Neprilysin 2 catalyses the degradation of natriuretic peptides despite sacubitrilat Inhibition

Figures

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  Fig. 1. Electrophoretic analysis of RT-PCR products obtained from RNA isolated from HUVECs, neurons, and cardiomyocytes. RPL, NEP, and NEP2 RT-PCR products were obtained using primers specific to the RPL13A, NEP, and NEP2 genes, respectively. The expected lengths of the reaction products were 185 bp for RPL13A, 184 bp for NEP, and 165 bp for NEP2.

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  Fig. 2. Analysis of ANP and BNP cleavage by NEP and NEP2 in vitro. The NPs were incubated with NEP, NEP2 or without enzymes in the presence or absence of inhibitors: phosphoramidon (probes NEP + Ph. and NEP2 + Ph.) and sacubitrilat (NEP + Sac. and NEP2 + Sac.). (A and C). ANP immunoreactivity was assessed using intact-ring ANP-s-IA. BNP immunoreactivity assessed with intact-ring BNP s-IA. Error bars represent SD. In all cases, concentrations are expressed relative to the initial concentration of the peptide in the absence of the enzyme at each time point. Asterisks mark points where concentration difference from no enzyme probe was statistically significant (P < 0.05).

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  Fig. 3. Analysis of proBNP cleavage by NEP2. ProBNP was incubated with NEP, NEP2 or without enzymes in the presence or absence of phosphoramidon (probes NEP + Ph. and NEP2 + Ph.). (A) Immunoreactivity of proBNP, assessed with intact-ring BNP s-IA, shown as the percentage of peptide immunoreactivity in the enzyme-free sample. Error bars represent SD. In all cases, concentrations are expressed relative to the initial concentration of the peptide in the absence of the enzyme at each time point. Asterisks mark points where concentration difference from no enzyme probe was statistically significant (P < 0.005). (B) MS analysis of cleavage samples before (left panel) and after 2 h incubation (right panel).

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  Fig. 4. Analysis of the formation of neo-epitopes during BNP and proBNP cleavage by NEP and NEP2. The NPs were incubated with NEP, NEP2 or without enzymes in the presence or absence of inhibitors: phosphoramidon (probes NEP + Phosphoramidon and NEP2 + Phosphoramidon). (A and B) BNP proteolysis, neo5 and neo17 form generation, respectively. (C and D) proBNP proteolysis, neo5 and neo17 form generation, respectively. Error bars represent SD. Asterisks mark points where concentration difference from no enzyme probe was statistically significant (P < 0.005).