ABSTRACT
Cardiovascular diseases (CVD) are among the leading causes of death and disability worldwide. Pregnancy-associated plasma protein-A (PAPP-A) is a matrix metalloprotease localized on the cell surface. One of the substrates that PAPP-A cleaves is the insulin-like growth factor binding protein-4 (IGFBP-4), a member of the family of proteins that bind insulin-like growth factor (IGF). Proteolysis of IGFBP-4 by PAPP-A occurs at a specific site resulting in formation of two proteolytic fragments - N-terminal IGFBP-4 (NT-IGFBP-4) and C-terminal IGFBP-4 (CT-IGFBP-4), and leads to the release of IGF activating various cellular processes including migration, proliferation, and cell growth. Increased levels of the proteolytic IGFBP-4 fragments correlate with the development of CVD complications and increased risk of death in patients with the coronary heart disease, acute coronary syndrome, and heart failure. However, there is no direct evidence that PAPP-A specifically cleaves IGFBP-4 in the cardiac tissue under normal and pathological conditions. In the present study, using a primary culture of rat neonatal cardiomyocytes as a model, we have demonstrated that: 1) proteolysis of IGFBP-4 by PAPP-A occurs in the conditioned medium of cardiomyocytes, 2) PAPP-A-specific IGFBP-4 proteolysis is increased when cardiomyocytes are transformed to a hypertrophic state. Thus, it can be assumed that the enhancement of IGFBP-4 cleavage by PAPP-A and hypertrophic changes in cardiomyocytes accompanying CVD are interrelated, and PAPP-A appears to be one of the activators of the IGF-dependent processes in normal and hypertrophic-state cardiomyocytes.
Subject(s)
Cardiomegaly/enzymology , Insulin-Like Growth Factor Binding Protein 4/metabolism , Myocytes, Cardiac/enzymology , Pregnancy-Associated Plasma Protein-A/metabolism , Proteolysis , Animals , Animals, Newborn , Cardiomegaly/pathology , Cells, Cultured , Myocytes, Cardiac/pathology , RatsABSTRACT
BACKGROUND: Entresto™ is a new heart failure (HF) therapy that includes the neprilysin (NEP) inhibitor sacubitril. One of the NEP substrates is B-type natriuretic peptide (BNP); its augmentation by NEP inhibition is considered as a possible mechanism for the positive effects of Entresto. We hypothesized that the circulating products of BNP proteolysis by NEP might reflect NEP impact on the metabolism of active BNP. We suggest that NEP-based BNP cleavage at position 17-18 results in BNP ring opening and formation of a novel epitope with C-terminal Arg-17 (BNP-neo17 form). In this study, we use a specific immunoassay to explore BNP-neo17 in a rat model and HF patient plasma. METHODS: We injected BNP into rats, with or without NEP inhibition with sacubitril. BNP-neo17 in plasma samples at different time points was measured with a specific immunoassay with neglectable cross-reactivity to intact forms. BNP-neo17 and total BNP were measured in EDTA plasma samples of HF patients. RESULTS: BNP-neo17 generation in rat circulation was prevented by NEP inhibition. The maximum 13.2-fold difference in BNP-neo17 concentrations with and without sacubitril was observed at 2 min after injection. BNP-neo17 concentrations in 32 HF patient EDTA plasma samples ranged from 0 to 37 pg/mL (median, 5.4; interquartile range, 0-9.1). BNP-neo17/total BNP had no correlation with total BNP concentration (with r = -0.175, P = 0.680) and showed variability among individuals. CONCLUSIONS: BNP-neo17 formation is NEP dependent. Considering that BNP-neo17 is generated from the active form of BNP by NEP, we speculate that BNP-neo17 may reflect both the NEP activity and natriuretic potential and serve for HF therapy guidance.
Subject(s)
Heart Failure/blood , Immunoassay/methods , Natriuretic Peptide, Brain/metabolism , Neprilysin/metabolism , Aged , Aged, 80 and over , Aminobutyrates/pharmacology , Animals , Biphenyl Compounds , Cross Reactions , Drug Combinations , Epitopes/metabolism , Heart Failure/drug therapy , Humans , Male , Middle Aged , Natriuretic Peptide, Brain/immunology , Natriuretic Peptide, Brain/pharmacokinetics , Neprilysin/antagonists & inhibitors , Peptide Fragments , Rats, Wistar , Tetrazoles/pharmacology , ValsartanABSTRACT
BACKGROUND: The appearance of B-type natriuretic peptide (BNP) in the blood is ultimately caused by proteolytic processing of its precursor, proBNP. The mechanisms leading to the high plasma concentration of unprocessed proBNP are still poorly understood. The goals of the present study were to examine whether processing of proBNP takes place in the circulation and to evaluate the clearance rate of proBNP and proBNP-derived peptides. METHODS: We studied the processing of human proBNP in the circulation and the clearance rate of proBNP and proBNP-derived peptides (BNP and N-terminal fragment of proBNP, NT-proBNP) in rats by injecting the corresponding peptides and analyzing immunoreactivity at specific time points. Glycosylated and nonglycosylated proBNP and NT-proBNP were used in the experiments. We applied immunoassays, gel filtration, and mass spectrometry (MS) techniques to analyze the circulation-mediated processing of proBNP. RESULTS: ProBNP was effectively processed in the circulation into BNP (1-32) and various truncated BNP forms as confirmed by gel filtration and MS analysis. Glycosylation of proBNP close to the cleavage-site region suppressed its processing in the circulation. The terminal half-life for human glycosylated proBNP was 9.0 (0.5) min compared with 6.4 (0.5) min for BNP. For NT-proBNP, the terminal half-lives were 15.7 (1.4) min and 15.5 (1.3) min for glycosylated and nonglycosylated forms, respectively. CONCLUSIONS: In rats, processing of human proBNP to active BNP occurs in the circulation. The clearance rate of proBNP is quite similar to that of BNP. These observations suggest that peripheral proBNP processing may be an important regulatory step rather than mere degradation.
