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

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) (pK_i = 8.04 ± 0.06) and apelin-13(F13A) (pK_i = 8.07 ± 0.24) competed with [¹²⁵I]apelin-13 binding with nanomolar affinity, 4-fold lower than for [Pyr¹]apelin-13 (pK_i = 8.83 ± 0.06) whereas apelin-17 exhibited highest affinity (pK_i = 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.

Keywords: ACE2; [Pyr1]apelin-13(1–12); apelin; apelin receptor; biased signaling; forearm plethysmography; human heart; pulmonary arterial hypertension.

Figure 1

Aligned amino acid sequences of apelin peptides. Hydrophobic amino acids are shown in green, uncharged polar amino acids in yellow, basic amino acids in blue and pyroglutamate in red.

Figure 2

Selectivity of antisera determined by ELISA. (A) [Pyr¹]apelin-13_(1–12) antiserum demonstrated selectivity to the antigen CKGPMP (, corresponding to [Pyr¹]Apelin-13_(1–12)) relative to CKGPMPF (, corresponding to [Pyr¹]Apelin-13). (B) The apelin-12 antiserum demonstrated selectivity to [Pyr¹]Apelin-13 () relative to [Pyr¹]Apelin-13_(1–12) ().

Figure 3

Cleavage of [Pyr¹]apelin-13 to [Pyr¹]apelin-13_(1–12) by rhACE2 in vitro. Maldi-TOF spectra for (A) [Pyr¹]apelin-13, (B) [Pyr¹]apelin-13_(1–12), (C) [Pyr¹]apelin-13 incubated with rhACE2, (D) [Pyr¹]apelin-13_(1–12) incubated with rhACE2, (E) rhACE2 alone.

Figure 4

Detection of endogenous [Pyr¹]apelin-13_(1–12) peptide in human cardiovascular tissues. Bright field microphotographs of [Pyr¹]apelin-13_(1–12)-LI in (A) vascular and (B) endocardial endothelium and (C) vWF-LI in cardiomyopathy human heart. Confocal microphotographs of (D) [Pyr¹]apelin-13_(1–12)-LI (green), (E) vWF-LI (red) and (F) their overlay in normal human lung. (G) [Pyr¹]apelin-13_(1–12)-LI (green), (H) ACE2-LI (red) and (I) their overlay in normal human lung. Bright field microphotographs of (J) [Pyr¹]apelin-13_(1–12)-LI in normal human lung and (K,L) the absence of [Pyr¹]apelin-13_(1–12)-LI in PAH human lung tissue. LV, cardiomyopathy human heart; AP, [Pyr¹]apelin-13_(1–12); NL, normal human lung; PAHL, PAH human lung. Scale bar = 200 μm.

Figure 5

[Pyr¹]apelin-13_(1–12) binds to and activates the human apelin receptor. (A) Competition binding curve for [Pyr¹]apelin-13 () and [Pyr¹]apelin-13_(1–12) () in human left ventricle (n = 3). In cell based assays [Pyr¹]apelin-13 (), [Pyr¹]apelin-13_(1–12) (), and apelin-17 () (B) inhibited forskolin-stimulated cAMP production; (C) induced β-arrestin recruitment and (D) triggered apelin receptor internalization.

Figure 6

Responses to (A) [Pyr¹]apelin-13_(1–12) (, n = 9) and apelin-13(F13A) (, n = 9) in endothelium-denuded human saphenous vein in vitro. (B) Inotropic responses to (B) [Pyr¹]apelin-13_(1–12) (n = 6) and (C) isoprenaline (n = 4) in mouse paced right ventricle.

Figure 7

In vivo actions of [Pyr¹]apelin-13_(1–12) in rat. (A) Administration of intravenous [Pyr¹]apelin-13_(1–12) (, n = 5) resulted in a significant dose-dependent decrease in blood pressure in rat in vivo. Other parameters (B) stroke volume (SV), (C) cardiac output (CO), (D) peak velocity (PV) and velocity-time interval (VTI) were unaffected at any dose of [Pyr¹]apelin-13_(1–12). Significantly different from baseline; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. One-way ANOVA repeated measures compared to baseline with Dunnett's multiple comparison test.

Figure 8

In vivo actions of [Pyr¹]apelin-13_(1–12) in human volunteers. Infusion of [Pyr¹]apelin-13_(1–12) in healthy human volunteers (, n = 12) resulted in a dose-dependent increase in forearm blood flow. Significantly different from baseline; ^*P < 0.05, ^**P < 0.01. One-way ANOVA repeated measures compared to baseline with Dunnett's multiple comparison test.

Figure 9

Schematic diagram showing the interactions between apelin/apelin receptor with ACE2 of the renin angiotensin system. New contributions of this study are shown in red arrows. Ang, angiotensin; APJ, apelin receptor; AT1R, angiotensin receptor type I; MAS, Mas receptor.