardiovascular death or HF hospitalization (HR: 1.37; 95% confidence interval [CI]: 1.11 to 1.69; p = 0.003), cardiovascular death (HR: 1.60; 95% CI: 1.24 to 2.06; p < 0.001), and all-cause death (HR: 1.27; 95% CI: 1.06 to 1.53; p = 0.01). Figure 2 illustrates the diverging survival curves for the composite endpoint and for cardiovascular death relative to neprilysin median values.
As a continuous variable, neprilysin level was also significantly associated with the composite primary endpoint of cardiovascular death or HF hospitalization (HR: 1.17; 95% CI: 1.06 to 1.29; p = 0.001), and cardiovascular death (HR: 1.19; 95% CI: 1.06 to 1.32; p = 0.002), with a trend toward significance in all-cause death (HR: 1.09; 95% CI: 1.00 to 1.19; p = 0.06). In a comprehensive multivariable analysis that included NT-proBNP, soluble neprilysin remained significantly associated with the composite primary endpoint (HR: 1.18; 95% CI: 1.07 to 1.31; p = 0.001) and cardiovascular mortality (HR: 1.18; 95% CI: 1.05 to 1.32; p = 0.006) (Table 2) but not with all-cause mortality (HR: 1.05; 95% CI: 0.96 to 1.16; p = 0.28). Addition of soluble neprilysin to a comprehensive model that included 11 clinical variables and NT-proBNP (considered the baseline model) had little impact on discrimination but substantially improved calibration and reclassification (Table 3). Moreover, likelihood ratios showed a better global goodness of fit of the model that included neprilysin (p = 0.002 for composite primary endpoint and p = 0.007 for cardiovascular death, respectively).
On the other hand, in a multimarker approach that included neprilysin, NT-proBNP, ST2, and hsTnT, neprilysin remained significantly associated with the composite primary endpoint (HR: 1.15; 95% CI: 1.03 to 1.28; p = 0.02) and cardiovascular mortality (HR: 1.17; 95% CI: 1.03 to 1.32; p = 0.02). ST2 and hsTnT also remained independently associated with both the composite endpoint and cardiovascular mortality. In this multimarker model, the likelihood ratios also improved significantly in the model that included neprilysin (p = 0.02 for the composite endpoint and p = 0.04 for cardiovascular death, respectively).
A PARADIGM-like cohort from this real-life HF series was also examined, and circulating neprilysin levels remained significantly associated with the composite primary endpoint of cardiovascular death or HF hospitalization in the comprehensive multivariable analysis (HR: 1.23; 95% CI: 1.05 to 1.43; p = 0.008). In this cohort, neprilysin was not predictive of cardiovascular death (HR: 1.14; 95% CI: 0.95 to 1.36; p = 0.15) or all-cause mortality (HR: 1.07; 95% CI: 0.93 to 1.24; p = 0.36). In contrast, in patients with LVEF ≥45% (n = 184), neprilysin concentration remained prognostically meaningful in the multivariable analysis with both the composite primary endpoint of cardiovascular death or HF hospitalization (HR: 1.28; 95% CI: 1.06 to 1.54; p = 0.01) and cardiovascular death (HR: 1.31; 95% CI: 1.04 to 1.65; p = 0.02).
The identification of circulating neprilysin levels in patients with HF and the positive association of neprilysin with cardiovascular outcomes provides important pathobiological insights into HF syndrome and further supports the importance of neprilysin inhibition for augmenting natriuretic peptides as a therapeutic strategy.
Two major results must be highlighted from our data, including, for the first time, that circulating levels of soluble neprilysin were detected in sera from patients with chronic HF. The production of soluble/non–membrane-associated counterparts of membrane-bound proteins has been studied extensively and is known to occur as a consequence of ectodomain shedding, which involves the proteolytic cleavage of the extracellular domain, or release of non–membrane-associated enzyme from cells via exosomes. Catalytically active neprilysin levels have been detected in both the media of lymphoblastoid cell lines (10) and in serum from coal miners exposed to coal dust (19). This suggests the production of soluble neprilysin detected in these studies was likely mediated by ectodomain cleavage. Recent results suggest that the release of neprilysin from endothelial cells may also occur via exosomes, a process dependent on ADAM-17 (A Disintegrin And Metalloprotease-17) (12). All in all, these data indicate that neprilysin may exist as either a transmembrane or soluble protein, the latter being present in the sera of patients with HF, as shown here. In cardiovascular pathobiology, another example of transmembrane receptor and soluble circulating protein occurs with ST2 (20). Whether circulating neprilysin in the systemic circulation has the potential to cleave substrates throughout the body, leading to a manifestation of systemic effects of neprilysin in patients with HF, is uncertain, yet soluble neprilysin does retain its catalytic activity.
A second major finding of this study was the identification of a positive association between neprilysin levels and cardiovascular outcomes, particularly cardiovascular mortality and HF hospitalizations. In this large, real-life, consecutive cohort of 1,069 patients followed up for a mean of 4.1 years with 356 composite endpoint events, neprilysin was identified as a good pathobiological surrogate of cardiovascular mortality and morbidity, even after adjustment in a very comprehensive multivariable model that included 12 prognostically meaningful variables, including NT-proBNP. Thus, neprilysin inhibition appears to be a desirable target in HF.
The mechanism of action of neprilysin is quite complex and ubiquitous. In addition to natriuretic peptides, neprilysin breaks down other vasoactive peptides with opposing physiological actions, which indicates that neprilysin inhibition would increase the levels of natriuretic peptides and bradykinin (vasodilators) while increasing the levels of angiotensin II and endothelin-1 (vasoconstrictors) (Central Illustration). Remarkably, reports indicate that local infusion of neprilysin inhibitors alone caused predominantly vasoconstriction (21), which is the rationale for combining neprilysin inhibition and angiotensin II receptor blockade in the LCZ696 molecule. ARNIs are a new class of drug developed both to block the renin-angiotensin system and augment natriuretic peptides (4). LCZ696 is an ARNI that has been studied in the PARADIGM-HF trial, the largest, most contemporary, and most geographically diverse mortality and morbidity trial in patients with HFrEF (22). PARADIGM-HF tested the hypothesis that LCZ696 200 mg bid is superior to enalapril 10 mg bid in reducing mortality and morbidity in patients with HFrEF (7,22). The study was closed early because of an excess of reduced mortality in the treatment arm, thus suggesting that LCZ696 is a valuable alternative to an ACEI in patients with HFrEF. Because previous studies have shown that valsartan was not superior for an endpoint of all-cause mortality when combined with an ACEI (23), it is conceivable that the neprilysin inhibition component of LCZ696 played a major role in the clinical benefit found in PARADIGM-HF. Indeed, we demonstrate here that neprilysin remained strongly predictive for the composite endpoint of cardiovascular mortality and HF hospitalizations even in the subgroup of patients who fulfilled the main PARADIGM-HF criteria. Remarkably, the HR for the composite endpoint was even higher in the PARADIGM-like cohort than in the overall real-life cohort (1.23 vs. 1.18). Whether patients receiving LCZ696 in the PARADIGM-HF trial with higher neprilysin levels performed better compared than those with lower neprilysin is beyond the scope of this study.
The main objective of the present study was not to postulate neprilysin as a biomarker but rather to better understand soluble neprilysin pathobiology in HF and to put it in context of the results obtained in PARADIGM-HF. Nevertheless, our results show that soluble neprilysin indeed provides independent information to other biomarkers commonly used for HF risk stratification.
The catalytic activity of neprilysin toward individual natriuretic peptides is different. BNP is initially cleaved at a different site (Met4/Val5) than atrial or C-type natriuretic peptides (Cys7/Phe8) (24,25). Therefore, BNP might only be used as a diagnostic biomarker after extended treatment with LCZ696, whereas NT-proBNP, which is not a substrate for neprilysin, may therefore be used to assess the effect of LCZ696 on disease state, risk stratification, prognosis, and monitoring. This may also explain the lack of association found here between circulating neprilysin and NT-proBNP levels.
We only assessed circulating soluble neprilysin, but in humans, neprilysin is widely expressed in the kidney, lung, endothelial cells, vascular smooth muscle cells, cardiac myocytes, fibroblasts, neutrophils, adipocytes, testes, and brain, with the highest concentrations being present in the renal proximal tubules. In lymphocytes, neprilysin expression is developmentally regulated (26). Thus, the results reported here very likely underrepresent overall neprilysin expression and activity. Moreover, the experimental assay we describe for neprilysin determination has long incubation times, which makes it ill-suited at this time for daily clinical use. Additionally, the study population was a general HF population treated at a specific multidisciplinary HF unit in a tertiary care hospital; most patients were referred from the cardiology department and thus were relatively young men with HF of ischemic origin and reduced LVEF. As such, these results cannot necessarily be extrapolated to a global HF population.
We demonstrate for the first time that high levels of neprilysin are found in the circulation of patients with HF and that neprilysin concentrations are indicators of adverse outcomes for both cardiovascular mortality and morbidity. Neprilysin inhibition, provided by the ARNI LCZ696, is necessary for targeting novel pathophysiological contributors to HF and is crucial for improving patient outcomes.
COMPETENCY IN MEDICAL KNOWLEDGE: Neprilysin is a crucial enzyme that mediates the up-regulation of the natriuretic peptide axis in patients with HF, and inhibition of neprilysin as a therapeutic target is associated with reductions in cardiovascular morbidity and mortality.
TRANSLATIONAL OUTLOOK: Prospective trials are needed to assess tailored strategies of pharmacological neprilysin inhibition based on measurements of plasma neprilysin levels in patients with HF.
The authors thank Beatriz González, Roser Cabanes, Margarita Rodríguez, Nuria Benito, and Alba Ros for data collection and invaluable work in the heart failure clinic and Judit Peñafiel and Joan Vila from IMIM (Hospital del Mar Medical Research Institute), Barcelona, Spain, for their statistical help. The authors also acknowledge Redes Temáticas de Investigación Cooperativa en Salud (RETICS), Red Cardiovascular (RD12/0042/0047).
The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- angiotensin-converting enzyme inhibitor
- angiotensin II receptor blocker
- angiotensin receptor neprilysin inhibitor
- B-type natriuretic peptide
- confidence interval
- estimated glomerular filtration rate
- hazard ratio
- heart failure
- heart failure with reduced ejection fraction
- high-sensitivity troponin T
- left ventricular ejection fraction
- N-terminal pro-B-type natriuretic peptide
- New York Heart Association
- Received October 29, 2014.
- Accepted November 4, 2014.
- American College of Cardiology Foundation
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