Functional activity of apelin peptides in human saphenous vein

Aimee Brame, Janet Maguire, Rhoda Kuc, Anthony Davenport. University of Cambridge,Clinical Phramacology Unit,, CB2 0QQ, Cambridge, United Kingdom.

Apelin peptides are endogenous ligands for the G-protein coupled apelin receptor. We have shown that apelins function as endothelium-dependent vasodilators and are the most potent positive inotropic agents yet identified (Kleinz et al, 2004). In the absence of functional endothelium, apelins have a vascoconstrictor action which may have a role in cardiovascular pathology. We have previously localised the apelin receptor to human cardiomyocytes, vascular endothelial and smooth muscle cells. Apelin peptide expression was restricted to endothelial cells with [Pyr1]apelin-13 the most abundant form in human heart.

ACE-2 degrades angiotensin-II to angiotensin(1-7), is a viral receptor for SARS and the expression is modified in disease. Interestingly, ACE-2 has been identified as the only enzyme which cleaves apelin peptides in vitro (Vickers et al 2002). We hypothesized that [pyr1]apelin-13 released from endothelial cells may undergo further processing by cardiac ACE-2. Therefore our aim was to localise ACE-2 immunoreactivity to human cardiac tissues and to determine if the putative cleavage product [pyr1]apelin-12 retained biological activity in our human saphenous vein constrictor assay.

Human tissues were obtained with ethical approval and informed consent from patients undergoing cardiac surgery. Specific ACE-2-like immunoreactivity was determined in fresh frozen heart (left and right ventricle, n = 6) and visualised using the peroxidise anti-peroxidise method (Davenport 2005). Rings (4mm) of endothelium denuded saphenous vein (n = 6) were set up for isometric tension recordings in oxygenated Krebs solution (37°C). Cumulative concentration-response curves were constructed to apelin peptides (1.5pM-30nM) and experiments terminated with 100mM KCl. Peptide responses were expressed as %KCl. Data were fitted to a four parameter logistic equation (FigP, Biosoft, Cambridge, UK) to obtain values for potency (pD2) and maximum response (Emax) expressed as mean±s.e.mean. n-Values are the number of patients from whom tissue was obtained.

In sections of human heart, ACE-2-like immunoreactivity was observed in cardiomyocytes, endocardial endothelial cells and to vascular endothelium and smooth muscle of both intramyocardial and epicardial vessels.

Table 1. Vasoconstrictor responses to apelin peptides in human saphenous vein.

In saphenous vein, [pyr1]apelin-12 retained comparable agonist activity to [pyr1]apelin-13 (Table 1) suggesting that ACE-2 does not inactivate [pyr1]apelin-13.

The cardiac distribution of ACE-2 is similar to that of the apelin receptor. These data support the hypothesis that [pyr1]apelin-13 released from cardiac endothelial cells may undergo further processing by ACE-2 within the heart, furthermore our functional data indicate the cleaved peptide is biologically active, the significance of which is to be determined.

Davenport AP (editor), Receptor Binding Techniques, 2nd Edition, Humana Press, 2005.

Kleinz, M. J. and Davenport, A. P., (2004), Regul Pept, 118(3), 119-25

Vickers, C. et al., (2002), J Biol Chem, 277, 14838-14843