ACE2-Mediated Reduction of Oxidative Stress in the Central Nervous System Is Associated with Improvement of Autonomic Function

Oxidative stress in the central nervous system mediates the increase in sympathetic tone that precedes the development of hypertension. We hypothesized that by transforming Angiotensin-II (AngII) into Ang-(1–7), ACE2 might reduce AngII-mediated oxidative stress in the brain and prevent autonomic dysfunction. To test this hypothesis, a relationship between ACE2 and oxidative stress was first confirmed in a mouse neuroblastoma cell line (Neuro2A cells) treated with AngII and infected with Ad-hACE2. ACE2 overexpression resulted in a reduction of reactive oxygen species (ROS) formation. In vivo, ACE2 knockout (ACE2−/y) mice and non-transgenic (NT) littermates were infused with AngII (10 days) and infected with Ad-hACE2 in the paraventricular nucleus (PVN). Baseline blood pressure (BP), AngII and brain ROS levels were not different between young mice (12 weeks). However, cardiac sympathetic tone, brain NADPH oxidase and SOD activities were significantly increased in ACE2−/y. Post infusion, plasma and brain AngII levels were also significantly higher in ACE2−/y, although BP was similarly increased in both genotypes. ROS formation in the PVN and RVLM was significantly higher in ACE2−/y mice following AngII infusion. Similar phenotypes, i.e. increased oxidative stress, exacerbated dysautonomia and hypertension, were also observed on baseline in mature ACE2−/y mice (48 weeks). ACE2 gene therapy to the PVN reduced AngII-mediated increase in NADPH oxidase activity and normalized cardiac dysautonomia in ACE2−/y mice. Altogether, these data indicate that ACE2 gene deletion promotes age-dependent oxidative stress, autonomic dysfunction and hypertension, while PVN-targeted ACE2 gene therapy decreases ROS formation via NADPH oxidase inhibition and improves autonomic function. Accordingly, ACE2 could represent a new target for the treatment of hypertension-associated dysautonomia and oxidative stress

ACE2 overexpression in the PVN decreases NADPH oxidase activity in ACE2^−/y mice.

<p>NADPH oxidase activity in HT (A) and VLM (B) was significantly higher in ACE2^−/y mice on baseline (*<i>P</i><0.05 vs. NT, n = 4). This enzyme activity was significantly increased in NT following AngII infusion (*<i>P</i><0.05 vs. NT, n = 4) and remained more elevated in ACE2^−/y (†<i>P</i><0.05 vs. NT+AngII). Ad-ACE2 expression in the PVN of ACE2^−/y significantly decreased NADPH oxidase activity (‡<i>P</i><0.05 vs. KO+AngII+Ad-GFP). Baseline SOD activity was significantly higher in ACE2^−/y mice (C,D; *<i>P</i><0.05 vs. NT, n = 4). AngII infusion did not change SOD activity in either genotype (<i>P</i>>0.05, n = 4). Ad-ACE2 expression in the PVN of ACE2^−/y decreased SOD activity in HT (C; ‡<i>P</i><0.05 vs. KO+AngII+Ad-GFP) without changing its activity in VLM (D; <i>P</i>>0.05, n = 4).</p

ACE2 expression, activity and MAP in ACE2^−/y mice following ACE2 gene therapy to the PVN.

<p>Immunohistochemistry (A) showing green fluorescent protein (eGFP) expression targeted to the PVN, 7 days after unilateral administration of adenovirus. ACE2 activity in HT (B) and VLM (C) was significantly lower in ACE2^−/y than NT mice (*<i>P</i><0.01, n = 4). Seven days after ACE2 bilateral adenovirus injection to the PVN, ACE2 activity was significantly increased in HT and downstream VLM in KO mice (*<i>P</i><0.01 vs. NT, n = 4). Ad-hACE2 injected in the PVN of ACE2^−/y on the 3^rd day of AngII infusion did not alter the course of the developing hypertension (D, <i>P</i>>0.05, n = 8).</p

Effects of AngII and ACE2 on oxidative stress in Neuro2A cells.

<p>Western blots showed that AngII treatment (100 nmol/L, 24 h) decreased ACE2 (A; 100 KDa, n = 6, **<i>P</i><0.01 vs. vehicle), increased AT1R (43 KDa; B; n = 6, *<i>P</i><0.05 vs. vehicle) without changing AT2R (44 KDa; C; <i>P</i>>0.05 vs. vehicle) protein expression. Representative DHE staining (D) and quantified data (E), showing that AngII (100 nmol/L, 30 min) significantly increased ROS production (n = 6, **<i>P</i><0.01 vs. no treatment). Pre-treatment with Ad-ACE2-eGFP reduced AngII-stimulated ROS formation (n = 6, †<i>P</i><0.05 vs. AngII+Ad-eGFP, *<i>P</i><0.05 vs. no treatment). Blockade of AT1R with losartan completely prevented AngII-mediated ROS production (n = 6, †<i>P</i><0.05 vs. AngII+Ad-eGFP).</p

ROS and AngII levels in young ACE2^−/y mice.

<p>Representative DHE staining and quantified data showing no difference in baseline ROS levels in the PVN (A, B) or RVLM (C, D) between KO and NT mice (<i>P</i>>0.05, n = 8). AngII infusion significantly increased ROS production in these regions in both genotypes (A–D; *<i>P</i><0.05 vs. baseline, n = 8), with higher levels in KO (A–D; †<i>P</i><0.05 vs. NT+AngII). Baseline AngII levels in both plasma (E) and brain (F) were not different between mice (<i>P</i>>0.05, n = 8). While AngII infusion significantly increased plasma (E) AngII levels in both genotypes, only KO mice show elevated brain AngII levels (F) (*<i>P</i><0.05 vs. baseline, n = 8). KO mice exhibited higher plasma and central AngII levels following AngII infusion (†<i>P</i><0.05, vs. NT+AngII).</p

Normal blood pressure and increased autonomic dysfunction in young ACE2^−/y mice.

<p>Baseline blood pressure traces from young non-transgenic (NT) and ACE2^−/y (KO) mice over 3 days recording show no differences between genotypes (A). Mean arterial pressure (MAP) was similarly increased in both NT and KO mice during AngII infusion (B). KO mice exhibited enhanced bradycardia to propranolol (C; *<i>P</i><0.05 vs. NT; n = 8) and reduced tachycardia following atropine (D; *<i>P</i><0.05 vs. NT; n = 8). AngII infusion significantly increased sympathetic drive (C) and reduced vagal tone (D) in both NT and KO (†<i>P</i><0.05 vs. baseline, n = 8). Autonomic dysfunction remained more pronounced in KO compared to NT following AngII infusion. (C,D; ‡<i>P</i><0.05 vs. NT+AngII). Changes in BP following ganglionic blockade, an index of vascular sympathetic drive, were not different between NT and KO on baseline or following AngII infusion (E; <i>P</i>>0.05).</p

Increased BP, autonomic dysfunction and oxidative stress in mature ACE2^−/y mice.

<p>Baseline MAP was significantly increased in mature ACE2^−/y compared to age-matched NT mice (A). Increased sympathetic (B) and decreased parasympathetic (C) tones were also observed in ACE2^−/y mice (*<i>P</i><0.05 vs. NT). In addition, vascular sympathetic tone in ACE2^−/y mice was significantly elevated compared to NT (D; *<i>P</i><0.05 vs. NT). Moreover, oxidative stress was exacerbated in the several brain regions (SFO: subfornical organ, PVN: paraventricular ncleus, NTS: nucleus of the tractus solitarius, RVLM: rostral ventrolateral medulla and AP: area postrema) of mature ACE2^−/y compared to NT mice (E; *<i>P</i><0.05 vs. NT).</p