AUTHOR=Yang Peiran , Kuc Rhoda E. , Brame Aimée L. , Dyson Alex , Singer Mervyn , Glen Robert C. , Cheriyan Joseph , Wilkinson Ian B. , Davenport Anthony P. , Maguire Janet J. 

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

JOURNAL=Frontiers in Neuroscience

VOLUME=11

YEAR=2017

URL=https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.2017.00092

DOI=10.3389/fnins.2017.00092

ISSN=1662-453X

ABSTRACT=<p><bold>Aims:</bold> Apelin is a predicted substrate for ACE2, a novel therapeutic target. Our aim was to demonstrate the endogenous presence of the putative ACE2 product [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> in human cardiovascular tissues and to confirm it retains significant biological activity for the apelin receptor <italic>in vitro</italic> and <italic>in vivo</italic>. The minimum active apelin fragment was also investigated.</p><p><bold>Methods and Results:</bold> [Pyr<sup>1</sup>]apelin-13 incubated with recombinant human ACE2 resulted in de novo generation of [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> identified by mass spectrometry. Endogenous [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> 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 [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> (pK<sub>i</sub> = 8.04 ± 0.06) and apelin-13(F13A) (pK<sub>i</sub> = 8.07 ± 0.24) competed with [<sup>125</sup>I]apelin-13 binding with nanomolar affinity, 4-fold lower than for [Pyr<sup>1</sup>]apelin-13 (pK<sub>i</sub> = 8.83 ± 0.06) whereas apelin-17 exhibited highest affinity (pK<sub>i</sub> = 9.63 ± 0.17). The rank order of potency of peptides to inhibit forskolin-stimulated cAMP was apelin-17 (pD<sub>2</sub> = 10.31 ± 0.28) &gt; [Pyr<sup>1</sup>]apelin-13 (pD<sub>2</sub> = 9.67 ± 0.04) ≥ apelin-13(F13A) (pD<sub>2</sub> = 9.54 ± 0.05) &gt; [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> (pD<sub>2</sub> = 9.30 ± 0.06). The truncated peptide apelin-13(R10M) retained nanomolar potency (pD<sub>2</sub> = 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 (pD<sub>2</sub> = 10.26 ± 0.09) &gt;&gt; [Pyr<sup>1</sup>]apelin-13 (pD<sub>2</sub> = 8.43 ± 0.08) &gt; apelin-13(R10M) (pD<sub>2</sub> = 8.26 ± 0.17) &gt; apelin-13(F13A) (pD<sub>2</sub> = 7.98 ± 0.04) ≥ [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> (pD<sub>2</sub> = 7.84 ± 0.06) &gt;&gt; shorter fragments (pD<sub>2</sub> &lt; 6). [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> and apelin-13(F13A) contracted human saphenous vein with similar sub-nanomolar potencies and [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> was a potent inotrope in paced mouse right ventricle and human atria. [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> elicited a dose-dependent decrease in blood pressure in anesthetized rat and dose-dependent increase in forearm blood flow in human volunteers.</p><p><bold>Conclusions:</bold> We provide evidence that ACE2 cleaves [Pyr<sup>1</sup>]apelin-13 to [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> and this cleavage product is expressed in human cardiovascular tissues. We have demonstrated biological activity of [Pyr<sup>1</sup>]apelin-13<sub>(1–12)</sub> at the human and rodent apelin receptor <italic>in vitro</italic> and <italic>in vivo</italic>. 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.</p>