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

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

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

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

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