Characterisation of Proendothelin-1 Derived Peptides and Evaluation of Their Utility as Biomarkers of Vascular and Renal Pathologies

Abstract

Endothelin-1 (ET-1), the potent vasoconstrictor peptide, is synthesised from the 212 amino acid precursor preproendothelin-1 (ppET-1). ET-1 is strongly implicated in cardiac and renal pathologies. However, ET receptor antagonists demonstrated only limited efficacy in clinical trials of chronic heart failure (HF) and hypertension. ET-1 has a short circulating half-life and its plasma measurements have been inaccurate. Thus, alternative ppET-1 derived peptides may be more stable and could serve as better biomarkers of ET-1 synthesis. Moreover, alternative ppET-1 derived peptides may contribute to the biological effects resulting from EDN1 gene expression and may be interacting with the vasoconstrictor responses of ET-1 or other mediators. The aims of these investigations were to characterise ppET-1 derived peptide products and to evaluate their potential as biomarkers of ET-1 synthesis. A combination of specific immunoassays and HPLC were used to characterise ppET-1 processing in human endothelial (EA.hy 926) and epithelial (A549) cells in culture. NT-proET-1 (ppET-1[18 – 50]), endothelin-like domain peptide (ELDP, ppET-1[93 – 166]) and CT-proET-1 (ppET-1[169 – 212]) were identified in conditioned media samples as the main ppET-1 derived peptide products. The identities of ELDP and CT-proET-1 were confirmed by LC-MS/MS mass spectrometry. The three ppET-1 peptides were chemically synthesised and their in vivo clearance was investigated in male Wistar rats. Arterial plasma levels after intravenous administration of proET-1 peptides showed rapid clearance (<5 min) of NT-proET-1, while CT-proET-1 had the slowest clearance rate. Studies of proET-1 peptide stability in blood samples also showed NT-proET-1 had lower stability. Specific double-recognition site sandwich ELISAs optimised for plasma measurements were used to evaluate plasma concentrations of ELDP and CT-proET-1 in patients with chronic HF and chronic kidney disease (CKD). In conclusion, the results described in this thesis provide further evidence linking ET-1 to cardiac and renal disease processes. But the small differences between healthy individuals and patients with cardiovascular or renal disease indicate only a limited potential for proET-1 peptides as diagnostic biomarkers of EDN1-linked pathologies.