P1620Association of granulocyte neprilysin (CD10) expression with prognosis in heart failure with reduced ejection fraction
Authors: Arfsten H , Novak J , Cho A , Goliasch G , Bartko P E , Strunk G , Huelsmann M , Pavo N 
Published: October 2019

Abstract

Background
The exact mechanism of action of neprilysin inhibition (NEPi) is still a subject of debate. The soluble form of the enzyme (sNEP), detectable in plasma, is discussed controversially as a potential biomarker in heart failure with reduced ejection fraction (HFrEF). NEP is present on solid tissues but identically to CD10, expressed on the surface of leukocytes under physiological conditions. The possible impact of NEP expression on peripheral leukocytes on prognosis and its association with sNEP levels have not been investigated yet.

Methods
99 stable HFrEF patients were prospectively enrolled and clinically followed-up. Laboratory markers including NT-proBNP were assessed. NEP (CD10) expression on peripheral blood cells were measured by flow cytometry using a combination of six antibodies with fluorescence minus one samples as control [CD3 (#555339), CD19 (#555413), CD56 (#335826), CD16 (#561306), CD14 (#562692), +/−CD10 (#332777); BD Biosciences,USA]. sNEP levels were determined by a specific ELISA [SEB785Hu, USCN, China].Associations between NEP expression and heart failure severity, sNEP levels and all-cause mortality were determined.

Results
Median age was 65 years (IQR: 55–73), 75% were male. Median NT-proBNP level was 1700pg/ml (IQR: 794–4009).NEP was expressed on granulocytes with 94.8% (IQR: 90.5–97.4) of CD10+ cells and measurable on B-cells and monocytes with 8.5% (IQR: 5.3–13.5) and 0.8% (IQR: 0.4–1.5) of CD10+ cells of the respective leukocyte subtype. NEP expression on T-cells was not detectable. The mean fluorescence intensity (MFI) of CD10+ cells was 5461 (IQR: 4028–6904) for granulocytes, 640 (IQR: 535–740) for B-cells and 1589 (IQR: 1395–1975) for monocytes. Granulocyte NEP expression, but not NEP expression on B-cells or monocytes, correlated inversely with heart failure severity reflected by NT-proBNP level (r=−0.46, p<0.001) and NYHA class (p=0.013) (Figure A). sNEP concentrations correlated weakly with NEP expression on granulocytes (r=0.22, p=0.030) as well as the MFI of CD10+ granulocytes (r=0.31, p=0.003). 15% of the patients died during a median FUP of 24 (IQR: 23–28) months. Increased NEP expression on granulocytes was indicative for better overall survival even after adjustment for age and kidney function [adj. HR per 1-IQR increase of MFI 0.41 (95% CI: 0.18–0.94), p=0.035]. Kaplan-Meier analysis illustrates the impact of granulocyte NEP expression on outcome graphically (Figure B).
Figure 1

Conclusions
Albeit beneficial effects of NEPi by ARNI therapy, NEP expression on granulocytes is inversely correlated with heart failure severity and mortality. The results support the inverse relationship between BNP and plasma NEP activity reported for a mixed population of heart failure patients. The positive correlation of granulocyte NEP expression and sNEP indicates a possible contribution of shed membrane NEP molecules to plasma NEP levels as a surrogate marker. The utility of granulocyte NEP expression or sNEP as biomarkers in HFrEF have to be further evaluated.