[Pyr1]Apelin-13(1–12) Is a Biologically Active ACE2 Metabolite of the Endogenous Cardiovascular Peptide [Pyr1]Apelin-13

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.


INTRODUCTION
Apelins are a family of peptides that activate the apelin receptor (also known as APJ) and have an emerging importance in the physiology and pathophysiology of the cardiovascular system . Apelin peptides are present in human vascular and cardiac endothelial cells (Kleinz and Davenport, 2004) and plasma, with [Pyr 1 ]apelin-13 identified as the most abundant cardiovascular isoform Maguire et al., 2009;Zhen et al., 2013). Apelins mediate three major actions in vitro. Interaction with the apelin receptor on cardiac myocytes causes increased cardiac contractility and inotropic action, with apelin an order of magnitude more potent than endothelin-1. In vessels with an intact endothelium, apelin acts to release vasodilators that may oppose the actions of vasoconstrictors. We have also shown that removal of endothelium unmasks a constrictor response mediated by apelin receptors present on the vascular smooth muscle (Maguire et al., 2009). Importantly, 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(Japp et al., , 2010; Barnes et al., 2013;Brame et al., 2015). In heart failure patients, intracoronary [Pyr 1 ]apelin-13 caused coronary vasodilatation and increased cardiac contractility (Japp et al., 2010;Barnes et al., 2013). Systemic infusions of [Pyr 1 ]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) hypertrophy and right heart failure (Alastalo et al., 2011;Chandra et al., 2011). Therefore the apelin receptor may represent a novel target for future drug development.
Human angiotensin converting enzyme 2 (ACE2) has 40% sequence similarity with the C-terminal dipeptidyl-peptidase, ACE (Donoghue et al., 2000;Tipnis et al., 2000). ACE2 is expressed for example in heart, kidney and lung (Donoghue et al., 2000;Hamming et al., 2004) and is implicated in pathological conditions such as heart failure where it is up-regulated (Zisman et al., 2003;Goulter et al., 2004). A major role of ACE2 is to degrade angiotensin II to angiotensin (1-7) which then acts as a beneficial vasodilator and anti-proliferation agent, counterbalancing the actions of the vasoconstrictor angiotensin II (Santos et al., 2003(Santos et al., , 2008. ACE2 is also a viral receptor for the severe acute respiratory syndrome coronavirus  which down-regulates the enzyme from the cell surface resulting in angiotensin II-induced lung injury (Kuba et al., 2005). This has been the rational for the development of recombinant human ACE2 (rhACE2) in clinical trials for acute lung injury (Haschke et al., 2013). Interestingly, enhancing ACE2 activity pharmacologically or by gene transfer was effective in preventing or reversing PAH (Shenoy et al., 2011;Dai et al., 2015). Some beneficial actions of ACE2 are thought to be mediated by the conversion of angiotensin II to angiotensin(1-7). However, the possibility of interaction of ACE2 with other peptides was not clear until a screen of over 120 biologically active peptides reported only two others to be hydrolyzed with high catalytic efficiency by ACE2; dynorphin A 1-13, which has no reported vasoactivity and apelin-13, or apelin-36, resulting in the removal of the C-terminal phenylalanine, producing the metabolites apelin-13 (1-12) or apelin-36 (1-35) (Vickers et al., 2002). The loss of the terminal phenylalanine in apelin has been assumed to be a mechanism of degradation and inactivation of the peptide. Our aim was to understand the impact of this ACE2 cleavage reaction on the apelin signaling pathway.
Additional β-arrestin assay experiments were performed with [Pyr 1 ]apelin-13 that had been incubated with ACE2 as described above. In this assay control concentration-response curves were constructed to [Pyr 1 ]apelin-13 and [Pyr 1 ]apelin-13 (1-12) and these were compared to concentration-response curves constructed to both agonists following pre-incubation with rhACE2.

In vitro Functional Studies
Vascular smooth muscle apelin receptor-mediated contraction was exploited in a bioassay to compare the in vitro potency of apelin peptides. Experiments were carried out as previously described (Maguire, 2002) in endothelium-denuded saphenous vein with concentration-response curves constructed to [Pyr 1 ]apelin-13 (1-12) and apelin-13(F13A) (1 pmol/L-300 nmol/L). Agonist responses were expressed as a % of a terminal response to KCl (100 mmol/L). The inotropic action of [Pyr 1 ]apelin-13 (1-12) was determined in mouse paced RV (n = 6) and for comparison in two samples of human paced atrial appendage strips as described (Maguire et al., 2009). Data were expressed as % of the terminal response to CaCl 2 . Data from vascular and cardiac experiments were analyzed using a 4-prameter logistic curve (GraphPad Prism 6) to determine values of pD 2 and E MAX .

Systemic Infusions in Rat and Echocardiography
All experiments were performed according to local ethics committee (University College, London) and Home Office (UK) guidelines under the 1986 Scientific Procedures Act and conformed to the Directive 2010/63/EU. The effects of systemic infusion of [Pyr 1 ]apelin-13 (1-12) (incremental bolus doses 1-300 nmol/300 µL) on blood pressure, heart rate, stroke volume and cardiac output were assessed in male Wistar rats (300 ± 25 g body weight) as described (Brame et al., 2015), that were anesthetized with isoflurane (5% induction, 2% maintenance, continuous monitoring throughout). The left carotid artery and right jugular vein were cannulated (0.96 mm polyvinyl chloride tubing). Mean arterial pressure (MAP) was measured throughout the procedure via a pressure transducer (Powerlab AD Instruments, Chalgrove, UK) connected to the arterial line. Baseline hemodynamics were recorded using Chart 7.0 acquisition software and a 16 channel Powerlab system (AD Instruments, Chalgrove, UK) after a 30 min stabilization period. Thoracic echocardiography was performed at a scanning depth of 0-2 cm using a 14 MHz probe (Vivid 7 Dimension, GE Healthcare, Bedford, UK). Pulsed-wave Doppler was used to determine aortic blood flow velocities in the aortic arch. Stroke volume (SV) was determined as the product of the velocity-time integral (VTI) and vessel cross-sectional area.
Data from six consecutive cardiac cycles were used to determine heart rate (HR) and a marker of left ventricular contractility, peak velocity (PV). Values of SV and HR were used to calculate cardiac output (CO). Respiration rate was determined from movement of the diaphragm using time-motion (M)-model. At the end of the study rats were euthanized by intravenous pentobarbitone and exsanguination.

Forearm Venous Occlusion Plethysmography in Human Volunteers
Studies were performed in healthy volunteers (n = 12) in the University of Cambridge Vascular Research Unit, Addenbrooke's Hospital, Cambridge, UK. Volunteer characteristics are given in Table 1. This study was carried out in accordance with the recommendations of the National Research Ethics Service Committee East of England-Cambridge Central with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the National Research Ethics Service Committee East of England-Cambridge Central (REC 11/EE/0305). Changes in forearm blood flow (FBF) in response to [Pyr 1 ]apelin-13 (1-12) (1, 10, 100 nmol/min) were measured as previously described (Brame et al., 2015).
Exclusion criteria were ischemic heart disease, respiratory, renal or neurological disease, diabetes mellitus, hypertension, BMI > 30, BMI < 18; smoker; pregnant; use of vasoactive medication or NSAIDS/aspirin within 48 h of study; current involvement in other research studies. An Omron HEM705CP oscillimetric sphygmomanometer was used to measure blood pressure and heart rate at baseline and then every 6 min in the contralateral arm. Periodically, a cuff around the upper arm was inflated for ∼8 s to 40 mmHg then deflated for 4 s to interrupt venous return and during a 3 min measurement hand circulation was excluded by inflation of wrist cuffs to 200 mmHg. Changes in forearm volume were measured by mercury-in-silastic strain gauge with FBF subsequently expressed as ml/100 ml forearm volume per min. For infusion of peptides via a 16-gauge catheter (Portex, Kent, UK), the brachial artery (non-dominant arm) was cannulated (27-gauge needle, Cooper's Needle Works, Birmingham, UK) under local anesthesia (lignocaine 1%, Hameln Pharmaceuticals Ltd., Gloucester, UK). FBF was measured in both arms and the response to [Pyr 1 ]apelin-13 (1-12) presented as absolute change in forearm blood flow from the pre-infusion baseline value.
[Pyr 1 ]apelin-13 (1-12) , supplied in sealed glass vials and stored at −40 • C until required, was allowed to warm to room temperature and diluted with physiological saline to produce stock solutions that were then filtered (0.2 µm flat filter, Portex, Hythe, UK) before further dilution in saline. [Pyr 1 ]apelin-13 (1-12) was infused in three incremental doses during each visit. Doses were previously optimized in a pilot study. Each dose was infused for 6 min with a 20 min saline infusion washout period before the next dose was administered. At the end of the study sodium nitroprusside was infused at 3µg/min for 6 min as a positive control followed by a saline infusion as a negative control. Values are mean ± SEM.

Statistical Analyses
Measurements are mean ± standard error of the mean (SEM

Materials
All chemical reagents were purchased from Sigma (Poole, UK), unless otherwise stated. [Pyr 1 ]apelin-13 (1-12) was custom synthesized to GLP standard using Fmoc chemistry on a solid phase support matrix to 98% purity by Maldi-TOF Mass spectroscopy and RP-HPLC analysis. Peptides were tested for sterility and demonstrated to be pyrogen free and biological activity confirmed using the β-arrestin assay. All apelin peptides were synthesized by Severn Biotech (Kidderminster, UK).
Biological Activity of [Pyr 1 ]Apelin-13 (1-12) ACE2-mediated hydrolysis has been assumed to inactivate apelin peptides. Wang and colleagues have recently reported that the ACE2 product of [Pyr 1 ]apelin-13 exhibits reduced or absent cardiovascular actions compared to the parent molecule (Wang et al., 2016). However, emerging evidence from structure activity studies suggested that the C-terminal phenylalanine was not a critical residue for apelin biological activity (Fan et al., 2003;Medhurst et al., 2003) and our hypothesis was that compared to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) may retain significant activity. Indeed, our results showed that [Pyr 1 ]apelin-13 (1-12) has nanomolar affinity for the native human apelin receptor exhibiting only a 3-fold reduction in binding affinity compared the parent peptide. This is consistent with previous studies where the C-terminal phenylalanine of [Pyr 1 ]apelin-13 or apelin-13 was replaced with alanine (F13A) (Fan et al., 2003;Medhurst et al., 2003) or removed from apelin-17 (K16P) Iturrioz et al., 2010) with only minimal loss of receptor affinity. Similarly, substitution of the C-terminal phenylalanine of [Pyr 1 ]apelin-13 with D-phenylalanine resulted in only a 20-fold decrease in receptor binding affinity, which was modest compared to substitution of other residues known to be important for binding (Murza et al., 2012). Overall, these studies show that loss of the C-terminal phenylalanine from apelin isoforms does not significantly alter receptor binding.
As described above, amino acid substitution studies have proposed a consensus on those amino acids within apelin-13 that are important for receptor binding and activation (Narayanan et al., 2015). The C-terminal phenylalanine (F13), adjacent proline and the N-terminal pyroglutamic acid were not identified as important. We have investigated whether the truncated peptides apelin-13(R10M), apelin-13(R9P) and apelin-13(P9M) retained significant binding and functional activity with the hypothesis that apelin-13(R10M) was likely to represent the minimum active fragment as it was the shortest fragment containing all the identified important amino acids. Results of the competition binding experiments and β-arrestin recruitment assays supported this hypothesis as do data from previous publications showing diminished G αi -mediated signaling and calcium mobilization when the methionine (position 11 in apelin-13) was removed (Medhurst et al., 2003;Zhang et al., 2014), while others have demonstrated activity of apelin-12 (apelin-13 without the N-terminal pyroglutamic acid) in vivo (Pisarenko et al., 2011). Residues arginine 2 and methionine 11 are indispensable as they are required to form the crucial RPRL motif or provide steric volume (Langelaan et al., 2009;Macaluso and Glen, 2010;Gerbier et al., 2015). In contrast, although the C-terminal phenylalanine has been shown to make specific contacts within the binding pocket of the apelin receptor (Iturrioz et al., 2010), our experimental data suggested that its removal did not abolish binding or functional activity.
Apelin-17, despite its unclear biosynthetic pathway, has been reported to have equal or higher binding affinity and potency in inhibiting cAMP accumulation and inducing receptor internalization compared with [Pyr 1 ]apelin-13 (Medhurst et al., 2003;El Messari et al., 2004). We investigated apelin-17 in our assays and consistently found higher binding affinity and higher potency than [Pyr 1 ]apelin-13 in the cAMP and β-arrestin assays. Intriguingly, apelin-17 appeared to be more biased for βarrestin compared to G protein signaling relative to the reference agonist [Pyr 1 ]apelin-13. This suggests that N-terminal extension of apelin-13 may result in peptides that stabilize different conformations of the apelin receptor and may be a mechanism by which apelin receptor activation is fine-tuned at the cellular level.

AUTHOR CONTRIBUTIONS
All authors contributed either to the conception or design of the work (PY, AB, AD, MS, RG, JC, IW, APD, and JM), or acquired and analyzed data or interpreted data (PY, RK, AB, AD, MS, APD, and JM). All authors agree to be accountable for all aspects of the work.