Localisation and Receptor Pharmacology of (Pyr1)apelin-13(1-12), the ACE2 Metabolite of (Pyr1)apelin-13

Peiran Yang, Rhoda Kuc, Aimee Brame, Janet Maguire, Nicholas Morrell, Anthony Davenport. University of Cambridge, Cambridge, UK

Background: Apelin, the endogenous ligand of the G protein-coupled receptor APJ, elicits endothelium-dependent vasodilatation, endothelium-independent vasoconstriction in human blood vessels and positive inotropy in human heart, in vitro (Maguire et al., 2009). Isoforms of mature apelin range from apelin-36 to apelin-13, with (Pyr1)apelin-13 the predominant isoform in human heart. Angiotensin-converting enzyme 2 (ACE2), which metabolises angiotensin-II to angiotensin(1-7), has beneficial effects in the cardiopulmonary system and recombinant ACE2 or ACE2 activators may be promising in the treatment of pulmonary arterial hypertension (PAH) (Shenoy et al., 2011). Interestingly, ACE2 cleaves (Pyr1)apelin-13 to (Pyr1)apelin-13(1-12) (Vickers et al., 2002) but it is not known whether this reaction occurs under physiological conditions or whether (Pyr1)apelin-13(1-12) is biologically active. Activating the apelin/APJ system has also been implicated as a therapeutic target in PAH, (Anderson et al., 2011) therefore it is vital to understand the consequence of ACE2 activity on apelin pharmacology.

Hypotheses and Objectives: We hypothesized that (Pyr1)apelin-13(1-12) is generated under physiological conditions in tissues where (Pyr1)apelin-13 and ACE2 are present and that this may be altered in PAH. The first aim was to discover whether (Pyr1)apelin-13(1-12) is present in heart and lung tissues, and to identify co-localization with ACE2 in specific cell types. The second objective was to test if ACE2 inactivates (Pyr1)apelin-13, by confirming whether (Pyr1)apelin-13(1-12) is an APJ agonist using a β-arrestin recruitment assay.

Experimental Approach: Peroxidase-anti-peroxidase and dual-labelling fluorescent immunocytochemistry, using specific ACE2 and (Pyr1)apelin-13(1-12) antibodies, were performed using 30µm sections of heart and lung. These were normal (n=4) and PAH (n=4) human tissues and tissues from healthy male Sprague-Dawley rats (n=4) and the monocrotaline (60mg/kg, n=4)) rat model of PAH. β-Arrestin recruitment by (Pyr1)apelin-13 and (Pyr1)apelin-13(1-12) were compared using expressed human APJ and a β-galactosidase complementation assay. Concentration-response curves were analysed using GraphPad Prism 5 to determine values of pD2 and maximum response.

Key Results: (Pyr1)apelin-13(1-12) was detected in the endothelium of human and rat normal heart and lung and PAH heart, co-localizing with ACE2-like immunoreactivity. (Pyr1)apelin-13(1-12)-like immunoreactivity was reduced/absent in human and rat PAH lungs. In the β-arrestin assay, compared to (Pyr1)apelin-13 (pD2=8.51±0.08), (Pyr1)apelin-13(1-12) was a full agonist (pD2=7.84±0.07, n=5).

Conclusions and implications: This is the first report of endogenous (Pyr1)apelin-13(1-12) expression in human and rat heart and lung with confirmation that this product of ACE2 retains biological activity comparable to that of the parent molecule. These data show a reduction in pulmonary levels of (Pyr1)apelin-13(1-12) in PAH. Therefore, if activation of APJ is beneficial to cardiovascular function then, in addition to targeting this system directly, generation of (Pyr1)apelin-13(1-12) may contribute to the beneficial effects of ACE2 therapy in disease.

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