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. Author manuscript; available in PMC: 2021 Nov 1.
Published in final edited form as: Hypertension. 2020 Sep 8;76(5):1514–1525. doi: 10.1161/HYPERTENSIONAHA.120.15681

Overexpression of Central ACE2 Attenuates the Pressor Response to Chronic Central Infusion of Angiotensin II: A Potential Role for Nrf2

Anyun Ma 1, Lie Gao 1, Ahmed M Wafi 1, Li Yu 1, Tara Rudebush 1, Wenxian Zhou 1, Irving H Zucker 1
PMCID: PMC7544628  NIHMSID: NIHMS1620816  PMID: 32895018

Abstract

We investigated the mechanism by which ACE2 overexpression alters neurohumoral outflow and central oxidative stress is still unclear. Nuclear factor (erythroid-derived 2)-like2 (Nrf2) is a master antioxidant transcription factor that regulates cytoprotective and antioxidant genes. We hypothesized that upregulation of central ACE2 inhibits the pressor response to Angiotensin II (AngII) by reducing reactive oxygen species (ROS) through a Nrf2/antioxidant enzyme-mediated mechanism in the rostral ventrolateral medulla (RVLM). SynhACE2+/+ mice and their littermate controls synhACE2−/− were used to evaluate the consequence of intracerebroventricular (icv) infusion of AngII. In control mice, AngII infusion evoked a significant increase in blood pressure (BP) and norepinephrine (NE) excretion, along with polydipsia and polyuria. The pressor effect of central AngII was completely blocked in synhACE2+/+ mice. Polydipsia, NE excretion, and markers of oxidative stress in response to central AngII were also reduced in synhACE2+/+ mice. The Mas receptor (MasR) agonist Ang 1-7 and blocker A779 had no effects on BP. synhACE2+/+ mice showed enhanced expression of Nrf2 in the RVLM which was blunted following AngII infusion. AngII evoked nuclear translocation of Nrf2 in cultured Neuro2A (N2A) cells. In synhACE2−/− mice, the central AngII pressor response was attenuated by simultaneous icv-infusion of the Nrf2 activator Sulforaphane (SFN); BP was enhanced by knockdown of Nrf2 in the RVLM in Nrf2 floxed (Nrf2f/f) mice. These data suggest that the hypertensive effects of icv AngII are attenuated by selective overexpression of brain synhACE2, and may be mediated by Nrf2-upregulated antioxidant enzymes in the RVLM.

Keywords: Sympathetic nerve activity, oxidant stress, antioxidants, brainstem, Nrf2

Graphical Abstract

graphic file with name nihms-1620816-f0007.jpg

Introduction

Hypertension (HTN) is one of the most common causes of morbidity and mortality worldwide. Essential HTN is characterized, in part, by activation of the renin-angiotensin-aldosterone system (RAAS) and elevated SNA 1. There is a consensus that much of what is called “essential” HTN has a neurogenic component 2, 3. The circulating and central RAAS alter autonomic function and it is well accepted that central AngII increases arterial pressure (AP) by activating SNA and vasopressin secretion, indicating the primacy of central alterations in response to AngII 4. The areas of the central nervous system that regulate cardiovascular and sympathetic function are, to a large degree, centered around the RVLM and integrative areas such as the hypothalamus, the organum vasculosum of the lamina terminalis and the nucleus of the solitary tract (NTS), among others. Alterations in the function of these centers have been shown to play a critical role in cardiovascular disease 5, 6. Through binding to AT1R, AngII activates several downstream signals to elicit enhanced SNA and pressor effects7, 8.

The ACE homolog, ACE2 is a carboxy peptidase that cleaves the terminal phenylalanine from AngII to form Ang1-7. Ang1-7 is an important component of the RAAS system; the so-called “good arm of the RAAS” 9, 10. In previous work from this laboratory we have shown that rabbits with CHF exhibit increased ACE and decreased ACE2 expression in several central nuclei including the RVLM, the paraventricular nucleus (PVN) and the NTS11. We also demonstrated that SNA was significantly reduced in mice with CHF that overexpress ACE2 selectively in the brain12. However, the mechanisms by which central ACE2 reduces sympathetic outflow are not completely understood. In a HTN model, Sriramula et al.13 showed attenuation of the pressor response to peripheral infusion of AngII in mice that overexpress ACE2 in the brain. The same group also showed that peripheral AngII infusion increased oxidative stress in the PVN and RVLM significantly more in ACE2 knockout mice compared to their non-transgenic littermates14.

Oxidative stress is thought to be a key molecular mechanism that links the RAAS and SNA15. To reduce excess ROS, living organisms have developed a variety of antioxidant systems to prevent their harmful effects on cells. One of the most important of these is the redox sensitive transcription factor Nrf2, which is thought to play a critical role in up-regulating antioxidant genes to maintain redox balance in a variety of disease states16. Increased ROS activates Nrf2 by releasing it from its cytosolic tether, Kelch-like ECH-associated protein 1 (Keap1). We previously showed that selective Nrf2 gene deletion in the RVLM increases BP and sympathetic outflow in normal mice due to increases in oxidative stress17. A beneficial effect was observed in mice with CHF when Nrf2 was overexpressed in the RVLM via Nrf2 gene transfer or Keap1 gene knockdown18. Further evidence supporting a role for Nrf2 in BP regulation comes from studies using the Nrf2 activator SFN which lowered BP in spontaneously hypertensive stroke-prone (SHRSP) rats to a level comparable to normal Sprague Dawley rats 19, 20. It is unknown whether there is an interaction between Nrf2 and ACE2 in response to central AngII administration. To determine the potential role of Nrf2 in the beneficial effects of central ACE2 on autonomic regulation in HTN, we addressed the hypothesis that overexpression of brain ACE2 reduces SNA in the icv-AngII infusion mouse model through a modulatory effect of Nrf2 in the RVLM.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ICV infusion and implantation of osmotic minipump (see detailed methods in supplemental file at http://hyper.ahajournals.org)

Cell culture (see detailed methods in supplemental file at http://hyper.ahajournals.org)

Animals

All procedures were approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Center. Experiments were carried out consistent with the NIH Guide for the Care and Use of Laboratory Animals and conformed with the ARRIVE Guidelines to the extent possible21. Experiments were performed on two types of transgenic mouse models (3 month old, males). SynhACE2+/+ mice where brain (driven by the synapsin promoter) human ACE2 was overexpressed were used. Their non-transgenic littermates on the C57BL6 background were used as controls. These mice were bred from original heterozygotes obtained from the laboratory of Dr. Eric Lazartigues (The University of Iowa and the Louisiana State Health Sciences Center, New Orleans) 22. Nrf2f/f mice, where Nrf2 in the RVLM was selectively deleted by microinjection of a Lenti-Cre-GFP virus into the RVLM were also used in this study. This model was originally obtained from Dr. Shyam Biswal (the Johns Hopkins University) 23. Animals were housed in standard polypropylene cages in a facility with a 12:12 hour light-dark cycle (6am – 6pm lights on) and fed with standard mouse chow (Harlan Laboratories, Indianapolis, IN) and allowed water ad libitum.

Blood pressure and hemodynamics

Mice were instrumented with a radio telemetry unit (PA-C10; Data Sciences International, Minneapolis, MN) with the catheter inserted into the left carotid artery as previously described 24. After one week of recovery, pulsatile and mean arterial pressure (MAP) and heart rate (HR) were measured for 2 hours daily from noon to 2:00 PM. Following 3 days of baseline recording, a cannula was implanted into the right lateral ventricle and fixed to the skull with cyanoacrylate adhesive. AngII (100 ng/kg/min) or artificial cerebrospinal fluid (aCSF) was administered through an icv cannula via an osmotic minipump (Model 1002, Alzet, Inc. Cupertino, CA) for 14 days (0.25 ul/hr) or in some experiments for only 7 days (0.5 ul/hr). Co-administration of AngII with Ang1-7 (200 ng/kg/min) or the MasR antagonist A779 (400 ng/kg/min) was carried out in subsets of mice. Finally, co-administration of AngII with SFN (500 ng/kg/min) was performed on a subset of mice.

HR variability (HRV) was assessed by the standard deviation of all normal beats (SDRR). Power spectral density (PSD) and the ratio of low frequency (0.15 Hz-1.5 Hz) to high frequency (1.5 Hz-5 Hz) (LF/HF) was used as a surrogate index of cardiac sympatho-vagal balance25, 26. All data were acquired and analyzed using LabChart 8 software (ADInstruments, Inc. Colorado Springs, CO).

Water intake, urine flow, and urinary norepinephrine

Daily water intake and urine output were measured in metabolic cages (Harvard Apparatus, Holliston, MA) during the period when hemodynamic parameters were recorded. Mice were placed in the metabolic cage for 3 days prior to acquiring data. Urine samples were collected for the measurement of urinary NE concentration with an enzyme-linked immunoassay (EIA) kit (Rocky Mountains Diagnostics, Colorado Springs, CO). 24 hour NE excretion was calculated from 24h urine volume multiplied by urinary NE concentration.

Western blotting (see details in supplemental file at http://hyper.ahajournals.org)

Oxidative stress in the RVLM (see details in supplemental file at http://hyper.ahajournals.org)

Statistical analysis

Blood pressure and Western Blot results in the synhACE2 overexpression study were analyzed by a 2-way ANOVA followed by Tukey’s multiple comparisons. Sympathetic and oxidative stress parameters were analyzed by a 2-way ANOVA followed by Sidak’s multiple comparisons test. Blood pressure results from the Nrf2-Cre study were analyzed by a 1-way ANOVA followed by Tukey’s multiple comparisons test. Sulphorafane experimental results were analyzed using either a 1-way ANOVA followed by Dunnett’s multiple comparisons or 2-way ANOVA followed by Multiple t tests. Cell experiment data were analyzed by a 1-way ANOVA followed by Tukey’s multiple comparisons. All data were analyzed using Prism8 (GraphPad Software, San Diego, CA) and were expressed as mean ± SE. Differences were considered statistically significant at a p value of <0.05.

Results

Overexpression of Central ACE2 attenuates icv AngII-induced HTN

To evaluate the effect of overexpression of ACE2 on hemodynamic changes induced by central AngII, we determined AP and HR over 2 weeks before and during icv AngII administration in both synhACE2+/+ mice and their control littermates. As shown in Fig. 1A, in synhACE2−/− animals, chronic icv infusion of AngII significantly increased MAP [AngII(10-day average) 125.1±5.3 mmHg vs baseline (3-day average) 92.3±1.4 mmHg; Day 10 MAP 136.7±4.6 mmHg vs Day(−1) MAP 94.9±1.6 mmHg; n=6, p<0.05] as well as compared to animals infused with aCSF [AngII(10-day average) 125.1±5.3 mmHg, vs. aCSF(10-day average) 96.1±3.0 mmHg; Day 10 AngII group MAP 136.7±4.6 mmHg vs Day10 aCSF group MAP 95.5 ±1.1 mmHg; n=6, p<0.05]. The aCSF treated group did not exhibit any increase in MAP from baseline during the infusion period. ACE2 overexpression significantly attenuated AngII-induced HTN [MAP(10 day average) of synhACE2+/+/AngII 101.1±6.5 mmHg vs. synhACE2−/−/AngII 125.1±5.3 mmHg; Day 10 MAP synhACE2+/+/AngII 100.1±4.0 mmHg vs. synhACE2−/−/AngII 136.7±4.6 mmHg; n=6, p<0.05]. There were no effects on HR in any of the treatment groups (Fig. 1B). To determine if the inhibition of the hypertensive effect of AngII in synhACE2+/+ animals was mediated through a reduction in AngII-AT1R signaling, or an enhancement of the Ang1-7/MasR pathway, we co-administered Ang1-7 with AngII to synhACE2−/− mice, or the MasR antagonist, A779 with AngII to synhACE2+/+ mice, respectively. As is shown in Fig. 1A, Ang1-7 failed to blunt the pressor response induced by AngII in synhACE2−/− animals. In addition, the attenuated HTN in synhACE2+/+ animals was not prevented or reversed by co-infusion of A779 [MAP(10 day average) of synhACE2+/+/AngII/A779 101.0±4.8 mmHg vs synhACE2+/+/AngII 101.1±6.5 mmHg, p>0.05].

Figure 1. Central synhACE2 overexpression attenuates ICV-AngII induced BP increase.

Figure 1.

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A. Chronic icv AngII infusion induced a progressively increased MAP in the synhACE2−/− mice, which was completely blunted in the synhACE2+/+ mice. Chronic icv co-administration of AngII and Ang1-7 in synhACE2−/− mice did not attenuate the pressor response, nor did co-administration of AngII and MasR blocker A779 in synhACE2+/+ mice reverse the anti-hypertensive effect. Baseline BP was not altered by ACE2 overexpression. B. HR was not affected by icv-AngII in any of the genotypes. (n=5-7; *p<0.01 vs. baseline BP; #p<0.05 vs. baseline BP; @p<0.05 (10-day average) synhACE2+/+/AngII vs. synhACE2−/−/AngII; &p<0.05 (10-day average) synhACE2−/−/AngII/Ang1-7 vs. synhACE2−/−/aCSF; 2-way ANOVA followed by Tukey’s multiple comparisons)

The 24-hour diurnal hemodynamic data prior to and after AngII treatment are summarized in Supplemental Table S1 (http://hyper.ahajournals.org). and are separated by day and night averages. Circadian variability was clearly visible in both aCSF and AngII infusion groups. Fig. S1 summarizes the day and night data for MAP before and after icv infusion. Chronic icv AngII evoked a pressor response in both the day and night compared to aCSF in synhACE2−/− mice. However, this increase was markedly inhibited in synhACE2+/+ mice.

SynhACE2 attenuates central AngII infusion-induced polydipsia

AngII is well known for its ability to stimulate thirst behavior and fluid intake 27. To assess the effect of ACE2 overexpression on the central AngII-induced drinking response, we measured water intake and urine output for each group of animals (Fig. S2 http://hyper.ahajournals.org). Baseline water intake and urine production showed no differences among animals of all genotypes. In synhACE2−/− animals, AngII administration markedly increased water intake from a baseline of 5.6±0.2ml to 25.9±0.6 ml (n=5, p<0.05), with a significant increase in 24-hour urine excretion from 1.6±0.3 to 13.9±1.5 ml (n=5, p<0.05). This enhancement of fluid intake started gradually after AngII infusion and was sustained during the infusion. In synhACE2+/+ mice however, the drinking and urine volumes in response to icv AngII were attenuated (ΔH2O volume=10.5±2.1ml, ΔUrine volume=6.9±1.7ml n=5, p<0.05) compared to their non-transgenic littermates. There were no differences between synhACE2+/+ and synhACE2−/− mice infused with aCSF infusion.

Overexpression of ACE2 attenuates central AngII infusion-induced sympathetic overactivity

To determine if the inhibition of the BP response in synhACE2+/+ mice in response to central AngII was associated with a reduction in sympathetic outflow, we measured 24-hour urinary NE excretion and evaluated HRV and PSD. HRV was evaluated in both the time-domain, as reflected by SDRR, and the frequency-domain, as reflected by LF/HF using pulsatile BP data.

Fig. 2A shows changes in 24-hour NE excretion in each group. While baseline NE excretion was similar between all animals, icv AngII infusion markedly increased NE excretion in the synhACE2−/− group but not in synhACE2+/+ mice, suggesting that the increase of NE excretion due to AngII was prevented by central ACE2 overexpression. There was a clear increase in LF/HF as well as a decrease in SDRR in the AngII treated SynhACE2−/− group (Fig. 2B–C) indicating cardiac autonomic tone was skewed to an increased SNA. These changes were normalized in mice overexpressing central synhACE2. Taken together, these data suggest that selective overexpression of synhACE2 in the brain prevents AngII-induced sympatho-excitation.

Figure 2. synhACE2+/+ attenuates central AngII- induced sympathetic hyperactivity.

Figure 2.

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A. 24h NE excretion was reduced in synhACE2+/+ mice (n=4; *p<0.01 compared to synhACE2−/− /aCSF post-icv; #p<0.05 compared to synhACE2−/−/AngII post-icv). LF/HF(B) and SDRR(C) during baseline and post-infusion period showed ACE2 overexpression improved AngII induced HRV impairment. D. Representative PSD tracings. (n=3-4; *p<0.05 compared to synhACE2−/−/AngII-baseline; #p<0.05 compared to synhACE2−/−/AngII-post-icv; 2-way ANOVA followed by Sidak’s multiple comparisons test). Note different scales in each panel.

ACE2 overexpression attenuates central AngII infusion-induced oxidative stress

To determine if overexpression of ACE2 reduces central AngII-mediated ROS production, we evaluated oxidative stress using 8-hydroxydeoxyguanosine (8-OHdG) immunostaining of brainstem slices containing the RVLM. ROS oxidizes lipids, proteins and nucleic acids and causes cellular lesions. Since DNA is prevelant in both nuclei and mitochondria, evaluating DNA damage is widely used to assess cellular oxidative stress. As shown in Fig. 3, central AngII infusion markedly increased RVLM DNA oxidation in synhACE2−/− animals. This enhanced immunostaining intensity was reduced in SynhACE2+/+ mice, to the extent comparable to that of the aCSF group. ACE2 overexpression did not alter redox state in aCSF treated animals. Taken together, these data confirmed that central ACE2 overexpression effectively prevented icv AngII-induced oxidative stress in the RVLM.

Figure 3. synhACE2+/+ mice exhibit anti-oxidative property in response to central AngII.

Figure 3.

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Representative 8-OHdG immunofluorescence staining in the RVLM (A) and quantified data (B) showed that icv AngII infusion significantly increased DNA oxidation in the RVLM area of synhACE2−/− mice, but not in synhACE2+/+ group. ACE2 overexpression did not change ROS production during aCSF treatment (n=4, *p<0.05 vs. synhACE2−/−/AngII; #p<0.05 vs. synhACE2−/− /aCSF; 2-way ANOVA followed by Sidak’s multiple comparisons test).

Nrf2 and NAD(P)H quinone oxidoreductase 1 (NQO1) are upregulated in the RVLM of SynhACE2+/+ mice

In previous studies we showed that in animals with CHF, expression of both ACE2 and Nrf2 in the RVLM was significantly decreased 11, 18, 28. To further determine if there is a correlation between ACE2 and Nrf2, we measured Nrf2 protein in the RVLM of SynhACE2+/+ mice with or without central AngII administration. We found that icv AngII evoked an upregulation of Nrf2 in the RVLM of SynhACE2−/− animals that was inhibited by ACE2 overexpression (Fig. 4A). The antioxidant enzyme NQO1 (Fig. 4B) exhibited a similar trend as Nrf2. In order to determine if these changes in Nrf2 are specific to the RVLM, we also assessed tissues from the cerebral cortex and the hypothalamus, where no differences in Nrf2 levels were found (Fig. 4C–D).

Figure 4. Representative immunoblot of Nrf2 and NQO1 in the brain of synapsin human Angiotensin Converting Enzyme 2 positive (synhACE2+/+) with chronic central AngII infusion.

Figure 4.

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A. Western blot of whole tissue lysates from RVLM punches showed an increase of Nrf2 in response to icv-AngII infusion, which was restored by ACE2 overexpression. B. RVLM NQO1 was significantly increased by ACE2 overexpression, which was attenuated in the ACE2+/+/AngII group. C-D. Nrf2 was not different between groups in the visual cortex or hypothalamus. (n=4-6, *p<0.05; 2-way ANOVA followed by Tukey’s multiple comparisons test).

Selective knockdown of Nrf2 in the RVLM enhances the pressor effect of central infusion of AngII

Using Nrf2f/f mice, we evaluated the impact of RVLM Nrf2 deletion on central AngII infusion-induced HTN. Fig. 5A–C show immunofluorescence images and western blots confirming reduction of Nrf2 in the RVLM of Nrf2f/f mice following Cre-GFP viral injection (n=3, p<0.05). In a functional study, we show that depletion of Nrf2 in the RVLM increased baseline BP (mean ΔMAP= 21.0±1.8mmHg, n=3, p<0.05), which is consistent with our previous data 17. In addition, we found that the AngII infusion-evoked increase in MAP was greater in the Nrf2f/f mice compared to GFP-treated control group; also, the peak MAP of Nrf2f/f-Cre-AngII mice was increased to 145.2 ± 5.5 mmHg, although this was not significantly higher compared to their baseline peak MAP (Nrf2f/f-Cre-aCSF 132.4± 3.6 mmHg) (Fig. 5D).

Figure 5. Nrf2 knockdown in the RVLM enhances central AngII-induced MAP.

Figure 5.

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A. Representative immunofluorescence staining of Nrf2 knockdown in the RVLM. B. Staining of the neurons in the RVLM. RVLM Nrf2 protein quantification was validated by western immunoblot (C) (n=3, *p<0.05). D. 24-hour peak MAP at baseline and post icv-Ang II infusion showed that Nrf2 deletion in the RVLM enhanced BP increase (n=3, *p<0.05; 1-way ANOVA followed by Tukey’s multiple comparisons).

icv-infusion of SFN attenuates the pressor effect induced by central infusion of AngII

To further address if brain Nrf2 is involved in the regulation of BP, we determined the influence of Nrf2 activation on icv AngII infusion-evoked HTN. Central Nrf2 was activated by icv infusion of SFN, a sulfur-containing compound that has been shown to possess potent antioxidant and anti-inflammatory properties through activation of Nrf2 29. Indeed, we found that icv infusion of SFN significantly upregulated Nrf2 expression in the RVLM, however Nrf2 was not further increased when AngII was simultaneously administrered (Fig. 6A).

Figure 6. icv-SFN attenuates the pressor and sympathetic responses to central AngII.

Figure 6.

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A. Western blot using whole tissue lysates from RVLM punches show that co-administration of icv AngII and SFN increased Nrf2 protein in RVLM(n=5, *p<0.05; 1-way ANOVA followed by Dunnett’s multiple comparisons). B. icv-AngII infusion increases MAP, which was attenuated with subsequent infusion of SFN. SFN alone did not alter BP. Daily MAP was recorded for 2 hours around noon(n=6, *p<0.05 compared to baseline; #p<0.05, &p<0.05 compared to AngII; 2-way ANOVA followed by Multiple t tests). C-D. LF/HF and SDRR during baseline and post-icv infusion period. (n=4-6, *p<0.05, #p<0.05, **p<0.01; 2-way ANOVA followed by Sidak’s multiple comparisons test).

Fig. 6B shows BP data for this intervention. As can be seen icv SFN alone had no effect on baseline BP but abolished icv AngII-induced HTN when these two reagents were administrated together. In addition, HTN induced by pre-administration of AngII was significantly reduced by the subsequent addition of icv SFN, suggesting that central Nrf2 activation not only prevents but also decreases AngII-induced HTN.

Fig. 6C and 6D show LF/HF and SDRR data. After the first icv-infusion of AngII, LF/HF increased and SDRR decreased, suggesting increased sympathetic tone. The increased LF/HF due to icv-AngII was significantly restored by SFN. SFN plus AngII infusion trended towards a blunting in the decrease in SDRR, however this did not reach statistical significance. These data suggest that SFN, in part, improves autonomic balance prior to or during central AngII infusion.

Effect of AngII and ACE2 on intracellular Nrf2

As a transcription factor, Nrf2 regulates phase 2 anti-oxidant enzyme expression by nuclear translocation when activated by intracellular ROS30, 31. However, many redox protective agents, such as β-lactoglobulin peptide (BRP2) or SFN were found to exert their antioxidant effects by also promoting Nrf2 nuclear translocation32, 33. Therefore in cell experiments we also examined the effect of AngII on Nrf2 behavior. Using N2A cells, we assessed Nrf2 translocation after AngII at different concentrations and different time periods, using SFN treatment as a positive control. Fig. S3 and Fig. S4A (http://hyper.ahajournals.org) demonstrates western blot data for Nrf2 in cytoplasmic and nuclear fractions after AngII stimulation. There were no statistical differences in Nrf2 in the cytoplasmic fraction between different conditions, however in the nuclear fraction Nrf2 increased in a dose-dependent manner, with a peak level reached at 100nM AngII and 2 hours of treatment. GAPDH and Lamin-B were chosen for cytoplasmic and nuclear loading controls, respectively.

In order to determine the effect of ACE2 on Nrf2 after AngII stimulation, we transfected N2A cells with hACE2-eGFP adenovirus to overexpress intracellular ACE2 prior to AngII treatment. We found that ACE2 overexpression increases whole cell Nrf2 shown in the western immunoblot (Fig. S4B at http://hyper.ahajournals.org). Although the presence of either ACE2 or AngII upregulated Nrf2 in N2A cells, when combined together, Nrf2 increase was attenuated.

Discussion

The current study provides evidence that ACE2 upregulation in the brain abolishes the pressor effect in response to icv AngII in conscious mice. Because the attenuated response to AngII has been shown to be mediated by a reduction in oxidant stress14, we felt it important to evaluate hemodynamics and SNA during chronic infusion of central AngII. We also examined the notion that reduction of the pressor response to AngII in synhACE2 expressing mice was mediated and/or associated with increases in the antioxidant transcription factor Nrf2 in the RVLM, thereby resulting in a decrease in oxidative stress and sympathetic outflow. Furthermore, we hypothesized that deletion of Nrf2 in the RVLM would potentiate the pressor response to icv AngII. While our data clearly show marked inhibition of the pressor, sympathetic and polydipsic effects of central AngII infusion in synhACE2+/+ mice, they do not support a role for Ang1-7 or the MasR pathway in these responses. They do however show a modulatory role and association of Nrf2 and antioxidant enzyme expression in response to central AngII.

Increased circulating AngII has been shown in patients with severe essential HTN34. In addition to direct vasoconstriction and effects on salt and water balance, circulating AngII may also play a critical role in the pathogenesis of HTN by stimulating brainstem neurons. This may be mediated through regions lacking a brain blood barrier (BBB) or impaired BBB function due to chronic HTN35, 36. Furthermore, accumulating evidence suggests that de novo synthesis of AngII in autonomic regulatory regions of the brain plays a role in HTN37. The mechanism by which central AngII induces HTN may rely on the generation of ROS that consequently excites pre-sympathetic neurons through the modulation of ion channel function38–40. Our data provide new evidence to further support this concept (Figs. 2 and 3). It is important to note that since both synhACE2 overexpression and AngII infusion are directed to the brain globally, it is plausible that other areas may have elevated ROS as well. However, this study only focuses on RVLM due to its critical role in impacting sympathetic outflow and blood pressure. It is reasonable to speculate that targeting either central AngII or ROS would be a potential strategy to treat HTN. In this study using synhACE2+/+ mice to overexpress the human isoform of ACE2 in the brain 22, we explored a potential mechanism for the anti-hypertensive effect of hACE2 by focusing on the Nrf2-antioxidant signaling pathway.

Although it has been well accepted that the Ang1-7/MasR axis exerts a variety of protective effects on cardiovascular function in HTN41, 42, it remains to be elucidated if Ang1-7 is the key mediator for the anti-hypertensive effects of overexpression of ACE2 in response to icv AngII. Medullary and hypothalamic nuclei are interconnected and the degree to which excitatory and inhibitory neurons are activated or inhibited will determine the magnitude of sympathetic outflow and activation of the RVLM and spinal cord in response to a variety of neurotransmitters and neuronal modulators. The literature is confusing in this regard. Fontes et al. showed that microinjection of Ang1-7 directly into rat RVLM increased MAP at a dose of 25 pmol 43. Feng et al. reported that chronic subcutaneous (s.c.) infusion of A779 (600 ng/kg/min) reversed the anti-pressor effect of central ACE2 in response to peripheral administration of AngII (600 ng/kg/min, s.c.)22. These data imply a role for central Ang1-7 in cardiovascular regulation but are contradictory in terms of the direct effects of Ang1-7, most likely due to differences in dosage, route of administration and target location. Other studies have similar limitations. Feng et al. showed that selective overexpression of ACE2 in the subfornical organ attenuated the pressor effect of AngII (icv bolus injection), which was not reversed by the MasR antagonist, A779 44. Wysocki et al. 45 demonstrated that subcutaneous infusion of human recombinant ACE2 (rACE2) prevented the hypertensive effect of AngII (1000 ng/kg per minute, s.c.) and that this effect was not blocked by A779 (100 ng/kg/min, s.c.). In Wistar rats, Campagnole-Santos et al. observed that icv Ang1-7 failed to attenuate central AngII induced BP increases, but improved baroreflex sensitivity impairment 46. These studies appear to indicate discrepancies between central and peripheral routes of administration, or between different nuclei with regard to responses to RAAS components and interactions. In the current study, we found that Ang1-7 and A779 did not change the inhibitory effects of overexpression of brain synhACE2 on icv AngII-induced HTN, suggesting that AngII degradation may constitute the major mechanism by which central ACE2 overexpression inhibits this response. However, given the evidence that ACE2 can act as an antioxidant 47, other factors such as antioxidant defense systems may play an intermediate role.

In the current study we examined the role of Nrf2 as an antioxidant transcription factor on the responses to central AngII. This was based, in part, on the observation that overexpression of central Nrf2 exhibited a sympathoinhibitory effect in animals with CHF 48. To evaluate the potential interaction between the central RAAS and Nrf2 in modulating SNA, we selectively knocked down Nrf2 by deliverying Cre-virus into the RVLM of Nrf2-floxed mice, followed by an evaluation of the BP response to icv-AngII infusion. We found that deletion of Nrf2 in the RVLM significantly increased BP. In contrast, central administration of the Nrf2 activator SFN attenuated the pressor response to central AngII. It is unclear why our data showed both AngII and SFN increased RVLM Nrf2 whereas only the latter demonstrated an anti-hypertensive effect. It is possible that SFN may also exhibit independent anti-inflammatory effects such as suppression of NFkB49. The exact mechanism requires further study. These data suggest however that targeting Nrf2 expression in the RVLM may modulate the pathogenesis of AngII-induced neurogenic HTN.

Our data demonstrate AngII stimulated nuclear translocation of Nrf2 in a dose-dependent manner and provide direct evidence that intracellular Nrf2 responds to AngII. 100nM AngII for 2 hours triggered an acute maximal increase in nuclear translocation. ACE2 overexpression in N2A cells upregulated intracellular Nrf2 almost as much as AngII, and when treatment of ACE2 overexpressing cells with AngII this response was reduced, which are consistent with western blot data from RVLM tissue (Fig. 4). It is difficult to explain why AngII failed to trigger an increase in Nrf2 in ACE2 virally-transfected cells since the Nrf2-upregulating effect of ACE2 would be expected to exert a “synergistic” effect on Nrf2 expression. It is possible that the consumption of AngII by ACE2 in the brain is too rapid to allow the former to exert its Nrf2-inducing effect through increased ROS or that degradation of AngII by ACE2 overrides the Nrf2-inducing effects of AngII or ACE2 alone. In other words, changes in ROS levels may represent either a cause or an effect of Nrf2 upregulation, depending on the timing of measurement. Although the exact mechanism by which ACE2 upregulates Nrf2 is not completely clear, these results suggest that Nrf2 upregulation may impart antioxidant qualities to ACE2 that further reduces the response to AngII. Taken together, these data show that Nrf2 responds to both AngII and ACE2 in N2A cells separately; co-existence of AngII and ACE2 though does not trigger a synergistic increase of Nrf2.

Clearly, while the results of this study suggest that both ACE2 and Nrf2 play a role in central pressor responses mediated by exogenous AngII, there are several limitations that will await further studies. First, icv infusion may not deliver agents to the major sympatho-excitatory areas of the brain. While we did not measure tissue levels of AngII or Ang1-7, there is evidence that chronic infusion of AngII may reach a steady state by which levels of AngII reach most areas50. Second, while we measured Nrf2 in the RVLM, activation of this transcription factor and downstream antioxidant proteins may occur at several areas of the brain that are involved in autonomic regulation although cortical and hypothalamic Nrf2 was not changed. Third, the relationship between ACE2 and Nrf2 in the antioxidant effect of ACE2 overexpression can only really be conclusively tested by selectively knocking down Nrf2 in the RVLM in animals that overexpress ACE2 (i.e. synhACE2+/+ and Nrf2f/f). This will be a critical experiment that provides evidence for a cause-effect relationship. What is clear however, is that the dramatic effect of hACE2 overexpression is not mediated through the MasR and is unlikey to be mediated by increased levels of Ang1-7. Other AngII metabolites may be be more important than Ang1-7. For instance alamandine, perhaps working through the AT2R, may be important to investgate in regard to central oxidative stress 51, 52. Finally, no dose-response studies were carried out with AngII infusion or combined AngII and Ang1-7 or with A779. Therefore, it is not clear if higher doses would have been effective in reversing the pressor response to icv AngII.

In summary, the current study shows that selective overexpression of ACE2 in the brain abolished the pressor response to central administration of AngII. Our data also show that Nrf2 expression was significantly upregulated in the RVLM with ACE2 overexpression, suggesting that the antioxidant effect of ACE2 in the RVLM may involve Nrf2. However, our data did not support the idea that Ang1-7 or the MasR mediates the decreased pressor response to AngII in ACE2 overexpressing animals.

Selective deletion of Nrf2 in the RVLM resulted in elevated BP, whereas upregulation of Nrf2 using the pharmacologic agent SFN resulted in attenuation of the pressor response to central AngII. Overall, these results suggest that central Nrf2 may possess anti-hypertensive properties. The antioxidant signaling pathway of Nrf2 may contribute to the protective effects of ACE2 in response to chronic central AngII infusion.

Perspectives

While centrally acting drugs such as clonidine and moxonidine have been used in HF and HTN they are associated with negative side effects and are difficult to regulate their dose – response relationships53, 54. Because both disorders are associated with oxidative stress and increases in sympathetic outflow it is reasonable to evaluate therapies that have been shown to affect both mechanisms.

The two agents studied in this study have been shown to reduce oxidative stress in both CHF and HTN 13, 17, 55, 56. ACE2 or ACE2 activation and Ang1-7 have been used to treat HTN in animal models45, 57–59. The mechanism for the protective effects of these therapies has been assumed to be a reduction in AngII peptide and both a vasodilator and sympatho-inhibitory effect of Ang1-7 mediated, in part, through a reduction in oxidative stress 60, 61 primarily in the peripheral circulation. According to ClinicalTrials.gov there are several clinical trials where modulation of ACE2 is or has recently been investigated for the treatment of cardiovascular and metabolic diseases. Similarly there are numerous trials where Nrf2 activation is being used to treat a variety of diseases including cardiovascular disease.

The results presented here suggest that activation or overexpression of both ACE2 and Nrf2 reduce sympathetic outflow in HTN and CHF when the RVLM is targeted. What is less clear from this work is how this could be used in a translational way for human disease. Systemic administration of ACE2 or Nrf2 activators will, of course, target all tissues. It is highly likely that small molecule activators such as diminazene aceturate (DIZE; 62), sulforaphane63 and dimethyl fumarate64 are likely to gain entry through the blood brain barrier or via areas with no blood brain barrier such as the circumventricular organs65. Therefore, systemic administration may also target the CNS and potentially the sympatho-regulatory areas in the brain. In addition, new modalities of drug delivery have been developed using a variety of nanoparticles and extracellular vesicles for antioxidant therapy66–68.

Supplementary Material

Supplemental Material
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Novelty and Significance.

What is New?

  • Overexpression of human ACE2 in the central nervous system completely prevents the pressor, drinking and sympathetic responses to central Ang II infusion.

  • ACE2 overexpression reduces oxidative stress in the RVLM in response to central Ang II.

  • Increases in ACE2 expression correlate with increased Nrf2 and antioxidant enzyme expression.

  • The Nrf2 activator, sulphoraphane reduces the pressor response to central Ang II.

What is relevant?

  • The antioxidant effects of overexpression of ACE2 in the central nervous system may be mediated by increases in the antioxidant transcription factor, Nrf2 in response to central Ang II and oxidative stress.

  • Activation of Nrf2 may strategically lower arterial pressure and sympathetic tone in some forms of hypertension.

Summary

In this study we show that overexpression of ACE2 in the brain is associated with a reduction in oxidative stress in the RVLM, activation of Nrf2 and inhibition of the sympathetic and pressor responses to central Ang II. We conclude that activation of both central ACE2 and Nrf2 are therapeutic stratagies to manage neurogenic hypertension.

Acknowledgement

The authors than Dr. Eric Lazartigues from the LSU Health Sciences Center in New Orleans for the initial contribution of synhACE2 mice.

Sources of Funding

This work was supported by a grant from the NHLBI P01-HL62222. AM was supported by a UNMC pre-doctoral fellowship. AMW was supported by a pre-doctoral fellowship from Jazan University. IHZ was supported, in part, by the Theodore F. Hubbard Foundation.

Footnotes

Disclosures

The authors have nothing to disclose.

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