Abstract 18898: Characterization of Proendothelin-1 Derived Peptides Identifies a Co-secreted Modulator of ET-1 Vasoconstriction, and Provides Insights for Biomarker Measurements
Background: ET-1 is strongly implicated in cardiac and renal pathologies both as a potent vasoconstrictor, and as an inducer of tissue remodeling and fibrosis. However, in experimental and human studies ET antagonists are often ineffective. ET-1 biosynthesis occurs via a 212 amino acid peptide preproendothelin-1 (ppET-1); hence other peptides from this precursor may contribute to the biological effects of EDN1 gene expression.
Methods: Specific immunoassays were used to identify the peptide products of ppET-1 in conditioned media samples from EA.hy 926 and A549 cell lines. Secretion of proET-1 peptides was also investigated using primary cultures of human aortic endothelial (HAEC) and smooth muscle cells (HAVSMC), and cardiomyocytes (HCM). Synthetic proET-1 peptides were used to study their rate of clearance from the circulation in anaesthetized rats, and effects on ET-1 induced vasoconstrictor and blood pressure responses.
Results: Using specific antibodies 3 peptides were identified: NT-proET-1 (ppET-1[18-50]), MP-proET-1 (ppET-1[93-166]) and CT-proET-1 (ppET-1[169-212]). Specific sandwich ELISAs for these three peptides showed that they were secreted from all cell types studied with levels of release correlating with ET-1. Arterial blood measurements after i.v. administration (1 nmol/kg) of peptide showed markedly different elimination rates. NT-proET-1 had the most rapid clearance (<5 min) and CT-proET-1 the slowest (8% of initial values 40 min after injection). On rat mesenteric resistance arteries MP-proET-1 alone showed no significant vasoconstrictor effect up to 10 nM, but after pre-treatment with ET-1 (1 - 3 nM) it produced a concentration dependent vasoconstriction. Pre-incubation of rat mesenteric resistance arteries with 10 nM MP-proET-1 increased the response of 1 nM ET-1 by 5 fold (P <0.002). MP-proET-1 alone (3 nmol/kg) had no effect on blood pressure in anaesthetised rats, but it significantly increased the duration of the pressor response to ET-1 (0.3 nmol/kg, P <0.02).
Conclusions: ProET-1 derived peptides may play an important role in vascular pathologies by modulating ET-1 responses, or the actions of other mediators. The slower systemic clearance of CT-proET-1 favors its use as a biomarker of EDN1-linked pathologies.
- © 2012 by American Heart Association, Inc.