Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2 - PubMed (original) (raw)

Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2

Vaibhav B Patel et al. J Mol Med (Berl). 2015 Sep.

Abstract

Activation of the angiotensin 1-7/Mas receptor (MasR) axis counteracts angiotensin II (Ang II)-mediated cardiovascular disease. Recombinant human angiotensin-converting enzyme 2 (rhACE2) generates Ang 1-7 from Ang II. We hypothesized that the therapeutic effects of rhACE2 are dependent on Ang 1-7 action. Wild type male C57BL/6 mice (10-12 weeks old) were infused with Ang II (1.5 mg/kg/d) and treated with rhACE2 (2 mg/kg/d). The Ang 1-7 antagonist, A779 (200 ng/kg/min), was administered to a parallel group of mice. rhACE2 prevented Ang II-induced hypertrophy and diastolic dysfunction while A779 prevented these beneficial effects and precipitated systolic dysfunction. rhACE2 effectively antagonized Ang II-mediated myocardial fibrosis which was dependent on the action of Ang 1-7. Myocardial oxidative stress and matrix metalloproteinase 2 activity was further increased by Ang 1-7 inhibition even in the presence of rhACE2. Activation of Akt and endothelial nitric oxide synthase (eNOS) by rhACE2 were suppressed by the antagonism of Ang 1-7 while the activation of pathological signaling pathways was maintained. Blocking Ang 1-7 action prevents the therapeutic effects of rhACE2 in the setting of elevated Ang II culminating in systolic dysfunction. These results highlight a key cardioprotective role of Ang 1-7, and increased Ang 1-7 action represents a potential therapeutic strategy for cardiovascular diseases.

Key messages: Activation of the renin-angiotensin system (RAS) plays a key pathogenic role in cardiovascular disease. ACE2, a monocarboxypeptidase, negatively regulates pathological effects of Ang II. Antagonizing Ang 1-7 prevents the therapeutic effects of recombinant human ACE2. Our results highlight a key protective role of Ang 1-7 in cardiovascular disease.

Keywords: Angiotensin 1–7; Angiotensin-converting enzyme 2; PI3K/Akt signaling; Renin–angiotensin system.

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Figures

Fig. 1

Plasma ACE2 activity and angiotensin peptide levels in response to Ang II and treatment with rhACE2 and A779. Plasma ACE2 activity was markedly elevated in response to rhACE2 (a). Ang II infusion increased plasma Ang II levels which was prevented by treatment with rhACE2 (b) which also resulted in elevated plasma Ang 1–7 levels (c). n = 8 for the vehicle group and _n_=10 for all other groups. *p<0.05 compared to the vehicle group; #p<0.05 compared to the Ang II group; $p<0.05 compared with all other groups

Fig. 2

Antagonism of Ang 1–7 inhibits cardioprotective effects of rhACE2 and leads to systolic dysfunction in response to Ang II. Echocardiographic assessment of heart function as illustrated by M-mode images (a) and quantitative evaluation of heart function showing reduced LV ejection fraction (b) and LV fractional shortening (c), increased LV posterior wall thickness (LVPWT) (d) and isovolumic relaxation time (IVRT) (e) in response to Ang 1–7 blockade in the setting of elevated Ang II and treatment with rhACE2. n = 12 for each group. Taqman PCR analysis showing increased mRNA expression of atrial natriuretic factor (ANF, f), brain natriuretic peptide (BNP, g), β-myosin heavy chain (β-MHC, h), and α-skeletal actin (α-SKA, i) in response to Ang 1–7 blockade in the setting of elevated Ang II and treatment with rhACE2. _n_=6 for each group. *p<0.05 compared to the vehicle group; #p<0.05 compared to the Ang II group

Fig. 3

Antagonism of Ang 1–7 inhibits the anti-fibrotic effects of rhACE2 and exacerbates Ang II-induced cardiac fibrosis. Masson trichrome (a) and picrosirius red (b) staining showing Ang II-mediated increased perivascular and interstitial myocardial fibrosis which was inhibited by rhACE2 and further exacerbated by blocking Ang 1–7 action using A779. Quantitative evaluation of myocardial collagen levels derived from PSR staining images showing A779 co-treatment with rhACE2 ameliorated Ang II-mediated increase in myocardial collagen levels (c). n = 4 for each group. Similarly, higher mRNA expression of procollagen Iα (d), procollagen IIIα (e) and transforming growth factor betal (TGF-β1, f) normalized with 18S in response to Ang II was blocked and exacerbated by rhACE2 and A779, respectively, _n_=8 for each group. *p<0.05 compared to the vehicle group; #p<0.05 compared to the Ang II-treated group

Fig. 4

Inhibition of Ang 1–7 action inhibits the anti-fibrotic effects of rhACE2 and exacerbates Ang II-induced renal and lung fibrosis. Masson trichrome staining images (a, c) and picrosirius red staining images (b, d) showing enhanced renal (a, b) and lung fibrosis (c, d) in Ang II treated mice which was suppressed by rhACE2 treatment and exacerbated by blocking Ang 1–7 with A779 (a-d). Quantitative evaluation of renal (e) and lung (f) collagen levels derived from PSR staining images confirming that rhACE2 and A779 co-treatment suppressed and potentiated Ang II-mediated increase in tissue fibrosis, respectively. _n_=4 for each group. *p<0.05 compared to the vehicle group; #p<0.05 compared to the Ang II group

Fig. 5

Myocardial oxidative stress induced by Ang II is suppressed and exacerbated by rhACE2 and A779, respectively. Ang II treatment increased myocardial oxidative stress as shown by dihydroethidium (DHE) fluorescence imaging and quantification of superoxide levels (a, c), increased nitrotyrosine level (b, d) and increased NADPH oxidase activity (e). Recombinant human ACE2 prevented Ang II-induced oxidative stress while A779 restored nitrotyrosine levels and further exacerbates DHE fluorescence and NADPH oxidase activity (a–e). Apocynin (1 mM) were used to suppress NADPH oxidase activity (gray bars). In situ zymography (f) and quantification (g) showing increased myocardial gelatinase activity in response to Ang II, suppression by rhACE2 and a further increase in response to blocking Ang 1–7 action. Gelatin zymography (i) showing increased MMP9 (ii), pro and active MMP2 (iii and iv) in response to Ang II which was suppressed by rhACE2; blocking Ang 1–7 action prevented these effects and further increased the levels of active MMP2 (h). _n_=6 for each group. RE relative expression, AU arbitrary unit. *p<0.05 compared to the vehicle group; #p<0.05 compared to the Ang II group

Fig. 6

Blocking Ang 1–7 action results in loss of phosphorylation of Akt and eNOS and maintained activation of the MAPK signaling pathways. Phosphorylation of myocardial Akt (serine-473) (a) and Akt (threonrne-308) (b) and eNOS (serinel 177) (c) were reduced by Ang II which was restored in response to rhACE2 and further suppressed by A779 treatment. Phosphorylation of myocardial ERK1/2 (d), JNK1/2 (e) and p38 (f) signaling pathways were increased by Ang II, suppressed by rhACE2 which was loss when Ang 1–7 action was blocked with A779 treatment. RE relative expression. n = 6 for each group. *p<0.05 compared to the vehicle group; #p<0.05 compared to the Ang II group

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Antagonism of angiotensin 1-7 prevents the therapeutic effects of recombinant human ACE2 - PubMed (original) (raw)