CORD-19:671f1c2956296e84da394af512513ea165882eba JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
TextSentencer_T1 0-124 Sentence denotes [Pyr 1 ]Apelin-13 (1-12) Is a Biologically Active ACE2 Metabolite of the Endogenous Cardiovascular Peptide [Pyr 1 ]Apelin-13
TextSentencer_T1 0-124 Sentence denotes [Pyr 1 ]Apelin-13 (1-12) Is a Biologically Active ACE2 Metabolite of the Endogenous Cardiovascular Peptide [Pyr 1 ]Apelin-13
TextSentencer_T2 126-134 Sentence denotes Abstract
TextSentencer_T2 126-134 Sentence denotes Abstract
TextSentencer_T3 135-323 Sentence denotes We provide evidence that ACE2 cleaves [Pyr 1 ]apelin-13 to [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and this cleavage product is expressed in human cardiovascular tissues.
TextSentencer_T3 135-323 Sentence denotes We provide evidence that ACE2 cleaves [Pyr 1 ]apelin-13 to [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) and this cleavage product is expressed in human cardiovascular tissues.
TextSentencer_T4 324-487 Sentence denotes We have demonstrated biological activity of [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) at the human and rodent apelin receptor in vitro and in vivo.
TextSentencer_T4 324-487 Sentence denotes We have demonstrated biological activity of [Pyr 1 ]apelin-13 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) at the human and rodent apelin receptor in vitro and in vivo.
TextSentencer_T5 488-669 Sentence denotes 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.
TextSentencer_T5 488-669 Sentence denotes 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.
TextSentencer_T6 671-676 Sentence denotes Aims:
TextSentencer_T6 671-676 Sentence denotes Aims:
TextSentencer_T7 677-746 Sentence denotes Apelin is a predicted substrate for ACE2, a novel therapeutic target.
TextSentencer_T7 677-746 Sentence denotes Apelin is a predicted substrate for ACE2, a novel therapeutic target.
TextSentencer_T8 747-1031 Sentence denotes Our aim was to demonstrate the endogenous presence of the putative ACE2 product [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in human cardiovascular tissues and to confirm it retains significant biological activity for the apelin receptor in vitro and in vivo.
TextSentencer_T8 747-1031 Sentence denotes Our aim was to demonstrate the endogenous presence of the putative ACE2 product [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in human cardiovascular tissues and to confirm it retains significant biological activity for the apelin receptor in vitro and in vivo.
TextSentencer_T9 1032-1089 Sentence denotes The minimum active apelin fragment was also investigated.
TextSentencer_T9 1032-1089 Sentence denotes The minimum active apelin fragment was also investigated.
TextSentencer_T10 1090-1302 Sentence denotes Methods and Results: [Pyr 1 ]apelin-13 incubated with recombinant human ACE2 resulted in de novo generation of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) identified by mass spectrometry.
TextSentencer_T10 1090-1302 Sentence denotes Methods and Results: [Pyr 1 ]apelin-13 incubated with recombinant human ACE2 resulted in de novo generation of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) identified by mass spectrometry.
TextSentencer_T11 1303-1467 Sentence denotes Endogenous [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) was detected by immunostaining in human heart and lung localized to the endothelium.
TextSentencer_T11 1303-1467 Sentence denotes Endogenous [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) was detected by immunostaining in human heart and lung localized to the endothelium.
TextSentencer_T12 1468-1555 Sentence denotes Expression was undetectable in lung from patients with pulmonary arterial hypertension.
TextSentencer_T12 1468-1555 Sentence denotes Expression was undetectable in lung from patients with pulmonary arterial hypertension.
TextSentencer_T13 1556-1850 Sentence denotes In human heart [Pyr 1 ]apelin-13 (1-12) (pK i = 8.04 ± 0.06) and apelin-13(F13A) (pK i = 8.07 ± 0.24) competed with [ 125 I]apelin-13 binding with nanomolar affinity, 4-fold lower than for [Pyr 1 ]apelin-13 (pK i = 8.83 ± 0.06) whereas apelin-17 exhibited highest affinity (pK i = 9.63 ± 0.17).
TextSentencer_T13 1556-1850 Sentence denotes In human heart [Pyr 1 ]apelin-13 (1-12) (pK i = 8.04 ± 0.06) and apelin-13(F13A) (pK i = 8.07 ± 0.24) competed with [ 125 I]apelin-13 binding with nanomolar affinity, 4-fold lower than for [Pyr 1 ]apelin-13 (pK i = 8.83 ± 0.06) whereas apelin-17 exhibited highest affinity (pK i = 9.63 ± 0.17).
TextSentencer_T14 1851-2090 Sentence denotes The rank order of potency of peptides to inhibit forskolin-stimulated cAMP was apelin-17 (pD 2 = 10.31 ± 0.28) > [Pyr 1 ]apelin-13 (pD 2 = 9.67 ± 0.04) ≥ apelin-13(F13A) (pD 2 = 9.54 ± 0.05) > [Pyr 1 ]apelin-13 (1-12) (pD 2 = 9.30 ± 0.06).
TextSentencer_T14 1851-2090 Sentence denotes The rank order of potency of peptides to inhibit forskolin-stimulated cAMP was apelin-17 (pD 2 = 10.31 ± 0.28) > [Pyr 1 ]apelin-13 (pD 2 = 9.67 ± 0.04) ≥ apelin-13(F13A) (pD 2 = 9.54 ± 0.05) > [Pyr 1 ]apelin-13 (1-12) (pD 2 = 9.30 ± 0.06).
TextSentencer_T15 2091-2232 Sentence denotes The truncated peptide apelin-13(R10M) retained nanomolar potency (pD 2 = 8.70 ± 0.04) but shorter fragments exhibited low micromolar potency.
TextSentencer_T15 2091-2232 Sentence denotes The truncated peptide apelin-13(R10M) retained nanomolar potency (pD 2 = 8.70 ± 0.04) but shorter fragments exhibited low micromolar potency.
TextSentencer_T16 2233-2955 Sentence denotes In a β-arrestin recruitment assay the rank order of potency was apelin-17 (pD 2 = 10.26 ± 0.09) >> [Pyr 1 ]apelin-13 (pD 2 = 8.43 ± 0.08) > apelin-13(R10M) (pD 2 = 8.26 ± 0.17) > apelin-13(F13A) (pD 2 = 7.98 ± 0.04) ≥ [Pyr 1 ]apelin-13 (1-12) (pD 2 = 7.84 ± 0.06) >> shorter fragments (pD 2 < 6). [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) and apelin-13(F13A) contracted human saphenous vein with similar sub-nanomolar potencies and [Pyr 1 ]apelin-13 (1-12) was a potent inotrope in paced mouse right ventricle and human atria. [Pyr 1 ]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.
TextSentencer_T16 2233-2955 Sentence denotes In a β-arrestin recruitment assay the rank order of potency was apelin-17 (pD 2 = 10.26 ± 0.09) >> [Pyr 1 ]apelin-13 (pD 2 = 8.43 ± 0.08) > apelin-13(R10M) (pD 2 = 8.26 ± 0.17) > apelin-13(F13A) (pD 2 = 7.98 ± 0.04) ≥ [Pyr 1 ]apelin-13 (1-12) (pD 2 = 7.84 ± 0.06) >> shorter fragments (pD 2 < 6). [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) and apelin-13(F13A) contracted human saphenous vein with similar sub-nanomolar potencies and [Pyr 1 ]apelin-13 (1-12) was a potent inotrope in paced mouse right ventricle and human atria. [Pyr 1 ]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.
TextSentencer_T17 2956-3143 Sentence denotes 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 .
TextSentencer_T17 2956-3143 Sentence denotes 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 .
TextSentencer_T18 3144-3381 Sentence denotes 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) .
TextSentencer_T18 3144-3381 Sentence denotes 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) .
TextSentencer_T19 3382-3427 Sentence denotes Apelins mediate three major actions in vitro.
TextSentencer_T19 3382-3427 Sentence denotes Apelins mediate three major actions in vitro.
TextSentencer_T20 3428-3610 Sentence denotes 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.
TextSentencer_T20 3428-3610 Sentence denotes 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.
TextSentencer_T21 3611-3734 Sentence denotes In vessels with an intact endothelium, apelin acts to release vasodilators that may oppose the actions of vasoconstrictors.
TextSentencer_T21 3611-3734 Sentence denotes In vessels with an intact endothelium, apelin acts to release vasodilators that may oppose the actions of vasoconstrictors.
TextSentencer_T22 3735-3904 Sentence denotes 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) .
TextSentencer_T22 3735-3904 Sentence denotes 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) .
TextSentencer_T23 3905-4159 Sentence denotes 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) .
TextSentencer_T23 3905-4159 Sentence denotes 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) .
TextSentencer_T24 4160-4328 Sentence denotes In heart failure patients, intracoronary [Pyr 1 ]apelin-13 caused coronary vasodilatation and increased cardiac contractility (Japp et al., 2010; Barnes et al., 2013) .
TextSentencer_T24 4160-4328 Sentence denotes In heart failure patients, intracoronary [Pyr 1 ]apelin-13 caused coronary vasodilatation and increased cardiac contractility (Japp et al., 2010; Barnes et al., 2013) .
TextSentencer_T25 4329-4543 Sentence denotes 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) .
TextSentencer_T25 4329-4543 Sentence denotes 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) .
TextSentencer_T26 4544-4853 Sentence denotes 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) .
TextSentencer_T26 4544-4853 Sentence denotes 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) .
TextSentencer_T27 4854-4941 Sentence denotes Therefore the apelin receptor may represent a novel target for future drug development.
TextSentencer_T27 4854-4941 Sentence denotes Therefore the apelin receptor may represent a novel target for future drug development.
TextSentencer_T28 4942-5107 Sentence denotes 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) .
TextSentencer_T28 4942-5107 Sentence denotes 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) .
TextSentencer_T29 5108-5347 Sentence denotes 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) .
TextSentencer_T29 5108-5347 Sentence denotes 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) .
TextSentencer_T30 5348-5605 Sentence denotes 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 .
TextSentencer_T30 5348-5605 Sentence denotes 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 .
TextSentencer_T31 5606-5813 Sentence denotes 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) .
TextSentencer_T31 5606-5813 Sentence denotes 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) .
TextSentencer_T32 5814-5961 Sentence denotes This has been the rational for the development of recombinant human ACE2 (rhACE2) in clinical trials for acute lung injury (Haschke et al., 2013) .
TextSentencer_T32 5814-5961 Sentence denotes This has been the rational for the development of recombinant human ACE2 (rhACE2) in clinical trials for acute lung injury (Haschke et al., 2013) .
TextSentencer_T33 5962-6125 Sentence denotes Interestingly, enhancing ACE2 activity pharmacologically or by gene transfer was effective in preventing or reversing PAH (Shenoy et al., 2011; Dai et al., 2015) .
TextSentencer_T33 5962-6125 Sentence denotes Interestingly, enhancing ACE2 activity pharmacologically or by gene transfer was effective in preventing or reversing PAH (Shenoy et al., 2011; Dai et al., 2015) .
TextSentencer_T34 6126-6241 Sentence denotes Some beneficial actions of ACE2 are thought to be mediated by the conversion of angiotensin II to angiotensin(1-7).
TextSentencer_T34 6126-6241 Sentence denotes Some beneficial actions of ACE2 are thought to be mediated by the conversion of angiotensin II to angiotensin(1-7).
TextSentencer_T35 6242-6689 Sentence denotes 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) .
TextSentencer_T35 6242-6689 Sentence denotes 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) .
TextSentencer_T36 6690-6821 Sentence denotes The loss of the terminal phenylalanine in apelin has been assumed to be a mechanism of degradation and inactivation of the peptide.
TextSentencer_T36 6690-6821 Sentence denotes The loss of the terminal phenylalanine in apelin has been assumed to be a mechanism of degradation and inactivation of the peptide.
TextSentencer_T37 6822-6922 Sentence denotes Our aim was to understand the impact of this ACE2 cleavage reaction on the apelin signaling pathway.
TextSentencer_T37 6822-6922 Sentence denotes Our aim was to understand the impact of this ACE2 cleavage reaction on the apelin signaling pathway.
TextSentencer_T38 6923-7209 Sentence denotes Specifically, our objectives were firstly to confirm that [Pyr 1 ]apelin-13 (1-12) (Figure 1) can be produced by ACE2 hydrolysis of [Pyr 1 ]apelin-13 and find evidence that [Pyr 1 ]apelin-13 (1-12) is an endogenous peptide and determine its distribution in human cardiovascular tissues.
TextSentencer_T38 6923-7209 Sentence denotes Specifically, our objectives were firstly to confirm that [Pyr 1 ]apelin-13 (1-12) (Figure 1) can be produced by ACE2 hydrolysis of [Pyr 1 ]apelin-13 and find evidence that [Pyr 1 ]apelin-13 (1-12) is an endogenous peptide and determine its distribution in human cardiovascular tissues.
TextSentencer_T39 7210-7407 Sentence denotes Secondly, to demonstrate that [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) binds to the apelin receptor and can activate down-stream signaling pathways in cell based assays.
TextSentencer_T39 7210-7407 Sentence denotes Secondly, to demonstrate that [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) binds to the apelin receptor and can activate down-stream signaling pathways in cell based assays.
TextSentencer_T40 7408-7580 Sentence denotes Thirdly, we have determined that [Pyr 1 ]apelin-13 (1-12) retains significant biological activity, compared with FIGURE 1 | Aligned amino acid sequences of apelin peptides.
TextSentencer_T40 7408-7580 Sentence denotes Thirdly, we have determined that [Pyr 1 ]apelin-13 (1-12) retains significant biological activity, compared with FIGURE 1 | Aligned amino acid sequences of apelin peptides.
TextSentencer_T41 7581-7715 Sentence denotes Hydrophobic amino acids are shown in green, uncharged polar amino acids in yellow, basic amino acids in blue and pyroglutamate in red.
TextSentencer_T41 7581-7715 Sentence denotes Hydrophobic amino acids are shown in green, uncharged polar amino acids in yellow, basic amino acids in blue and pyroglutamate in red.
TextSentencer_T42 7716-8322 Sentence denotes Frontiers in Neuroscience | www.frontiersin.org our previously reported data (Maguire et al., 2009; Brame et al., 2015) for [Pyr 1 ]apelin-13, in vitro using vascular and cardiac human and rodent tissues and by systemic infusions of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in vivo in the rat; finally, since the predominant action of apelin infused into the human forearm is vasodilatation, we have performed first-in-man studies with [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) to explore the physiological action of the peptide in healthy volunteers.
TextSentencer_T42 7716-8322 Sentence denotes Frontiers in Neuroscience | www.frontiersin.org our previously reported data (Maguire et al., 2009; Brame et al., 2015) for [Pyr 1 ]apelin-13, in vitro using vascular and cardiac human and rodent tissues and by systemic infusions of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in vivo in the rat; finally, since the predominant action of apelin infused into the human forearm is vasodilatation, we have performed first-in-man studies with [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) to explore the physiological action of the peptide in healthy volunteers.
TextSentencer_T43 8323-8660 Sentence denotes We also wished to explore the structure activity relationship (SAR) of the apelin peptides and therefore additional experiments were performed with the N-terminal extended putative endogenous peptide apelin-17, alanine substituted apelin-13(F13A), and the shorter fragments apelin-13(R10M), apelin-13(R9P) and apelin-13(P9M) (Figure 1) .
TextSentencer_T43 8323-8660 Sentence denotes We also wished to explore the structure activity relationship (SAR) of the apelin peptides and therefore additional experiments were performed with the N-terminal extended putative endogenous peptide apelin-17, alanine substituted apelin-13(F13A), and the shorter fragments apelin-13(R10M), apelin-13(R9P) and apelin-13(P9M) (Figure 1) .
TextSentencer_T44 8661-8894 Sentence denotes Our data expand our knowledge on the structure activity relationship of apelin peptides and demonstrate the significant biological activity of the ACE2 metabolite [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T44 8661-8894 Sentence denotes Our data expand our knowledge on the structure activity relationship of apelin peptides and demonstrate the significant biological activity of the ACE2 metabolite [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T45 8895-9233 Sentence denotes These data support the hypothesis that therapeutic strategies enhancing ACE2 activity or up-regulation of ACE2 in cardiovascular disease, both of which may result in enhanced breakdown of [Pyr 1 ]apelin-13, may not significantly compromise the beneficial effects of endogenous apelin signaling via generation of [Pyr 1 ]apelin-13 (1-12) .
TextSentencer_T45 8895-9233 Sentence denotes These data support the hypothesis that therapeutic strategies enhancing ACE2 activity or up-regulation of ACE2 in cardiovascular disease, both of which may result in enhanced breakdown of [Pyr 1 ]apelin-13, may not significantly compromise the beneficial effects of endogenous apelin signaling via generation of [Pyr 1 ]apelin-13 (1-12) .
TextSentencer_T46 9234-9393 Sentence denotes Human tissues samples were obtained with informed consent (Papworth Hospital Research Tissue Bank REC08/H0304/56) and local ethical approval (REC05/Q0104/142).
TextSentencer_T46 9234-9393 Sentence denotes Human tissues samples were obtained with informed consent (Papworth Hospital Research Tissue Bank REC08/H0304/56) and local ethical approval (REC05/Q0104/142).
TextSentencer_T47 9394-9819 Sentence denotes Synthesis of [Pyr 1 ]apelin-13 (1-12) from [Pyr 1 ]apelin-13 was confirmed by incubating [Pyr 1 ]apelin-13 (5 nmol) with rhACE2 enzyme (5 pmol, GSK, Ware, UK) in buffer (pH 6.5) containing 2-(N-morpholino)ethanesulfonic acid (MES, 500 mmol/L), NaCl (300 mmol/L) and ZnCl 2 (10 µmol/L) for 2 h at 37 • C followed by quenching with 10 µmol ethylenediaminetetraacetic acid (EDTA) (see Vickers et al., 2002 for similar protocol).
TextSentencer_T47 9394-9819 Sentence denotes Synthesis of [Pyr 1 ]apelin-13 (1-12) from [Pyr 1 ]apelin-13 was confirmed by incubating [Pyr 1 ]apelin-13 (5 nmol) with rhACE2 enzyme (5 pmol, GSK, Ware, UK) in buffer (pH 6.5) containing 2-(N-morpholino)ethanesulfonic acid (MES, 500 mmol/L), NaCl (300 mmol/L) and ZnCl 2 (10 µmol/L) for 2 h at 37 • C followed by quenching with 10 µmol ethylenediaminetetraacetic acid (EDTA) (see Vickers et al., 2002 for similar protocol).
TextSentencer_T48 9820-10107 Sentence denotes The reaction mixture was analyzed by Maldi-TOF mass spectrometry after samples were desalted using Millipore µC18ZipTip (MA, USA), washed with 5% acetic acid and eluted with CHCA matrix (in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid) to a stainless steel sample slide.
TextSentencer_T48 9820-10107 Sentence denotes The reaction mixture was analyzed by Maldi-TOF mass spectrometry after samples were desalted using Millipore µC18ZipTip (MA, USA), washed with 5% acetic acid and eluted with CHCA matrix (in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid) to a stainless steel sample slide.
TextSentencer_T49 10108-10216 Sentence denotes Samples were air dried and analyzed using a Waters Maldi MicroMX time-of-flight mass spectrometer (MA, USA).
TextSentencer_T49 10108-10216 Sentence denotes Samples were air dried and analyzed using a Waters Maldi MicroMX time-of-flight mass spectrometer (MA, USA).
TextSentencer_T50 10217-10268 Sentence denotes Calibration was external using polyethylene glycol.
TextSentencer_T50 10217-10268 Sentence denotes Calibration was external using polyethylene glycol.
TextSentencer_T51 10269-10316 Sentence denotes The 10 Hz-laser power was just above threshold.
TextSentencer_T51 10269-10316 Sentence denotes The 10 Hz-laser power was just above threshold.
TextSentencer_T52 10317-10406 Sentence denotes The spectra were the sum of 1,000 shots collected from a spiral track of the sample area.
TextSentencer_T52 10317-10406 Sentence denotes The spectra were the sum of 1,000 shots collected from a spiral track of the sample area.
TextSentencer_T53 10407-10468 Sentence denotes Data were processed using Waters MassLynx software (MA, USA).
TextSentencer_T53 10407-10468 Sentence denotes Data were processed using Waters MassLynx software (MA, USA).
TextSentencer_T54 10469-10701 Sentence denotes Control reactions included incubation of [Pyr 1 ]apelin-13 (1-12) (5 nmol) with rhACE2, incubation of [Pyr 1 ]apelin-13 (5 nmol) or [Pyr 1 ]apelin-13 (1-12) (5 nmol) without rhACE2 and incubation of rhACE2 (5 pmol) without peptides.
TextSentencer_T54 10469-10701 Sentence denotes Control reactions included incubation of [Pyr 1 ]apelin-13 (1-12) (5 nmol) with rhACE2, incubation of [Pyr 1 ]apelin-13 (5 nmol) or [Pyr 1 ]apelin-13 (1-12) (5 nmol) without rhACE2 and incubation of rhACE2 (5 pmol) without peptides.
TextSentencer_T55 10702-10936 Sentence denotes Peroxidase-anti-peroxidase and dual-labeling immunofluorescent staining were conducted as described (Kleinz et al., 2005) using frozen sections of human cardiomyopathy heart (n = 4), histologically normal (n = 6) and PAH (n = 4) lung.
TextSentencer_T55 10702-10936 Sentence denotes Peroxidase-anti-peroxidase and dual-labeling immunofluorescent staining were conducted as described (Kleinz et al., 2005) using frozen sections of human cardiomyopathy heart (n = 4), histologically normal (n = 6) and PAH (n = 4) lung.
TextSentencer_T56 10937-11302 Sentence denotes Affinity purified rabbit-anti-[Pyr 1 ]apelin-13 (1-12) antiserum (1:25-1:100 dilution) was custom synthesized (antibody raised against the relevant apelin peptide fragment CKGPMP, that lacks the C-terminal phenylalanine) and selectivity confirmed by comparison with the corresponding fragment containing the C-terminal phenylalanine (CKGPMPF) by ELISA (Figure 2A) .
TextSentencer_T56 10937-11302 Sentence denotes Affinity purified rabbit-anti-[Pyr 1 ]apelin-13 (1-12) antiserum (1:25-1:100 dilution) was custom synthesized (antibody raised against the relevant apelin peptide fragment CKGPMP, that lacks the C-terminal phenylalanine) and selectivity confirmed by comparison with the corresponding fragment containing the C-terminal phenylalanine (CKGPMPF) by ELISA (Figure 2A) .
TextSentencer_T57 11303-11390 Sentence denotes In contrast the commercially available apelin-12 antibody (Phoenix Pharmaceuticals Inc.
TextSentencer_T57 11303-11390 Sentence denotes In contrast the commercially available apelin-12 antibody (Phoenix Pharmaceuticals Inc.
TextSentencer_T58 11391-11486 Sentence denotes CA, USA) crossed reacted with [Pyr 1 ]apelin-13 but not [Pyr 1 ]apelin-13 (1-12) ( Figure 2B ).
TextSentencer_T58 11391-11486 Sentence denotes CA, USA) crossed reacted with [Pyr 1 ]apelin-13 but not [Pyr 1 ]apelin-13 (1-12) ( Figure 2B ).
TextSentencer_T59 11487-11577 Sentence denotes ACE2 (1:50, R&D Systems, MN, USA and 1:200, Abcam, Cambridge, UK) antisera were also used.
TextSentencer_T59 11487-11577 Sentence denotes ACE2 (1:50, R&D Systems, MN, USA and 1:200, Abcam, Cambridge, UK) antisera were also used.
TextSentencer_T60 11578-11672 Sentence denotes Von Willebrand factor (vWF) (1:50, Dako, Glostrup, Denmark) was used as an endothelial marker.
TextSentencer_T60 11578-11672 Sentence denotes Von Willebrand factor (vWF) (1:50, Dako, Glostrup, Denmark) was used as an endothelial marker.
TextSentencer_T61 11673-11986 Sentence denotes The peroxidase stained sections were examined with a bright field microscope (Olympus, Southend-on-Sea, UK) and imaged using a CC12 camera and CellD Soft Imaging System (Olympus), whereas fluorescent staining were imaged using a Leica Scanning Confocal Microscope (TCS SP2, Leica Microsystems, Milton Keynes, UK).
TextSentencer_T61 11673-11986 Sentence denotes The peroxidase stained sections were examined with a bright field microscope (Olympus, Southend-on-Sea, UK) and imaged using a CC12 camera and CellD Soft Imaging System (Olympus), whereas fluorescent staining were imaged using a Leica Scanning Confocal Microscope (TCS SP2, Leica Microsystems, Milton Keynes, UK).
TextSentencer_T62 11987-12149 Sentence denotes Image processing with rolling ball method and histogram redistribution were applied equally to the entire image and channel overlay were carried out using ImageJ.
TextSentencer_T62 11987-12149 Sentence denotes Image processing with rolling ball method and histogram redistribution were applied equally to the entire image and channel overlay were carried out using ImageJ.
TextSentencer_T63 12150-12233 Sentence denotes Assays were performed in human heart as previously described (Brame et al., 2015) .
TextSentencer_T63 12150-12233 Sentence denotes Assays were performed in human heart as previously described (Brame et al., 2015) .
TextSentencer_T64 12234-12678 Sentence denotes Briefly, homogenate of human left ventricle (LV) was incubated for 90 min with 0.1 nmol/L [Glp 65 , Nle 75 , Tyr 77 ][ 125 I]apelin-13 in assay buffer (mmol/L; Tris 50, MgCl 2 5, pH 7.4, 22 • C), in the presence of increasing concentrations of [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) , apelin-13(F13A), and apelin-17 (0.01 nmol/L-100 µmol/L) or, for SAR, a single concentration (1 µmol/L) apelin-13(R10M), apelin-13(R9P) and apelin-13(P9M).
TextSentencer_T64 12234-12678 Sentence denotes Briefly, homogenate of human left ventricle (LV) was incubated for 90 min with 0.1 nmol/L [Glp 65 , Nle 75 , Tyr 77 ][ 125 I]apelin-13 in assay buffer (mmol/L; Tris 50, MgCl 2 5, pH 7.4, 22 • C), in the presence of increasing concentrations of [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) , apelin-13(F13A), and apelin-17 (0.01 nmol/L-100 µmol/L) or, for SAR, a single concentration (1 µmol/L) apelin-13(R10M), apelin-13(R9P) and apelin-13(P9M).
TextSentencer_T65 12679-12745 Sentence denotes Non-specific binding was defined using 2 µmol/L [Pyr 1 ]apelin-13.
TextSentencer_T65 12679-12745 Sentence denotes Non-specific binding was defined using 2 µmol/L [Pyr 1 ]apelin-13.
TextSentencer_T66 12746-12956 Sentence denotes Equilibrium was broken by centrifugation (20,000 g for 10 min, 4 • C) and pellets washed with Tris-HCl buffer (50 mmol/L, pH 7.4, 4 • C), re-centrifuged and pellets counted for detection of bound radioactivity.
TextSentencer_T66 12746-12956 Sentence denotes Equilibrium was broken by centrifugation (20,000 g for 10 min, 4 • C) and pellets washed with Tris-HCl buffer (50 mmol/L, pH 7.4, 4 • C), re-centrifuged and pellets counted for detection of bound radioactivity.
TextSentencer_T67 12957-13043 Sentence denotes Competition binding data were analyzed using GraphPad Prism 6 (GraphPad Software, Inc.
TextSentencer_T67 12957-13043 Sentence denotes Competition binding data were analyzed using GraphPad Prism 6 (GraphPad Software, Inc.
TextSentencer_T68 13044-13278 Sentence denotes La Jolla, CA, USA) to obtain values of pK i (the negative log 10 of the dissociation constant derived from the IC 50 for the competing ligands, the radioligand concentration and radioligand affinity by the Cheng and Prusoff equation).
TextSentencer_T68 13044-13278 Sentence denotes La Jolla, CA, USA) to obtain values of pK i (the negative log 10 of the dissociation constant derived from the IC 50 for the competing ligands, the radioligand concentration and radioligand affinity by the Cheng and Prusoff equation).
TextSentencer_T69 13279-13320 Sentence denotes Experiments were performed in triplicate.
TextSentencer_T69 13279-13320 Sentence denotes Experiments were performed in triplicate.
TextSentencer_T70 13321-13587 Sentence denotes Inhibition of cAMP accumulation, β-arrestin recruitment and receptor internalization by apelin isoforms, modified and truncated apelin peptides were studied using cells expressing the human apelin receptor (DiscoverX, CA, USA) as per the manufacturer's instructions.
TextSentencer_T70 13321-13587 Sentence denotes Inhibition of cAMP accumulation, β-arrestin recruitment and receptor internalization by apelin isoforms, modified and truncated apelin peptides were studied using cells expressing the human apelin receptor (DiscoverX, CA, USA) as per the manufacturer's instructions.
TextSentencer_T71 13588-13746 Sentence denotes In all assays the resulting chemiluminescent signal was measured as relative light units (RLU) using a LumiLITE TM Microplate Reader (DiscoveRx, Fremont, CA).
TextSentencer_T71 13588-13746 Sentence denotes In all assays the resulting chemiluminescent signal was measured as relative light units (RLU) using a LumiLITE TM Microplate Reader (DiscoveRx, Fremont, CA).
TextSentencer_T72 13747-14053 Sentence denotes In the cAMP assay 15 µmol/L forskolin was used to stimulate cAMP production and concentration-response curves were constructed to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) , apelin-17, apelin-13(F13A) (all 1 pmol/L-30 nmol/L), apelin-13(R10M), apelin-13 (R9P) and apelin-13(P9M) (all 1 nmol/L-10 µmol/L).
TextSentencer_T72 13747-14053 Sentence denotes In the cAMP assay 15 µmol/L forskolin was used to stimulate cAMP production and concentration-response curves were constructed to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) , apelin-17, apelin-13(F13A) (all 1 pmol/L-30 nmol/L), apelin-13(R10M), apelin-13 (R9P) and apelin-13(P9M) (all 1 nmol/L-10 µmol/L).
TextSentencer_T73 14054-14120 Sentence denotes Agonist responses were expressed as a % of the forskolin response.
TextSentencer_T73 14054-14120 Sentence denotes Agonist responses were expressed as a % of the forskolin response.
TextSentencer_T74 14121-14417 Sentence denotes In the β-arrestin and internalization assays concentration-response curves were constructed to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) , apelin-13(F13A), apelin-13(R10M), (all 10 pmol/L-1 µmol/L), apelin-17 (1 pmol/L-100 nmol/L), apelin-13 (R9P) and apelin-13(P9M) (both 1 nmol/L-300 µmol/L).
TextSentencer_T74 14121-14417 Sentence denotes In the β-arrestin and internalization assays concentration-response curves were constructed to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) , apelin-13(F13A), apelin-13(R10M), (all 10 pmol/L-1 µmol/L), apelin-17 (1 pmol/L-100 nmol/L), apelin-13 (R9P) and apelin-13(P9M) (both 1 nmol/L-300 µmol/L).
TextSentencer_T75 14418-14503 Sentence denotes Agonist responses were expressed as a % of the maximum response to [Pyr 1 ]apelin-13.
TextSentencer_T75 14418-14503 Sentence denotes Agonist responses were expressed as a % of the maximum response to [Pyr 1 ]apelin-13.
TextSentencer_T76 14504-14800 Sentence denotes Data were analyzed using a 4-parameter logistic equation using GraphPad Prism 6 to determine values of potency, pD 2 (−log 10 EC 50 , where EC 50 is the concentration producing half maximal response) and maximum response (E MAX ). n-Values are given as number of replicates/number of experiments.
TextSentencer_T76 14504-14800 Sentence denotes Data were analyzed using a 4-parameter logistic equation using GraphPad Prism 6 to determine values of potency, pD 2 (−log 10 EC 50 , where EC 50 is the concentration producing half maximal response) and maximum response (E MAX ). n-Values are given as number of replicates/number of experiments.
TextSentencer_T77 14801-15164 Sentence denotes Using the data from the cAMP and β-arrestin assays with the predominant cardiac isoform [Pyr 1 ]apelin-13 used as the reference ligand, the relative activation of G proteindependent and -independent signaling pathways by [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) and apelin-17 were compared using bias analysis as described by van der Westhuizen et al. (2014) .
TextSentencer_T77 14801-15164 Sentence denotes Using the data from the cAMP and β-arrestin assays with the predominant cardiac isoform [Pyr 1 ]apelin-13 used as the reference ligand, the relative activation of G proteindependent and -independent signaling pathways by [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) and apelin-17 were compared using bias analysis as described by van der Westhuizen et al. (2014) .
TextSentencer_T78 15165-15296 Sentence denotes Additional β-arrestin assay experiments were performed with [Pyr 1 ]apelin-13 that had been incubated with ACE2 as described above.
TextSentencer_T78 15165-15296 Sentence denotes Additional β-arrestin assay experiments were performed with [Pyr 1 ]apelin-13 that had been incubated with ACE2 as described above.
TextSentencer_T79 15297-15539 Sentence denotes 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.
TextSentencer_T79 15297-15539 Sentence denotes 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.
TextSentencer_T80 15540-15679 Sentence denotes Vascular smooth muscle apelin receptor-mediated contraction was exploited in a bioassay to compare the in vitro potency of apelin peptides.
TextSentencer_T80 15540-15679 Sentence denotes Vascular smooth muscle apelin receptor-mediated contraction was exploited in a bioassay to compare the in vitro potency of apelin peptides.
TextSentencer_T81 15680-15904 Sentence denotes 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).
TextSentencer_T81 15680-15904 Sentence denotes 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).
TextSentencer_T82 15905-15988 Sentence denotes Agonist responses were expressed as a % of a terminal response to KCl (100 mmol/L).
TextSentencer_T82 15905-15988 Sentence denotes Agonist responses were expressed as a % of a terminal response to KCl (100 mmol/L).
TextSentencer_T83 15989-16189 Sentence denotes 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 ).
TextSentencer_T83 15989-16189 Sentence denotes 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 ).
TextSentencer_T84 16190-16251 Sentence denotes Data were expressed as % of the terminal response to CaCl 2 .
TextSentencer_T84 16190-16251 Sentence denotes Data were expressed as % of the terminal response to CaCl 2 .
TextSentencer_T85 16252-16401 Sentence denotes 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 .
TextSentencer_T85 16252-16401 Sentence denotes 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 .
TextSentencer_T86 16402-16613 Sentence denotes 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.
TextSentencer_T86 16402-16613 Sentence denotes 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.
TextSentencer_T87 16614-16982 Sentence denotes 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).
TextSentencer_T87 16614-16982 Sentence denotes 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).
TextSentencer_T88 16983-17082 Sentence denotes The left carotid artery and right jugular vein were cannulated (0.96 mm polyvinyl chloride tubing).
TextSentencer_T88 16983-17082 Sentence denotes The left carotid artery and right jugular vein were cannulated (0.96 mm polyvinyl chloride tubing).
TextSentencer_T89 17083-17248 Sentence denotes Mean arterial pressure (MAP) was measured throughout the procedure via a pressure transducer (Powerlab AD Instruments, Chalgrove, UK) connected to the arterial line.
TextSentencer_T89 17083-17248 Sentence denotes Mean arterial pressure (MAP) was measured throughout the procedure via a pressure transducer (Powerlab AD Instruments, Chalgrove, UK) connected to the arterial line.
TextSentencer_T90 17249-17423 Sentence denotes 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.
TextSentencer_T90 17249-17423 Sentence denotes 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.
TextSentencer_T91 17424-17563 Sentence denotes Thoracic echocardiography was performed at a scanning depth of 0-2 cm using a 14 MHz probe (Vivid 7 Dimension, GE Healthcare, Bedford, UK).
TextSentencer_T91 17424-17563 Sentence denotes Thoracic echocardiography was performed at a scanning depth of 0-2 cm using a 14 MHz probe (Vivid 7 Dimension, GE Healthcare, Bedford, UK).
TextSentencer_T92 17564-17654 Sentence denotes Pulsed-wave Doppler was used to determine aortic blood flow velocities in the aortic arch.
TextSentencer_T92 17564-17654 Sentence denotes Pulsed-wave Doppler was used to determine aortic blood flow velocities in the aortic arch.
TextSentencer_T93 17655-17772 Sentence denotes Stroke volume (SV) was determined as the product of the velocity-time integral (VTI) and vessel cross-sectional area.
TextSentencer_T93 17655-17772 Sentence denotes Stroke volume (SV) was determined as the product of the velocity-time integral (VTI) and vessel cross-sectional area.
TextSentencer_T94 17773-17920 Sentence denotes Data from six consecutive cardiac cycles were used to determine heart rate (HR) and a marker of left ventricular contractility, peak velocity (PV).
TextSentencer_T94 17773-17920 Sentence denotes Data from six consecutive cardiac cycles were used to determine heart rate (HR) and a marker of left ventricular contractility, peak velocity (PV).
TextSentencer_T95 17921-17984 Sentence denotes Values of SV and HR were used to calculate cardiac output (CO).
TextSentencer_T95 17921-17984 Sentence denotes Values of SV and HR were used to calculate cardiac output (CO).
TextSentencer_T96 17985-18076 Sentence denotes Respiration rate was determined from movement of the diaphragm using time-motion (M)-model.
TextSentencer_T96 17985-18076 Sentence denotes Respiration rate was determined from movement of the diaphragm using time-motion (M)-model.
TextSentencer_T97 18077-18171 Sentence denotes At the end of the study rats were euthanized by intravenous pentobarbitone and exsanguination.
TextSentencer_T97 18077-18171 Sentence denotes At the end of the study rats were euthanized by intravenous pentobarbitone and exsanguination.
TextSentencer_T98 18172-18319 Sentence denotes Studies were performed in healthy volunteers (n = 12) in the University of Cambridge Vascular Research Unit, Addenbrooke's Hospital, Cambridge, UK.
TextSentencer_T98 18172-18319 Sentence denotes Studies were performed in healthy volunteers (n = 12) in the University of Cambridge Vascular Research Unit, Addenbrooke's Hospital, Cambridge, UK.
TextSentencer_T99 18320-18368 Sentence denotes Volunteer characteristics are given in Table 1 .
TextSentencer_T99 18320-18368 Sentence denotes Volunteer characteristics are given in Table 1 .
TextSentencer_T100 18369-18567 Sentence denotes 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.
TextSentencer_T100 18369-18567 Sentence denotes 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.
TextSentencer_T101 18568-18658 Sentence denotes All subjects gave written informed consent in accordance with the Declaration of Helsinki.
TextSentencer_T101 18568-18658 Sentence denotes All subjects gave written informed consent in accordance with the Declaration of Helsinki.
TextSentencer_T102 18659-18786 Sentence denotes The protocol was approved by the National Research Ethics Service Committee East of England-Cambridge Central (REC 11/EE/0305).
TextSentencer_T102 18659-18786 Sentence denotes The protocol was approved by the National Research Ethics Service Committee East of England-Cambridge Central (REC 11/EE/0305).
TextSentencer_T103 18787-18945 Sentence denotes 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) .
TextSentencer_T103 18787-18945 Sentence denotes 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) .
TextSentencer_T104 18946-19224 Sentence denotes 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.
TextSentencer_T104 18946-19224 Sentence denotes 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.
TextSentencer_T105 19225-19382 Sentence denotes 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.
TextSentencer_T105 19225-19382 Sentence denotes 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.
TextSentencer_T106 19383-19608 Sentence denotes 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.
TextSentencer_T106 19383-19608 Sentence denotes 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.
TextSentencer_T107 19609-19753 Sentence denotes Changes in forearm volume were measured by mercury-in-silastic strain gauge with FBF subsequently expressed as ml/100 ml forearm volume per min.
TextSentencer_T107 19609-19753 Sentence denotes Changes in forearm volume were measured by mercury-in-silastic strain gauge with FBF subsequently expressed as ml/100 ml forearm volume per min.
TextSentencer_T108 19754-20018 Sentence denotes 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).
TextSentencer_T108 19754-20018 Sentence denotes 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).
TextSentencer_T109 20019-20182 Sentence denotes 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.
TextSentencer_T109 20019-20182 Sentence denotes 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.
TextSentencer_T110 20183-20565 Sentence denotes [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.
TextSentencer_T110 20183-20565 Sentence denotes [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.
TextSentencer_T111 20566-20615 Sentence denotes Doses were previously optimized in a pilot study.
TextSentencer_T111 20566-20615 Sentence denotes Doses were previously optimized in a pilot study.
TextSentencer_T112 20616-20731 Sentence denotes Each dose was infused for 6 min with a 20 min saline infusion washout period before the next dose was administered.
TextSentencer_T112 20616-20731 Sentence denotes Each dose was infused for 6 min with a 20 min saline infusion washout period before the next dose was administered.
TextSentencer_T113 20732-20884 Sentence denotes 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.
TextSentencer_T113 20732-20884 Sentence denotes 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.
TextSentencer_T114 20885-20907 Sentence denotes Values are mean ± SEM.
TextSentencer_T114 20885-20907 Sentence denotes Values are mean ± SEM.
TextSentencer_T115 20908-20963 Sentence denotes Measurements are mean ± standard error of the mean (SEM
TextSentencer_T115 20908-20963 Sentence denotes Measurements are mean ± standard error of the mean (SEM
TextSentencer_T116 20965-21234 Sentence denotes 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.
TextSentencer_T116 20965-21234 Sentence denotes 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.
TextSentencer_T117 21235-21367 Sentence denotes Peptides were tested for sterility and demonstrated to be pyrogen free and biological activity confirmed using the β-arrestin assay.
TextSentencer_T117 21235-21367 Sentence denotes Peptides were tested for sterility and demonstrated to be pyrogen free and biological activity confirmed using the β-arrestin assay.
TextSentencer_T118 21368-21443 Sentence denotes All apelin peptides were synthesized by Severn Biotech (Kidderminster, UK).
TextSentencer_T118 21368-21443 Sentence denotes All apelin peptides were synthesized by Severn Biotech (Kidderminster, UK).
TextSentencer_T119 21444-21514 Sentence denotes [Pyr 1 ]Apelin-13 (1-12) Is Synthesized from [Pyr 1 ]Apelin-13 by ACE2
TextSentencer_T119 21444-21514 Sentence denotes [Pyr 1 ]Apelin-13 (1-12) Is Synthesized from [Pyr 1 ]Apelin-13 by ACE2
TextSentencer_T120 21515-21680 Sentence denotes As shown in the mass spectra, [Pyr 1 ]apelin-13 ( Figure 3A) and [Pyr 1 ]apelin-13 (1-12) (Figure 3B ) alone produced signals at 1533.8 and 1386.7 m/z, respectively.
TextSentencer_T120 21515-21680 Sentence denotes As shown in the mass spectra, [Pyr 1 ]apelin-13 ( Figure 3A) and [Pyr 1 ]apelin-13 (1-12) (Figure 3B ) alone produced signals at 1533.8 and 1386.7 m/z, respectively.
TextSentencer_T121 21681-21923 Sentence denotes Incubating [Pyr 1 ]apelin-13 with rhACE2 resulted in a signal at the mass-to-charge ratio of 1386.7, corresponding to de novo generation of [Pyr 1 ]apelin-13 (1-12) , with a weak signal representing the remaining parent peptide ( Figure 3C ).
TextSentencer_T121 21681-21923 Sentence denotes Incubating [Pyr 1 ]apelin-13 with rhACE2 resulted in a signal at the mass-to-charge ratio of 1386.7, corresponding to de novo generation of [Pyr 1 ]apelin-13 (1-12) , with a weak signal representing the remaining parent peptide ( Figure 3C ).
TextSentencer_T122 21924-22040 Sentence denotes In contrast, incubating [Pyr 1 ]apelin-13 (1-12) with rhACE2 ( Figure 3D ) did not produce shorter apelin fragments.
TextSentencer_T122 21924-22040 Sentence denotes In contrast, incubating [Pyr 1 ]apelin-13 (1-12) with rhACE2 ( Figure 3D ) did not produce shorter apelin fragments.
TextSentencer_T123 22041-22149 Sentence denotes Finally, rhACE2 enzyme alone ( Figure 3E ) did not result in interfering signals in the relevant mass range.
TextSentencer_T123 22041-22149 Sentence denotes Finally, rhACE2 enzyme alone ( Figure 3E ) did not result in interfering signals in the relevant mass range.
TextSentencer_T124 22150-22372 Sentence denotes [Pyr 1 ]Apelin-13 (1-12) Is an Endogenous Apelin Peptide Localized to the Endothelium Endogenous [Pyr 1 ]apelin-13 (1-12) peptide was detectable in human cardiovascular tissues and localized to the endothelium (Figure 4) .
TextSentencer_T124 22150-22372 Sentence denotes [Pyr 1 ]Apelin-13 (1-12) Is an Endogenous Apelin Peptide Localized to the Endothelium Endogenous [Pyr 1 ]apelin-13 (1-12) peptide was detectable in human cardiovascular tissues and localized to the endothelium (Figure 4) .
TextSentencer_T125 22373-22826 Sentence denotes [Pyr 1 ]apelin-13 (1-12) -like immunoreactivity (-LI) was detected in vascular ( Figure 4A ) and endocardial ( Figure 4B ) endothelium identified by positive staining with vWF ( Figure 4C ) in sections of human cardiomyopathy heart, where ACE2 expression has been reported to be increased (Zisman et al., 2003; Goulter et al., 2004) . [Pyr 1 ]apelin-13 (1-12) -LI ( Figure 4D ) and vWF-LI ( Figure 4E) were also co-expressed ( Figure 4F ) in human lung.
TextSentencer_T125 22373-22826 Sentence denotes [Pyr 1 ]apelin-13 (1-12) -like immunoreactivity (-LI) was detected in vascular ( Figure 4A ) and endocardial ( Figure 4B ) endothelium identified by positive staining with vWF ( Figure 4C ) in sections of human cardiomyopathy heart, where ACE2 expression has been reported to be increased (Zisman et al., 2003; Goulter et al., 2004) . [Pyr 1 ]apelin-13 (1-12) -LI ( Figure 4D ) and vWF-LI ( Figure 4E) were also co-expressed ( Figure 4F ) in human lung.
TextSentencer_T126 22827-22962 Sentence denotes Importantly, [Pyr 1 ]apelin-13 (1-12) -LI ( Figure 4G ) and ACE2-LI ( Figure 4H ) co-localized ( Figure 4I) in pulmonary blood vessels.
TextSentencer_T126 22827-22962 Sentence denotes Importantly, [Pyr 1 ]apelin-13 (1-12) -LI ( Figure 4G ) and ACE2-LI ( Figure 4H ) co-localized ( Figure 4I) in pulmonary blood vessels.
TextSentencer_T127 22963-23093 Sentence denotes Importantly, as apelin is reduced in PAH, the presence of [Pyr 1 ]apelin-13 (1-12) was investigated in sections of human PAH lung.
TextSentencer_T127 22963-23093 Sentence denotes Importantly, as apelin is reduced in PAH, the presence of [Pyr 1 ]apelin-13 (1-12) was investigated in sections of human PAH lung.
TextSentencer_T128 23094-23236 Sentence denotes Compared to normal lung (Figure 4J) , [Pyr 1 ]apelin-13 (1-12) -LI was not detectable in the vascular endothelium of PAH lung (Figures 4K,L) .
TextSentencer_T128 23094-23236 Sentence denotes Compared to normal lung (Figure 4J) , [Pyr 1 ]apelin-13 (1-12) -LI was not detectable in the vascular endothelium of PAH lung (Figures 4K,L) .
TextSentencer_T129 23237-23462 Sentence denotes [Pyr 1 ]apelin-13 (1-12) competed with [ 125 I]apelin-13 binding with nanomolar affinity, pK i = 8.04 ± 0.06 (n = 3), that was 4-fold lower than the parent molecule [Pyr 1 ]apelin-13 (pK i = 8.83 ± 0.06, n = 3) ( Figure 5A ).
TextSentencer_T129 23237-23462 Sentence denotes [Pyr 1 ]apelin-13 (1-12) competed with [ 125 I]apelin-13 binding with nanomolar affinity, pK i = 8.04 ± 0.06 (n = 3), that was 4-fold lower than the parent molecule [Pyr 1 ]apelin-13 (pK i = 8.83 ± 0.06, n = 3) ( Figure 5A ).
TextSentencer_T130 23463-23663 Sentence denotes Apelin-13(F13A) exhibited comparable affinity to [Pyr 1 ]apelin-13 (1-12) (pK i = 8.07 ± 0.24, n = 3) whereas the extended peptide apelin-17 competed with highest affinity (pK i = 9.63 ± 0.17, n = 3).
TextSentencer_T130 23463-23663 Sentence denotes Apelin-13(F13A) exhibited comparable affinity to [Pyr 1 ]apelin-13 (1-12) (pK i = 8.07 ± 0.24, n = 3) whereas the extended peptide apelin-17 competed with highest affinity (pK i = 9.63 ± 0.17, n = 3).
TextSentencer_T131 23664-23851 Sentence denotes Of the shorter fragments apelin-13(R10M) (1 µM) competed for 100% of specific binding whereas apelin-13(R9P) and apelin-13(P9M) were less effective competing for 38% and 62% respectively.
TextSentencer_T131 23664-23851 Sentence denotes Of the shorter fragments apelin-13(R10M) (1 µM) competed for 100% of specific binding whereas apelin-13(R9P) and apelin-13(P9M) were less effective competing for 38% and 62% respectively.
TextSentencer_T132 23852-24172 Sentence denotes In the signaling assays, [Pyr 1 ]apelin-13 (1-12) inhibited forskolin-stimulated cAMP production with sub-nanomolar potency, pD 2 = 9.30 ± 0.06 (n = 8/3) and was 2-fold less potent than the reference agonist [Pyr 1 ]apelin-13, pD 2 = 9.67 ± 0.04 (n = 7/5) but comparable to apelin-13(F13A) (pD 2 = 9.54 ± 0.05, n = 2/1).
TextSentencer_T132 23852-24172 Sentence denotes In the signaling assays, [Pyr 1 ]apelin-13 (1-12) inhibited forskolin-stimulated cAMP production with sub-nanomolar potency, pD 2 = 9.30 ± 0.06 (n = 8/3) and was 2-fold less potent than the reference agonist [Pyr 1 ]apelin-13, pD 2 = 9.67 ± 0.04 (n = 7/5) but comparable to apelin-13(F13A) (pD 2 = 9.54 ± 0.05, n = 2/1).
TextSentencer_T133 24173-24262 Sentence denotes Apelin-17 (pD 2 = 10.31 ± 0.28, n = 5/2) was ∼5 times more potent than [Pyr 1 ]apelin-13.
TextSentencer_T133 24173-24262 Sentence denotes Apelin-17 (pD 2 = 10.31 ± 0.28, n = 5/2) was ∼5 times more potent than [Pyr 1 ]apelin-13.
TextSentencer_T134 24263-24321 Sentence denotes All ligands fully inhibited cAMP production ( Figure 5B ).
TextSentencer_T134 24263-24321 Sentence denotes All ligands fully inhibited cAMP production ( Figure 5B ).
TextSentencer_T135 24322-24476 Sentence denotes Apelin-13(R10M) retained nanomolar potency (pD 2 = 8.70 ± 0.04, n = 3/1) whereas inhibition was incomplete for apelin-13(R9P) and apelin-13(P9M) at 10 µM.
TextSentencer_T135 24322-24476 Sentence denotes Apelin-13(R10M) retained nanomolar potency (pD 2 = 8.70 ± 0.04, n = 3/1) whereas inhibition was incomplete for apelin-13(R9P) and apelin-13(P9M) at 10 µM.
TextSentencer_T136 24477-24837 Sentence denotes In the G protein-independent β-arrestin recruitment assay [Pyr 1 ]apelin-13 (pD 2 = 8.43 ± 0.08, n = 25/10), [Pyr 1 ]apelin-13 (1-12) (pD 2 = 7.84 ± 0.06, n = 17/6) and apelin-13(F13A) (pD 2 = 7.98 ± 0.04, n = 6/2) were 15-40-fold less potent than in the cAMP assay, with [Pyr 1 ]apelin-13 ∼4-fold more potent than [Pyr 1 ]apelin-13 (1-12) and apelin-13(F13A).
TextSentencer_T136 24477-24837 Sentence denotes In the G protein-independent β-arrestin recruitment assay [Pyr 1 ]apelin-13 (pD 2 = 8.43 ± 0.08, n = 25/10), [Pyr 1 ]apelin-13 (1-12) (pD 2 = 7.84 ± 0.06, n = 17/6) and apelin-13(F13A) (pD 2 = 7.98 ± 0.04, n = 6/2) were 15-40-fold less potent than in the cAMP assay, with [Pyr 1 ]apelin-13 ∼4-fold more potent than [Pyr 1 ]apelin-13 (1-12) and apelin-13(F13A).
TextSentencer_T137 24838-25026 Sentence denotes Interestingly, unlike the shorter peptides, apelin-17 exhibited comparable potency as an agonist in both the β-arrestin and cAMP assays with a pD 2 = 10.26 ± 0.09 (n = 11/4) ( Figure 5C ).
TextSentencer_T137 24838-25026 Sentence denotes Interestingly, unlike the shorter peptides, apelin-17 exhibited comparable potency as an agonist in both the β-arrestin and cAMP assays with a pD 2 = 10.26 ± 0.09 (n = 11/4) ( Figure 5C ).
TextSentencer_T138 25027-25194 Sentence denotes Apelin-13(R10M) (pD 2 = 8.26 ± 0.17, n = 9/3) was ∼2-fold less potent than [Pyr 1 ]apelin-13 and curves were incomplete for apelin-13(R9P) and aplin-13(P9M) at 300 µM.
TextSentencer_T138 25027-25194 Sentence denotes Apelin-13(R10M) (pD 2 = 8.26 ± 0.17, n = 9/3) was ∼2-fold less potent than [Pyr 1 ]apelin-13 and curves were incomplete for apelin-13(R9P) and aplin-13(P9M) at 300 µM.
TextSentencer_T139 25195-25370 Sentence denotes Similar to the β-arrestin assay, in the internalization assay [Pyr 1 ]apelin-13 (1-12) (pD 2 = 8.19 ± 0.06) was 5-fold less potent than [Pyr 1 ]apelin-13 (pD 2 = 8.94 ± 0.17).
TextSentencer_T139 25195-25370 Sentence denotes Similar to the β-arrestin assay, in the internalization assay [Pyr 1 ]apelin-13 (1-12) (pD 2 = 8.19 ± 0.06) was 5-fold less potent than [Pyr 1 ]apelin-13 (pD 2 = 8.94 ± 0.17).
TextSentencer_T140 25371-25503 Sentence denotes Both peptides were full agonists with comparable efficacy values (E MAX values were 97 ± 2% and 99 ± 3% respectively) ( Figure 5D ).
TextSentencer_T140 25371-25503 Sentence denotes Both peptides were full agonists with comparable efficacy values (E MAX values were 97 ± 2% and 99 ± 3% respectively) ( Figure 5D ).
TextSentencer_T141 25504-25877 Sentence denotes Comparing the cAMP and β-arrestin data for the three endogenous peptides with [Pyr 1 ]apelin-13 as the reference agonist, analysis ( Table 2 ) demonstrated a bias factor of 0.24 for [Pyr 1 ]apelin-13 (1-12) and 68 for apelin-17 indicating that compared to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) was 4-fold G protein biased and apelin-17 was markedly β-arrestin biased.
TextSentencer_T141 25504-25877 Sentence denotes Comparing the cAMP and β-arrestin data for the three endogenous peptides with [Pyr 1 ]apelin-13 as the reference agonist, analysis ( Table 2 ) demonstrated a bias factor of 0.24 for [Pyr 1 ]apelin-13 (1-12) and 68 for apelin-17 indicating that compared to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1-12) was 4-fold G protein biased and apelin-17 was markedly β-arrestin biased.
TextSentencer_T142 25878-26121 Sentence denotes In the β-arrestin recruitment assay [Pyr 1 ]apelin-13 was significantly more potent than [Pyr 1 ]apelin-13 (1-12) (p < 0.0001), [Pyr 1 ]apelin-13 incubated with rhACE2 (p < 0.001) or [Pyr 1 ]apelin-13 (1-12) incubated with rhACE2 (p < 0.0001).
TextSentencer_T142 25878-26121 Sentence denotes In the β-arrestin recruitment assay [Pyr 1 ]apelin-13 was significantly more potent than [Pyr 1 ]apelin-13 (1-12) (p < 0.0001), [Pyr 1 ]apelin-13 incubated with rhACE2 (p < 0.001) or [Pyr 1 ]apelin-13 (1-12) incubated with rhACE2 (p < 0.0001).
TextSentencer_T143 26122-26234 Sentence denotes Both of the rhACE2 combinations exhibited comparable potency to [Pyr 1 ]apelin-13 (1-12) (p > 0.05) ( Table 3) .
TextSentencer_T143 26122-26234 Sentence denotes Both of the rhACE2 combinations exhibited comparable potency to [Pyr 1 ]apelin-13 (1-12) (p > 0.05) ( Table 3) .
TextSentencer_T144 26235-26369 Sentence denotes Therefore the reaction product of [Pyr 1 ]apelin-13 and rhACE2 more closely resembled [Pyr 1 ]apelin-13 (1-12) than [Pyr 1 ]apelin-13.
TextSentencer_T144 26235-26369 Sentence denotes Therefore the reaction product of [Pyr 1 ]apelin-13 and rhACE2 more closely resembled [Pyr 1 ]apelin-13 (1-12) than [Pyr 1 ]apelin-13.
TextSentencer_T145 26370-26709 Sentence denotes [Pyr 1 ]apelin-13 (1-12) (pD 2 = 9.63 ± 0.35, E MAX = 24 ± 6%, n = 9) and apelin-13(F13A) (pD 2 = 9.72 ± 0.36, E MAX = 23 ± 4%, n = 9) contracted saphenous vein with comparable subnanomolar potencies and maximum responses ( Figure 6A ) to the data that we had previously obtained with [Pyr 1 ]apelin-13 in this assay (Brame et al., 2015) .
TextSentencer_T145 26370-26709 Sentence denotes [Pyr 1 ]apelin-13 (1-12) (pD 2 = 9.63 ± 0.35, E MAX = 24 ± 6%, n = 9) and apelin-13(F13A) (pD 2 = 9.72 ± 0.36, E MAX = 23 ± 4%, n = 9) contracted saphenous vein with comparable subnanomolar potencies and maximum responses ( Figure 6A ) to the data that we had previously obtained with [Pyr 1 ]apelin-13 in this assay (Brame et al., 2015) .
TextSentencer_T146 26710-26988 Sentence denotes In mouse paced RV [Pyr 1 ]apelin-13 (1-12) produced a concentration-dependent increase in force of contraction with pD 2 = 11.68 ± 0.33, E MAX 49 ± 16% CaCl 2 (n = 6) ( Figure 6B ), compared to isoprenaline with pD 2 = 7.88 ± 0.42, E MAX = 78 ± 17% CaCl 2 (n = 4) ( Figure 6C ).
TextSentencer_T146 26710-26988 Sentence denotes In mouse paced RV [Pyr 1 ]apelin-13 (1-12) produced a concentration-dependent increase in force of contraction with pD 2 = 11.68 ± 0.33, E MAX 49 ± 16% CaCl 2 (n = 6) ( Figure 6B ), compared to isoprenaline with pD 2 = 7.88 ± 0.42, E MAX = 78 ± 17% CaCl 2 (n = 4) ( Figure 6C ).
TextSentencer_T147 26989-27148 Sentence denotes Limited human atrial appendage strips were available to test for the inotropic action of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T147 26989-27148 Sentence denotes Limited human atrial appendage strips were available to test for the inotropic action of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T148 27149-27308 Sentence denotes In tissue from two patients [Pyr 1 ]apelin-13 (1-12) acted as a positive inotrope with a sub-nanomolar potency value (pD 2 = 9.61, E MAX = 38% CaCl 2 , n = 2).
TextSentencer_T148 27149-27308 Sentence denotes In tissue from two patients [Pyr 1 ]apelin-13 (1-12) acted as a positive inotrope with a sub-nanomolar potency value (pD 2 = 9.61, E MAX = 38% CaCl 2 , n = 2).
TextSentencer_T149 27309-27416 Sentence denotes In anesthetized rat, [Pyr 1 ]apelin-13 (1-12) (n = 5) elicited a dosedependent decrease in BP (Figure 7A) .
TextSentencer_T149 27309-27416 Sentence denotes In anesthetized rat, [Pyr 1 ]apelin-13 (1-12) (n = 5) elicited a dosedependent decrease in BP (Figure 7A) .
TextSentencer_T150 27417-27515 Sentence denotes The decrease in BP was significantly different (P < 0.0001) from baseline at doses of 10-300 nmol.
TextSentencer_T150 27417-27515 Sentence denotes The decrease in BP was significantly different (P < 0.0001) from baseline at doses of 10-300 nmol.
TextSentencer_T151 27516-27666 Sentence denotes Other cardiac parameters, SV, CO, PV and VTI were not altered by [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (Figures 7B-E) .
TextSentencer_T151 27516-27666 Sentence denotes Other cardiac parameters, SV, CO, PV and VTI were not altered by [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (Figures 7B-E) .
TextSentencer_T152 27667-27873 Sentence denotes In human volunteers (n = 12), [Pyr 1 ]apelin-13 (1-12) (Figure 8 ) produced comparable dose-dependent increases in forearm blood flow to that which we have shown for [Pyr 1 ]apelin-13 (Brame et al., 2015) .
TextSentencer_T152 27667-27873 Sentence denotes In human volunteers (n = 12), [Pyr 1 ]apelin-13 (1-12) (Figure 8 ) produced comparable dose-dependent increases in forearm blood flow to that which we have shown for [Pyr 1 ]apelin-13 (Brame et al., 2015) .
TextSentencer_T153 27874-27990 Sentence denotes No significant effects of either peptide on heart rate or blood pressure were observed at any dose (data not shown).
TextSentencer_T153 27874-27990 Sentence denotes No significant effects of either peptide on heart rate or blood pressure were observed at any dose (data not shown).
TextSentencer_T154 27991-28140 Sentence denotes Administration of intravenous [Pyr 1 ]apelin-13 (1-12) ( , n = 5) resulted in a significant dose-dependent decrease in blood pressure in rat in vivo.
TextSentencer_T154 27991-28140 Sentence denotes Administration of intravenous [Pyr 1 ]apelin-13 (1-12) ( , n = 5) resulted in a significant dose-dependent decrease in blood pressure in rat in vivo.
TextSentencer_T155 28141-28319 Sentence denotes 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 [Pyr 1 ]apelin-13 (1-12).
TextSentencer_T155 28141-28319 Sentence denotes 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 [Pyr 1 ]apelin-13 (1-12).
TextSentencer_T156 28320-28395 Sentence denotes Significantly different from baseline; *P < 0.05, **P < 0.01, ***P < 0.001.
TextSentencer_T156 28320-28395 Sentence denotes Significantly different from baseline; *P < 0.05, **P < 0.01, ***P < 0.001.
TextSentencer_T157 28396-28489 Sentence denotes One-way ANOVA repeated measures compared to baseline with Dunnett's multiple comparison test.
TextSentencer_T157 28396-28489 Sentence denotes One-way ANOVA repeated measures compared to baseline with Dunnett's multiple comparison test.
TextSentencer_T158 28490-28619 Sentence denotes Apelin-13 and apelin-36 peptides were previously reported to be substrates of purified human ACE2 enzyme (Vickers et al., 2002) .
TextSentencer_T158 28490-28619 Sentence denotes Apelin-13 and apelin-36 peptides were previously reported to be substrates of purified human ACE2 enzyme (Vickers et al., 2002) .
TextSentencer_T159 28620-28858 Sentence denotes In this study, we confirmed (Wang et al., 2016) that ACE2 also catalyzed the conversion of [Pyr 1 ]apelin-13, the predominant cardiovascular form of apelin, to [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in vitro.
TextSentencer_T159 28620-28858 Sentence denotes In this study, we confirmed (Wang et al., 2016) that ACE2 also catalyzed the conversion of [Pyr 1 ]apelin-13, the predominant cardiovascular form of apelin, to [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in vitro.
TextSentencer_T160 28859-29072 Sentence denotes This result was not unexpected since the PMPF sequence of [Pyr 1 ]apelin-13 (Figure 1) conforms to the consensus sequence for ACE2-mediated hydrolysis; Pro-X (1-3residues) -Pro-Hydrophobic (Vickers et al., 2002) .
TextSentencer_T160 28859-29072 Sentence denotes This result was not unexpected since the PMPF sequence of [Pyr 1 ]apelin-13 (Figure 1) conforms to the consensus sequence for ACE2-mediated hydrolysis; Pro-X (1-3residues) -Pro-Hydrophobic (Vickers et al., 2002) .
TextSentencer_T161 29073-29230 Sentence denotes In support, in the β-arrestin assay the product of de novo ACE2 metabolism demonstrated comparable potency to synthetic [Pyr 1 ]apelin-13 (1−12) ( Table 3) .
TextSentencer_T161 29073-29230 Sentence denotes In support, in the β-arrestin assay the product of de novo ACE2 metabolism demonstrated comparable potency to synthetic [Pyr 1 ]apelin-13 (1−12) ( Table 3) .
TextSentencer_T162 29231-29449 Sentence denotes Our results are consistent with a study reporting that a small proportion of [Pyr 1 ]apelin-13 was cleaved into [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in rat in vivo (Murza et al., 2014) .
TextSentencer_T162 29231-29449 Sentence denotes Our results are consistent with a study reporting that a small proportion of [Pyr 1 ]apelin-13 was cleaved into [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in rat in vivo (Murza et al., 2014) .
TextSentencer_T163 29450-29666 Sentence denotes We have previously demonstrated endothelial expression of apelin (Kleinz and Davenport, 2004; Kleinz et al., 2005) using an antibody specific for apelin isoforms containing the C-terminal phenylalanine ( Figure 2B) .
TextSentencer_T163 29450-29666 Sentence denotes We have previously demonstrated endothelial expression of apelin (Kleinz and Davenport, 2004; Kleinz et al., 2005) using an antibody specific for apelin isoforms containing the C-terminal phenylalanine ( Figure 2B) .
TextSentencer_T164 29667-29934 Sentence denotes This is consistently reported by others using alternative strategies to detect apelin Infusion of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in healthy human volunteers ( , n = 12) resulted in a dose-dependent increase in forearm blood flow.
TextSentencer_T164 29667-29934 Sentence denotes This is consistently reported by others using alternative strategies to detect apelin Infusion of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in healthy human volunteers ( , n = 12) resulted in a dose-dependent increase in forearm blood flow.
TextSentencer_T165 29935-29996 Sentence denotes Significantly different from baseline; *P < 0.05, **P < 0.01.
TextSentencer_T165 29935-29996 Sentence denotes Significantly different from baseline; *P < 0.05, **P < 0.01.
TextSentencer_T166 29997-30090 Sentence denotes One-way ANOVA repeated measures compared to baseline with Dunnett's multiple comparison test.
TextSentencer_T166 29997-30090 Sentence denotes One-way ANOVA repeated measures compared to baseline with Dunnett's multiple comparison test.
TextSentencer_T167 30091-30103 Sentence denotes expression .
TextSentencer_T167 30091-30103 Sentence denotes expression .
TextSentencer_T168 30104-30346 Sentence denotes ACE2 is also expressed in the endothelium (Donoghue et al., 2000; Hamming et al., 2004) , raising the possibility of endothelial processing of [Pyr 1 ]apelin-13 to produce [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T168 30104-30346 Sentence denotes ACE2 is also expressed in the endothelium (Donoghue et al., 2000; Hamming et al., 2004) , raising the possibility of endothelial processing of [Pyr 1 ]apelin-13 to produce [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T169 30347-30810 Sentence denotes For this study, we generated an antibody that cross-reacts with apelin isoforms without the Cterminal phenylalanine that has allowed selective identification of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in human heart and lung. [Pyr 1 ]apelin-13 (1-12) -LI was specifically localized to the endothelium, where the apelin receptor was also expressed, suggesting the possibility of autocrine signaling by this metabolite in these tissues.
TextSentencer_T169 30347-30810 Sentence denotes For this study, we generated an antibody that cross-reacts with apelin isoforms without the Cterminal phenylalanine that has allowed selective identification of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in human heart and lung. [Pyr 1 ]apelin-13 (1-12) -LI was specifically localized to the endothelium, where the apelin receptor was also expressed, suggesting the possibility of autocrine signaling by this metabolite in these tissues.
TextSentencer_T170 30811-31000 Sentence denotes Importantly, in human PAH lung, where endothelial apelin expression is known to be reduced (Alastalo et al., 2011; Kim et al., 2013) , no staining for [Pyr 1 ]apelin-13 (1-12) was detected.
TextSentencer_T170 30811-31000 Sentence denotes Importantly, in human PAH lung, where endothelial apelin expression is known to be reduced (Alastalo et al., 2011; Kim et al., 2013) , no staining for [Pyr 1 ]apelin-13 (1-12) was detected.
TextSentencer_T171 31001-31121 Sentence denotes Biological Activity of [Pyr 1 ]Apelin-13 (1-12) ACE2-mediated hydrolysis has been assumed to inactivate apelin peptides.
TextSentencer_T171 31001-31121 Sentence denotes Biological Activity of [Pyr 1 ]Apelin-13 (1-12) ACE2-mediated hydrolysis has been assumed to inactivate apelin peptides.
TextSentencer_T172 31122-31311 Sentence denotes 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) .
TextSentencer_T172 31122-31311 Sentence denotes 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) .
TextSentencer_T173 31312-31631 Sentence denotes 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.
TextSentencer_T173 31312-31631 Sentence denotes 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.
TextSentencer_T174 31632-31832 Sentence denotes 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.
TextSentencer_T174 31632-31832 Sentence denotes 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.
TextSentencer_T175 31833-32118 Sentence denotes 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.
TextSentencer_T175 31833-32118 Sentence denotes 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.
TextSentencer_T176 32119-32399 Sentence denotes 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) .
TextSentencer_T176 32119-32399 Sentence denotes 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) .
TextSentencer_T177 32400-32537 Sentence denotes Overall, these studies show that loss of the C-terminal phenylalanine from apelin isoforms does not significantly alter receptor binding.
TextSentencer_T177 32400-32537 Sentence denotes Overall, these studies show that loss of the C-terminal phenylalanine from apelin isoforms does not significantly alter receptor binding.
TextSentencer_T178 32538-32741 Sentence denotes The apelin receptor is G αi -coupled (Habata et al., 1999) therefore we next showed that [Pyr 1 ]apelin-13 (1-12) inhibited forskolin-stimulated cAMP with potency only 2-fold less than [Pyr 1 ]apelin-13.
TextSentencer_T178 32538-32741 Sentence denotes The apelin receptor is G αi -coupled (Habata et al., 1999) therefore we next showed that [Pyr 1 ]apelin-13 (1-12) inhibited forskolin-stimulated cAMP with potency only 2-fold less than [Pyr 1 ]apelin-13.
TextSentencer_T179 32742-32975 Sentence denotes This is in agreement with the previous reports of alanine and D-phenylalanine substituted [Pyr 1 ]apelin-13 (Medhurst et al., 2003; Murza et al., 2012) and K16P (El Messari et al., 2004; Iturrioz et al., 2010; Ceraudo et al., 2014) .
TextSentencer_T179 32742-32975 Sentence denotes This is in agreement with the previous reports of alanine and D-phenylalanine substituted [Pyr 1 ]apelin-13 (Medhurst et al., 2003; Murza et al., 2012) and K16P (El Messari et al., 2004; Iturrioz et al., 2010; Ceraudo et al., 2014) .
TextSentencer_T180 32976-33105 Sentence denotes Therefore, our study confirms that G αi -induced signaling is preserved in response to [Pyr 1 ]apelin-13 (1-12) receptor binding.
TextSentencer_T180 32976-33105 Sentence denotes Therefore, our study confirms that G αi -induced signaling is preserved in response to [Pyr 1 ]apelin-13 (1-12) receptor binding.
TextSentencer_T181 33106-33267 Sentence denotes In addition to G protein-dependent signaling, activation of the apelin receptor causes β-arrestin recruitment and receptor internalization (Evans et al., 2001) .
TextSentencer_T181 33106-33267 Sentence denotes In addition to G protein-dependent signaling, activation of the apelin receptor causes β-arrestin recruitment and receptor internalization (Evans et al., 2001) .
TextSentencer_T182 33268-33441 Sentence denotes In these assays, [Pyr 1 ]apelin-13 (1-12) again behaved as a full agonist at the human apelin receptor with only a 5-fold reduction in potency compared to [Pyr 1 ]apelin-13.
TextSentencer_T182 33268-33441 Sentence denotes In these assays, [Pyr 1 ]apelin-13 (1-12) again behaved as a full agonist at the human apelin receptor with only a 5-fold reduction in potency compared to [Pyr 1 ]apelin-13.
TextSentencer_T183 33442-33787 Sentence denotes This ability to induce receptor internalization without the C-terminal phenylalanine was reported for apelin-13(F13A) (Fan et al., 2003) , however, K16P was shown by a second group to exhibit markedly reduced potency and efficacy in β-arrestin recruitment and internalization of rat apelin receptor Iturrioz et al., 2010; Ceraudo et al., 2014) .
TextSentencer_T183 33442-33787 Sentence denotes This ability to induce receptor internalization without the C-terminal phenylalanine was reported for apelin-13(F13A) (Fan et al., 2003) , however, K16P was shown by a second group to exhibit markedly reduced potency and efficacy in β-arrestin recruitment and internalization of rat apelin receptor Iturrioz et al., 2010; Ceraudo et al., 2014) .
TextSentencer_T184 33788-33975 Sentence denotes Importantly, compared to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) is not a biased agonist in G protein-dependent and -independent signaling.
TextSentencer_T184 33788-33975 Sentence denotes Importantly, compared to [Pyr 1 ]apelin-13, [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) is not a biased agonist in G protein-dependent and -independent signaling.
TextSentencer_T185 33976-34167 Sentence denotes Apelins are modulators of vascular tone and cardiac contractility in vitro and in vivo (Japp et al., 2008 (Japp et al., , 2010 Maguire et al., 2009; Barnes et al., 2013; Brame et al., 2015) .
TextSentencer_T185 33976-34167 Sentence denotes Apelins are modulators of vascular tone and cardiac contractility in vitro and in vivo (Japp et al., 2008 (Japp et al., , 2010 Maguire et al., 2009; Barnes et al., 2013; Brame et al., 2015) .
TextSentencer_T186 34168-34514 Sentence denotes Therefore, as expected we found that [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) contracted human saphenous vein with equal potency and efficacy compared with our previous data for [Pyr 1 ]apelin-13 (Maguire et al., 2009; Brame et al., 2015) and was a potent inotrope in mouse paced right ventricle and human paced atria.
TextSentencer_T186 34168-34514 Sentence denotes Therefore, as expected we found that [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) contracted human saphenous vein with equal potency and efficacy compared with our previous data for [Pyr 1 ]apelin-13 (Maguire et al., 2009; Brame et al., 2015) and was a potent inotrope in mouse paced right ventricle and human paced atria.
TextSentencer_T187 34515-34629 Sentence denotes We next investigated the in vivo actions of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T187 34515-34629 Sentence denotes We next investigated the in vivo actions of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) .
TextSentencer_T188 34630-35223 Sentence denotes In anesthetized rat compared to our previously published data for [Pyr 1 ]apelin-13 (Brame et al., 2015) , the ACE2 metabolite caused a smaller but significant drop in blood pressure which contrasts with Wang and colleagues (Wang et al., 2016) who reported that [Pyr 1 ]apelin-13, but not [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , showed a sustained effect on blood pressure for up to 60 min after administration to mice in vivo, although both peptides appear to show an initial comparable reduction in blood pressure over the first 10-15 min in these experiments.
TextSentencer_T188 34630-35223 Sentence denotes In anesthetized rat compared to our previously published data for [Pyr 1 ]apelin-13 (Brame et al., 2015) , the ACE2 metabolite caused a smaller but significant drop in blood pressure which contrasts with Wang and colleagues (Wang et al., 2016) who reported that [Pyr 1 ]apelin-13, but not [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , showed a sustained effect on blood pressure for up to 60 min after administration to mice in vivo, although both peptides appear to show an initial comparable reduction in blood pressure over the first 10-15 min in these experiments.
TextSentencer_T189 35224-35704 Sentence denotes Interestingly, these authors also showed that [Pyr 1 ]apelin-13 is rapidly cleaved by ACE2 in vitro and in vivo suggesting that the ACE2 cleavage product may contribute to their observed sustained response to [Pyr 1 ]apelin-13 and as might be expected the half-life of the cleavage product is likely to be short and therefore the response to infused [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) is observed for only the initial 10∼15 min of the experiment.
TextSentencer_T189 35224-35704 Sentence denotes Interestingly, these authors also showed that [Pyr 1 ]apelin-13 is rapidly cleaved by ACE2 in vitro and in vivo suggesting that the ACE2 cleavage product may contribute to their observed sustained response to [Pyr 1 ]apelin-13 and as might be expected the half-life of the cleavage product is likely to be short and therefore the response to infused [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) is observed for only the initial 10∼15 min of the experiment.
TextSentencer_T190 35705-35975 Sentence denotes In agreement with this hypothesis, in our rat studies (Brame et al., 2015) the halflife of [Pyr 1 ]apelin-13 was less than 3 min and in humans FIGURE 9 | Schematic diagram showing the interactions between apelin/apelin receptor with ACE2 of the renin angiotensin system.
TextSentencer_T190 35705-35975 Sentence denotes In agreement with this hypothesis, in our rat studies (Brame et al., 2015) the halflife of [Pyr 1 ]apelin-13 was less than 3 min and in humans FIGURE 9 | Schematic diagram showing the interactions between apelin/apelin receptor with ACE2 of the renin angiotensin system.
TextSentencer_T191 35976-36032 Sentence denotes New contributions of this study are shown in red arrows.
TextSentencer_T191 35976-36032 Sentence denotes New contributions of this study are shown in red arrows.
TextSentencer_T192 36033-36164 Sentence denotes Ang, angiotensin; APJ, apelin receptor; AT1R, angiotensin receptor type I; MAS, Mas receptor. less than 8 min (Japp et al., 2008) .
TextSentencer_T192 36033-36164 Sentence denotes Ang, angiotensin; APJ, apelin receptor; AT1R, angiotensin receptor type I; MAS, Mas receptor. less than 8 min (Japp et al., 2008) .
TextSentencer_T193 36165-36456 Sentence denotes Importantly, in a firstin-man study, infusion of [Pyr 1 ]apelin-13 (1-12) resulted in an increase in forearm blood flow that was the same magnitude as we had previously obtained with [Pyr 1 ]apelin-13 (Brame et al., 2015) , confirming that this metabolite is a vasodilator in humans in vivo.
TextSentencer_T193 36165-36456 Sentence denotes Importantly, in a firstin-man study, infusion of [Pyr 1 ]apelin-13 (1-12) resulted in an increase in forearm blood flow that was the same magnitude as we had previously obtained with [Pyr 1 ]apelin-13 (Brame et al., 2015) , confirming that this metabolite is a vasodilator in humans in vivo.
TextSentencer_T194 36457-36719 Sentence denotes The 55-amino acid proapelin, derived from the 77-residue prepropeptide, contains a number of paired basic amino acids residues that are possible cleavage sites for endopeptidases to produce 13 to 36-residue isoforms (Tatemoto et al., 1998; Habata et al., 1999) .
TextSentencer_T194 36457-36719 Sentence denotes The 55-amino acid proapelin, derived from the 77-residue prepropeptide, contains a number of paired basic amino acids residues that are possible cleavage sites for endopeptidases to produce 13 to 36-residue isoforms (Tatemoto et al., 1998; Habata et al., 1999) .
TextSentencer_T195 36720-36968 Sentence denotes While the identities of these peptidases remain unknown, a study reported the direct cleavage of proapelin to apelin-13 by proprotein convertase subtilisin/kexin 3 (PCSK3, or furin), bypassing the generation of longer isoforms (Shin et al., 2013) .
TextSentencer_T195 36720-36968 Sentence denotes While the identities of these peptidases remain unknown, a study reported the direct cleavage of proapelin to apelin-13 by proprotein convertase subtilisin/kexin 3 (PCSK3, or furin), bypassing the generation of longer isoforms (Shin et al., 2013) .
TextSentencer_T196 36969-37183 Sentence denotes Post-translational modification results in the predominant apelin isoform detected in human cardiovascular tissue and plasma, [Pyr 1 ]apelin-13 Kleinz and Davenport, 2004; Maguire et al., 2009; Zhen et al., 2013) .
TextSentencer_T196 36969-37183 Sentence denotes Post-translational modification results in the predominant apelin isoform detected in human cardiovascular tissue and plasma, [Pyr 1 ]apelin-13 Kleinz and Davenport, 2004; Maguire et al., 2009; Zhen et al., 2013) .
TextSentencer_T197 37184-37569 Sentence denotes The putative ACE2 metabolite of this cardiac peptide, [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , is the focus of our study but we have also explored the pharmacology of several other important apelin isoforms to better understand the apelin structure activity relationships, namely the minimum active fragment apelin-13(R10M), apelin-13(F13A) and apelin-17.
TextSentencer_T197 37184-37569 Sentence denotes The putative ACE2 metabolite of this cardiac peptide, [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , is the focus of our study but we have also explored the pharmacology of several other important apelin isoforms to better understand the apelin structure activity relationships, namely the minimum active fragment apelin-13(R10M), apelin-13(F13A) and apelin-17.
TextSentencer_T198 37570-37767 Sentence denotes 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) .
TextSentencer_T198 37570-37767 Sentence denotes 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) .
TextSentencer_T199 37768-37891 Sentence denotes The C-terminal phenylalanine (F13), adjacent proline and the N-terminal pyroglutamic acid were not identified as important.
TextSentencer_T199 37768-37891 Sentence denotes The C-terminal phenylalanine (F13), adjacent proline and the N-terminal pyroglutamic acid were not identified as important.
TextSentencer_T200 37892-38225 Sentence denotes 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.
TextSentencer_T200 37892-38225 Sentence denotes 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.
TextSentencer_T201 38226-38685 Sentence denotes 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) .
TextSentencer_T201 38226-38685 Sentence denotes 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) .
TextSentencer_T202 38686-38892 Sentence denotes 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) .
TextSentencer_T202 38686-38892 Sentence denotes 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) .
TextSentencer_T203 38893-39156 Sentence denotes 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.
TextSentencer_T203 38893-39156 Sentence denotes 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.
TextSentencer_T204 39157-39389 Sentence denotes Early alanine scanning studies indicated that replacing the C-terminal phenylalanine with alanine did not abolish G αimediated signaling, calcium mobilization (Medhurst et al., 2003) and receptor internalization (Fan et al., 2003) .
TextSentencer_T204 39157-39389 Sentence denotes Early alanine scanning studies indicated that replacing the C-terminal phenylalanine with alanine did not abolish G αimediated signaling, calcium mobilization (Medhurst et al., 2003) and receptor internalization (Fan et al., 2003) .
TextSentencer_T205 39390-39648 Sentence denotes However, conflicting data have been reported for F13A, suggesting that it was an antagonist of the apelin receptor in terms of G αimediated signaling ([Pyr 1 ]apelin-13(F13A); De Mota et al., 2000) and hypotensive effect (apelin-13(F13A); Lee et al., 2005) .
TextSentencer_T205 39390-39648 Sentence denotes However, conflicting data have been reported for F13A, suggesting that it was an antagonist of the apelin receptor in terms of G αimediated signaling ([Pyr 1 ]apelin-13(F13A); De Mota et al., 2000) and hypotensive effect (apelin-13(F13A); Lee et al., 2005) .
TextSentencer_T206 39649-39845 Sentence denotes In this study, we found that apelin-13(F13A) resembled [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in receptor binding, cell based assays and the vasoconstrictor bioassay.
TextSentencer_T206 39649-39845 Sentence denotes In this study, we found that apelin-13(F13A) resembled [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in receptor binding, cell based assays and the vasoconstrictor bioassay.
TextSentencer_T207 39846-39952 Sentence denotes Based on our results apelin-13(F13A) behaves as an apelin agonist with no evidence of receptor antagonism.
TextSentencer_T207 39846-39952 Sentence denotes Based on our results apelin-13(F13A) behaves as an apelin agonist with no evidence of receptor antagonism.
TextSentencer_T208 39953-40229 Sentence denotes 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) .
TextSentencer_T208 39953-40229 Sentence denotes 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) .
TextSentencer_T209 40230-40393 Sentence denotes 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.
TextSentencer_T209 40230-40393 Sentence denotes 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.
TextSentencer_T210 40394-40543 Sentence denotes Intriguingly, apelin-17 appeared to be more biased for βarrestin compared to G protein signaling relative to the reference agonist [Pyr 1 ]apelin-13.
TextSentencer_T210 40394-40543 Sentence denotes Intriguingly, apelin-17 appeared to be more biased for βarrestin compared to G protein signaling relative to the reference agonist [Pyr 1 ]apelin-13.
TextSentencer_T211 40544-40777 Sentence denotes 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.
TextSentencer_T211 40544-40777 Sentence denotes 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.
TextSentencer_T212 40778-40890 Sentence denotes Past studies have suggested a possible link between apelin, its receptor, ACE2 and the renin angiotensin system.
TextSentencer_T212 40778-40890 Sentence denotes Past studies have suggested a possible link between apelin, its receptor, ACE2 and the renin angiotensin system.
TextSentencer_T213 40891-41181 Sentence denotes For example, apelin knockout mice showed aging or stress-associated cardiac contractility defects, similar to the cardiac phenotype of ACE2 knockout mice (Kuba et al., 2007) and apelin receptor knockout mice are more sensitive to the pressor effect of angiotensin II (Ishida et al., 2004) .
TextSentencer_T213 40891-41181 Sentence denotes For example, apelin knockout mice showed aging or stress-associated cardiac contractility defects, similar to the cardiac phenotype of ACE2 knockout mice (Kuba et al., 2007) and apelin receptor knockout mice are more sensitive to the pressor effect of angiotensin II (Ishida et al., 2004) .
TextSentencer_T214 41182-41362 Sentence denotes Interestingly, ACE2 expression is reduced in apelin knockout mice and apelin up-regulates ACE2 expression, indicating that apelin may reciprocally impact ACE2 (Sato et al., 2013) .
TextSentencer_T214 41182-41362 Sentence denotes Interestingly, ACE2 expression is reduced in apelin knockout mice and apelin up-regulates ACE2 expression, indicating that apelin may reciprocally impact ACE2 (Sato et al., 2013) .
TextSentencer_T215 41363-41610 Sentence denotes At the receptor level, the apelin receptor has been shown to physically interact with the angiotensin receptor type I (AT1R) , forcing it into a low affinity state and reducing the binding and signaling of angiotensin II (Siddiquee et al., 2013) .
TextSentencer_T215 41363-41610 Sentence denotes At the receptor level, the apelin receptor has been shown to physically interact with the angiotensin receptor type I (AT1R) , forcing it into a low affinity state and reducing the binding and signaling of angiotensin II (Siddiquee et al., 2013) .
TextSentencer_T216 41611-41748 Sentence denotes Reduced apelin expression due to heart failure or angiotensin II administration can be restored by AT1R blockade (Iwanaga et al., 2006) .
TextSentencer_T216 41611-41748 Sentence denotes Reduced apelin expression due to heart failure or angiotensin II administration can be restored by AT1R blockade (Iwanaga et al., 2006) .
TextSentencer_T217 41749-42063 Sentence denotes The opposing effects of apelin/ACE2 and angiotensin II have been shown in health and diseases such as heart failure, atherosclerosis and obesity/diabetes (Ashley et al., 2006; Gurzu et al., 2006; Iwanaga et al., 2006; Zhong et al., 2007; Chun et al., 2008; Barnes et al., 2013; Siddiquee et al., 2013) (Figure 9 ).
TextSentencer_T217 41749-42063 Sentence denotes The opposing effects of apelin/ACE2 and angiotensin II have been shown in health and diseases such as heart failure, atherosclerosis and obesity/diabetes (Ashley et al., 2006; Gurzu et al., 2006; Iwanaga et al., 2006; Zhong et al., 2007; Chun et al., 2008; Barnes et al., 2013; Siddiquee et al., 2013) (Figure 9 ).
TextSentencer_T218 42064-42371 Sentence denotes Our study contributes further evidence that ACE2, that is up-regulated in disease, acting on [Pyr 1 ]apelin-13 may result in the generation of the active metabolite, [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , and therefore a compensatory maintenance of apelin receptor signaling.
TextSentencer_T218 42064-42371 Sentence denotes Our study contributes further evidence that ACE2, that is up-regulated in disease, acting on [Pyr 1 ]apelin-13 may result in the generation of the active metabolite, [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) , and therefore a compensatory maintenance of apelin receptor signaling.
TextSentencer_T219 42372-42690 Sentence denotes However, a limitation of our study is that we have not measured levels of apelin peptides in plasma of patients receiving rhACE2 therapy, but we would hypothesize that levels of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) would be elevated and [Pyr 1 ]apelin-13 decreased compared to controls.
TextSentencer_T219 42372-42690 Sentence denotes However, a limitation of our study is that we have not measured levels of apelin peptides in plasma of patients receiving rhACE2 therapy, but we would hypothesize that levels of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) would be elevated and [Pyr 1 ]apelin-13 decreased compared to controls.
TextSentencer_T220 42691-43046 Sentence denotes In conclusion, this study confirmed that ACE2 cleaves [Pyr 1 ]apelin-13 into [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) and has demonstrated biological activity of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) at the human apelin receptor in vitro and in the cardiovascular system of rat and human in vivo.
TextSentencer_T220 42691-43046 Sentence denotes In conclusion, this study confirmed that ACE2 cleaves [Pyr 1 ]apelin-13 into [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) and has demonstrated biological activity of [Pyr 1 ]apelin-13 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) at the human apelin receptor in vitro and in the cardiovascular system of rat and human in vivo.
TextSentencer_T221 43047-43209 Sentence denotes The results also clarify R10M as the shortest active apelin fragment, apelin-13(F13A) as an agonist and apelin-17 as a more potent agonist than [Pyr 1 ]apelin-13.
TextSentencer_T221 43047-43209 Sentence denotes The results also clarify R10M as the shortest active apelin fragment, apelin-13(F13A) as an agonist and apelin-17 as a more potent agonist than [Pyr 1 ]apelin-13.
TextSentencer_T222 43210-43404 Sentence denotes Therefore, our study shows that reported enhanced ACE2 activity in cardiovascular disease should not significantly compromise the beneficial effects of apelin based therapies for example in PAH.
TextSentencer_T222 43210-43404 Sentence denotes Therefore, our study shows that reported enhanced ACE2 activity in cardiovascular disease should not significantly compromise the beneficial effects of apelin based therapies for example in PAH.
TextSentencer_T223 43405-43603 Sentence denotes 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).
TextSentencer_T223 43405-43603 Sentence denotes 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).
TextSentencer_T224 43604-43668 Sentence denotes All authors agree to be accountable for all aspects of the work.
TextSentencer_T224 43604-43668 Sentence denotes All authors agree to be accountable for all aspects of the work.