Abstract
Aims: Apelin is a predicted substrate for ACE2, a novel therapeutic target. Our aim was to demonstrate the endogenous presence of the putative ACE2 product [Pyr1]apelin-13(1–12) in human cardiovascular tissues and to confirm it retains significant biological activity for the apelin receptor in vitro and in vivo. The minimum active apelin fragment was also investigated.Methods and Results: [Pyr1]apelin-13 incubated with recombinant human ACE2 resulted in de novo generation of [Pyr1]apelin-13(1–12) identified by mass spectrometry. Endogenous [Pyr1]apelin-13(1–12) was detected by immunostaining in human heart and lung localized to the endothelium. Expression was undetectable in lung from patients with pulmonary arterial hypertension. In human heart [Pyr1]apelin-13(1–12) (pKi = 8.04 ± 0.06) and apelin-13(F13A) (pKi = 8.07 ± 0.24) competed with [125I]apelin-13 binding with nanomolar affinity, 4-fold lower than for [Pyr1]apelin-13 (pKi = 8.83 ± 0.06) whereas apelin-17 exhibited highest affinity (pKi = 9.63 ± 0.17). The rank order of potency of peptides to inhibit forskolin-stimulated cAMP was apelin-17 (pD2 = 10.31 ± 0.28) > [Pyr1]apelin-13 (pD2 = 9.67 ± 0.04) ≥ apelin-13(F13A) (pD2 = 9.54 ± 0.05) > [Pyr1]apelin-13(1–12) (pD2 = 9.30 ± 0.06). The truncated peptide apelin-13(R10M) retained nanomolar potency (pD2 = 8.70 ± 0.04) but shorter fragments exhibited low micromolar potency. In a β-arrestin recruitment assay the rank order of potency was apelin-17 (pD2 = 10.26 ± 0.09) >> [Pyr1]apelin-13 (pD2 = 8.43 ± 0.08) > apelin-13(R10M) (pD2 = 8.26 ± 0.17) > apelin-13(F13A) (pD2 = 7.98 ± 0.04) ≥ [Pyr1]apelin-13(1–12) (pD2 = 7.84 ± 0.06) >> shorter fragments (pD2 < 6). [Pyr1]apelin-13(1–12) and apelin-13(F13A) contracted human saphenous vein with similar sub-nanomolar potencies and [Pyr1]apelin-13(1–12) was a potent inotrope in paced mouse right ventricle and human atria. [Pyr1]apelin-13(1–12) elicited a dose-dependent decrease in blood pressure in anesthetized rat and dose-dependent increase in forearm blood flow in human volunteers.Conclusions: We provide evidence that ACE2 cleaves [Pyr1]apelin-13 to [Pyr1]apelin-13(1–12) and this cleavage product is expressed in human cardiovascular tissues. We have demonstrated biological activity of [Pyr1]apelin-13(1–12) at the human and rodent apelin receptor in vitro and in vivo. Our data show that reported enhanced ACE2 activity in cardiovascular disease should not significantly compromise the beneficial effects of apelin based therapies for example in PAH.
Highlights
Apelins are a family of peptides that activate the apelin receptor and have an emerging importance in the physiology and pathophysiology of the cardiovascular system (Yang et al, 2015)
Synthesis of [Pyr1]apelin-13(1–12) from [Pyr1]apelin-13 was confirmed by incubating [Pyr1]apelin-13 (5 nmol) with recombinant human ACE2 (rhACE2) enzyme (5 pmol, GSK, Ware, UK) in buffer containing 2-(N-morpholino)ethanesulfonic acid (MES, 500 mmol/L), NaCl (300 mmol/L) and ZnCl2 (10 μmol/L) for 2 h at 37◦C followed by quenching with 10 μmol ethylenediaminetetraacetic acid (EDTA)
In human volunteers (n = 12), [Pyr1]apelin-13(1–12) (Figure 8) produced comparable dose-dependent increases in forearm blood flow to that which we have shown for [Pyr1]apelin-13 (Brame et al, 2015)
Summary
Apelins are a family of peptides that activate the apelin receptor ( known as APJ) and have an emerging importance in the physiology and pathophysiology of the cardiovascular system (Yang et al, 2015). In healthy volunteers and heart failure patients, the major effect of apelin infused into the forearm in vivo was nitric oxide dependent arterial dilatation (Japp et al, 2008, 2010; Barnes et al, 2013; Brame et al, 2015). Intracoronary [Pyr1]apelin-13 caused coronary vasodilatation and increased cardiac contractility (Japp et al, 2010; Barnes et al, 2013). Systemic infusions of [Pyr1]apelin-13 in both volunteers and patients increased cardiac index and lowered mean arterial blood pressure and peripheral vascular resistance (Japp et al, 2010; Barnes et al, 2013). Apelin is down-regulated in pulmonary arterial hypertension (PAH), a devastating disease characterized by vascular remodeling resulting in progressive obliteration of the pulmonary circulation, leading to right ventricle (RV)
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