Relevance of Neutrophil Neprilysin in Heart Failure

Abstract

Significant expression of neprilysin (NEP) is found on neutrophils, which present the transmembrane integer form of the enzyme. This study aimed to investigate the relationship of neutrophil transmembrane neprilysin (mNEP) with disease severity, adverse remodeling, and outcome in HFrEF. In total, 228 HFrEF, 30 HFpEF patients, and 43 controls were enrolled. Neutrophil mNEP was measured by flow-cytometry. NEP activity in plasma and blood cells was determined for a subset of HFrEF patients using mass-spectrometry. Heart failure (HF) was characterized by reduced neutrophil mNEP compared to controls (p < 0.01). NEP activity on peripheral blood cells was almost 4-fold higher compared to plasma NEP activity (p = 0.031) and correlated with neutrophil mNEP (p = 0.006). Lower neutrophil mNEP was associated with increasing disease severity and markers of adverse remodeling. Higher neutrophil mNEP was associated with reduced risk for mortality, total cardiovascular hospitalizations, and the composite endpoint of both (p < 0.01 for all). This is the first report describing a significant role of neutrophil mNEP in HFrEF. The biological relevance of neutrophil mNEP and exact effects of angiotensin-converting-enzyme inhibitors (ARNi) at the neutrophil site have to be determined. However, the results may suggest early initiation of ARNi already in less severe HF disease, where effects of NEP inhibition may be more pronounced.

Keywords: CD10; biomarker; heart failure; neprilysin; neutrophils.

Figure 1

(A) Neutrophil membrane-associated neprilysin (mNEP) expression in HFrEF, HFpEF and controls. (B) Timely variance of neutrophil mNEP expression. (C) Mass spectrometry-based determination of the enzymatic activity of NEP in whole blood, peripheral cells, and plasma (n = 40). (D) Scatter plot with linear regression analysis and the Spearman rho correlation coefficient for neutrophil mNEP expression with peripheral cell mNEP activity. Comparison between groups was assessed by using the Kruskal–Wallis test, Mann–Whitney test, and paired Wilcoxon test. Levels of significance are indicated in the respective plots. Tukey boxplots are shown in (A,B); (C) displays geometric means and 95% confidence intervals.

Figure 2

Relationship of neutrophil membrane-associated neprilysin (mNEP) expression with heart failure (HF) severity, reflected by (A) New York Heart Association (NYHA) class and tertiles of N-terminal pro-brain natriuretic peptide (NT-proBNP), (B) HF etiology (non-ischemic vs. ischemic) and renin-angiotensin-system (RAS) inhibitor therapy, and (C) adverse remodeling indicated by echocardiographic indices of left heart function and right heart function. Comparison between groups was assessed by using the Kruskal–Wallis test, levels of significance are indicated in the respective plots. The respective plots display individuals values as well as median and interquartile range. LVF—left ventricular function; LVEDD—left ventricular end-diastolic diameter; MR—mitral regurgitation; RVF—right ventricular function; sPAP—systolic pulmonary artery pressure; TR—tricuspid regurgitation.

Figure 3

Neutrophil membrane-associated neprilysin (mNEP) expression and outcome. Unadjusted and adjusted effects of neutrophil mNEP expression on all-cause mortality, total cardiovascular (CV) hospitalizations, and the composite endpoint of both in HFrEF patients. Hazard ratios (HR) refer to a 1-interquartile range (IQR) increase in continuous variables. Hazard ratios (HR) are adjusted (adj.) for influencing variables, i.e., age, glomerular filtration rate (GFR), sex, and N-terminal pro-brain natriuretic peptide (NT-proBNP). Kaplan–Meier analysis for the primary outcome all-cause mortality with low and high neutrophil mNEP expression with the median MFI as the cut-off value. Comparison was calculated by the log-rank test.

Figure 4

Central illustration. Neprilysin (NEP) can be released in the circulation by ectodomain shedding resulting in a soluble form of NEP. Besides the non-membrane associated form of NEP, NEP can be detected on neutrophils (cellular transmembrane NEP [mNEP]) by flow cytometry. Low neutrophil mNEP is significantly associated with progressing heart failure severity, reflected by New York Heart Association classification and N-terminal pro-brain natriuretic peptide, adverse remodeling, and poor prognosis. NEP activity of the circulating cellular compartment determined by liquid chromatography-tandem mass spectrometry (LC-MS) is markedly increased compared with plasma and correlates with neutrophil mNEP expression.