Mas1 Receptor Activation is Necessary and Sufficient to Transduce ACE2 Effect in PAH, But Ang(1-7) Alone is Insufficient

Abstract

ACE2 has shown effectiveness in treating pulmonary hypertension in multiple animal models and has some promise in early human trials. The key barrier to translation is that enzymatically active ACE2 is difficult to manufacture and exhibits a short half-life in humans, making chronic administration challenging. Understanding the mechanism of effect is thus key to finding ways to bypass ACE2 while still reproducing therapeutic effects. In this study, we test the hypotheses that ACE2 produces its therapeutic effect through increased Mas1 signaling and that Ang(1-7) is sufficient as the Mas1 ligand. We found that the ACE2 effect is blocked in Mas1 knockout mice and that the Mas1 agonist AR234960 reproduces the ACE2 effect, indicating that Mas1 activation is necessary and sufficient for the ACE2 therapeutic effect. However, neither AlbudAb-stabilized Ang(1-7) nor Ang(1-7) stabilized through the use of protease inhibitors were capable of reproducing ACE2 effectiveness, indicating that Ang(1-7) alone does not activate Mas1 in this context. RNA-seq suggests that the key mechanisms downstream of Mas1 responsible for the therapeutic effect of ACE2 and AR234960 are the rescue of cytoskeletal and microtubule defects. Together, these findings indicate that direct activation of Mas1 will likely be effective in treating pulmonary arterial hypertension, but raise the question of the identity of the endogenous ligand(s).

Figure 1

Initial hypothesis for ACE2 therapeutic effect in pulmonary hypertension; ACE2 cleaves one amino acid from angiotensin II to produce Ang(1‐7), which signals through the Mas1 receptor to produce beneficial effects. This study demonstrates that this is partially incorrect; ACE2 and Mas1 are required, but Ang(1‐7) is not the ligand.

Figure 2

Mas1 activation is necessary and sufficient to reproduce the ACE2 effect on the pulmonary vasculature (A) Right Ventricular Systolic Pressure. Each symbol indicates pressure from one animal. The diamonds in the first two columns are Mas1^−/− mice; filled symbols in the normoxic group were treated with ACE2. Error bars are SEM. p values are Tukey HSD after ANOVA. (B) Right ventricular muscularization. Symbols, error bars, and statistics are as per (A). (C) Muscularized vessels. Each symbol is an average of vessel counts in ten 20× fields in a single mouse; each symbol is thus an average from an individual animal. Error bars are SEM. p values are nonparametric Wilcoxon values after ANOVA (overall ANOVA p = 0.0003). (D) Representative immunohistochemistry images for muscularized vessels. Blue is nuclear stain (DAPI); red is alpha‐smooth muscle actin.

Figure 3

Ang(1‐7) does not reproduce the effects of ACE2 or Mas1 activation on the pulmonary vasculature. (A) Analysis of the effect of ACE2 and Alt00 + Lisinopril on Angiotensin system components by mass spectrometry, in vivo in serum. The area of circles is proportional to quantity; red indicates reduced from control, and green indicates increased and is an average of treated animals. (B) Right Ventricular Systolic Pressure. Each symbol indicates pressure from one animal. Error bars are SEM. p values are Tukey HSD after ANOVA. (C) Right ventricular muscularization. Symbols, error bars, and statistics are as per (B). (D) Muscularized vessels. Each symbol is an average of vessel counts in ten 20× fields in a single mouse; each symbol is thus an average from an individual animal. Error bars are SEM. p values are nonparametric Wilcoxon values after ANOVA (overall ANOVA p = 0.0003). (E) Representative immunohistochemistry images for muscularized vessels. Blue is the nuclear stain (DAPI); red is alpha‐smooth muscle actin.

Figure 4

RNASeq (A) Venn diagram indicating overlap between genes differentially regulated by ACE2 (blue circle), AR234960 (purple circle), and Alt00/Lisi (green ellipse). Numbers indicate the number of genes regulated in common when these overlap. Sizes are roughly proportional to numbers. (B) Examples of genes differentially regulated in common with ACE2 and AR234960. (C) Examples of genes differentially regulated in common with ACE2 and Alt00/Lisi. (D) Examples of genes differentially regulated only by AR234960. For B–D, error bars are SEM, circles are values from individual animals, bar heights are averages, and *p < 0.05 by Wilcoxon nonparametric test compared to SuHx with vehicle only. (E) Western blot for LIMK1. Numbers are densitometry, normalized to Gapdh, with densitometry plotted at right. *p < 0.05 by Wilcoxon nonparametric test.