Angiotensin-Converting Enzyme 2 Metabolizes and Partially Inactivates Pyr-Apelin-13 and Apelin-17

The apelin and angiotensin family of peptides have a wide range of related physiological and pathophysiological effects on the heart and vasculature. Apelin is synthesized as a precursor 77 amino acid pre-pro-peptide and is subsequently processed into a family of apelin peptides, with pyrapelin 13 and apelin 17 being the dominant apelin peptides found in vivo. Apelin acts on the apelin receptor and regulates vascular homeostasis, angiogenesis, myocardial adaptation to stress, and fluid balance, thereby playing a key role in vascular diseases, such as systemic and pulmonary arterial hypertension, myocardial infarction, and heart failure. The C-terminal region of the apelin peptide is central for its overall biological activity. N-terminal deletions of apelin-17 reveal that the 12 C-terminal amino acids are core requirements for the internalization and biological potency of apelin action on the apelin receptor. Indeed, apelin-17-induced internalization of the apelin receptor decreases with every N-terminal deletion to apelin-12. Similarly, the N-terminal residues 2–5 of apelin 13 are critical for functional potency, and the C-terminal sequence consisting of residues 8–11 is important for binding activity and receptor internalization. The C-terminal residue of apelin, phenylalanine, is critical for binding of the apelin peptide to the apelin receptor. However, the functional relevance of the C-terminal phenylalanine Abstract—Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia–reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart. (Hypertension. 2016;68:365-377. DOI: 10.1161/HYPERTENSIONAHA.115.06892.) • Online Data Supplement