Brain-targeted ACE2 overexpression attenuates neurogenic hypertension by inhibiting COX mediated inflammation

Abstract

Overactivity of the renin angiotensin system (RAS), oxidative stress, and cyclooxygenases (COX) in the brain are implicated in the pathogenesis of hypertension. We previously reported that Angiotensin-Converting Enzyme 2 (ACE2) overexpression in the brain attenuates the development of DOCA-salt hypertension, a neurogenic hypertension model with enhanced brain RAS and sympathetic activity. To elucidate the mechanisms involved, we investigated whether oxidative stress, mitogen activated protein kinase signaling and cyclooxygenase (COX) activation in the brain are modulated by ACE2 in neurogenic hypertension. DOCA-salt hypertension significantly increased expression of Nox-2 (+61 ±5 %), Nox-4 (+50 ±13 %) and nitrotyrosine (+89 ±32 %) and reduced activity of the antioxidant enzymes, catalase (−29 ±4 %) and SOD (−31 ±7 %), indicating increased oxidative stress in the brain of non-transgenic mice. This increased oxidative stress was attenuated in transgenic mice overexpressing ACE2 in the brain. DOCA-salt-induced reduction of nNOS expression (−26 ±7 %) and phosphorylated eNOS/total eNOS (−30 ±3 %), and enhanced phosphorylation of Akt and ERK1/2 in the paraventricular nucleus (PVN), were reversed by ACE2 overexpression. In addition, ACE2 overexpression blunted the hypertension-mediated increase in gene and protein expression of COX-1 and COX-2 in the PVN. Furthermore, gene silencing of either COX-1 or COX-2 in the brain, reduced microglial activation and accompanied neuro-inflammation, ultimately attenuating DOCA-salt hypertension. Together, these data provide evidence that brain ACE2 overexpression reduces oxidative stress and COX-mediated neuro-inflammation, improves anti-oxidant and nitric oxide signaling, and thereby attenuates the development of neurogenic hypertension.

INTRODUCTION

The deoxycorticosterone acetate (DOCA)–salt model of hypertension is a widely used animal model of mineralocorticoid and salt sensitive hypertension characterized by elevated brain Angiotensin (Ang)-II levels without changes in peripheral Ang-II levels.^{1, 2} There is abundant evidence that DOCA-salt hypertension is mediated by increased oxidative stress. Oxidative stress involves an increased production of reactive oxygen species (ROS) and/or decreased antioxidant enzymes to metabolize them. Ang-II, the major actor of the renin-angiotensin system (RAS), stimulates the production of ROS. Previous evidence has shown the importance of the brain RAS in the regulation of blood pressure (BP) and in the development of hypertension.³ Accumulating evidence indicate that ROS are involved in mediating many of the effects of Ang-II, including autonomic dysfunction and sympatho-excitation, leading to neurogenic hypertension.^4–6 In the last decade, angiotensin converting enzyme 2 (ACE2) has emerged as a key player in compensatory mechanisms opposing the overactive RAS, a main contributor to the development of several cardiovascular diseases (CVD), including hypertension. It has been previously shown that Ang-II stimulation of Ang-II type 1 receptors (AT₁R) induces ROS formation^{4, 5, 7}-and reduces ACE2 activity and expression in the brain and at the periphery.⁸ Moreover, various genetic⁹ and experimental^{2, 10–12} models of hypertension are associated with elevated pro-inflammatory cytokines and chemokines in the hypothalamic paraventricular nucleus (PVN), an important cardiovascular regulatory center in the brain. Our group^{2, 7, 10, 13, 14} and others¹⁵ have shown that overexpression of ACE2 in the brain, or specifically within the PVN, results in attenuation of BP in Ang-II- and DOCA-salt-induced neurogenic hypertension models. In DOCA-salt hypertension, ACE2 activity was reduced by 50% in the hypothalamus, whereas transgenic mice with ACE2 overexpression had a 2-fold increase in ACE2 activity which was able to attenuate DOCA-salt hypertension.² However, the precise mechanisms by which increased brain RAS activity triggers hypertension and how ACE2 counteracts hypertension are unknown.

Cyclooxygenase (COX)-derived prostanoids are generated by either the constitutive isoform COX-1 or by the inducible form COX-2 and are implicated in BP regulation.^{16, 17} Both COX isoforms are expressed in the brain and by acting as key rate limiting enzymes, contribute substantially to the generation of prostaglandins.^{16, 17} Recent studies suggest a more diverse role for the prostanoid system components as both pro-hypertensive and anti-hypertensive roles coexist, affecting kidneys, blood vessels, endocrine organs and the brain.^17–20 Use of COX inhibitors and COX knockout mice resulted in contrasting results about the role of COX-1 and COX-2 in BP regulation.^17–20 Increasing evidence suggests considerable cross talk between COX isoforms and nitric oxide (NO) and that modulation of both pathways impacts the outcome of a physiological or pathological response.^{21, 22} While our group previously reported the participation of NO in the ACE2-mediated attenuation of neurogenic hypertension^{7, 13}, the impact on COX expression remains to be elucidated. We hypothesized that the attenuation of inflammation and hypertension by ACE2 overexpression is regulated by COX-mediated signaling mechanisms in the brain. Therefore, in this study we investigated the roles of oxidative stress, COX-isoforms, and the signaling mechanisms involved in the beneficial effects of brain ACE2 overexpression in neurogenic hypertension. Using a mouse model of DOCA-salt hypertension, and transgenic mice with neuron-targeted overexpression of ACE2, we examined whether oxidative stress and COX-isoforms in the brain contribute to the development of hypertension and whether ACE2 overexpression modulates these pathways in hypertension.

METHODS

A detailed Methods section is available in the online data supplement.

Experimental Animals

Experiments were performed in adult male (14–16 weeks old, 25–30 g) transgenic syn-hACE2 (SA) mice and non-transgenic (NT) littermates. SA mice were generated in collaboration with Dr. Curt D. Sigmund at The University of Iowa and back-crossed into the C57Bl/6 background for more than 9 generations. Animals were housed in a temperature- and humidity-controlled facility under a 12 hour dark/light cycle, fed standard mouse chow and water ad libitum. All procedures were approved by the LSU Health Sciences Center-NO Animal Care and Use Committee and are in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experimental Design

SA and NT mice first underwent uni-nephrectomy under 2.5% isoflurane anesthesia with oxygen flow (1 L/min). After 1 week of recovery, telemetry probes were implanted for conscious BP monitoring as described previously.^{2, 13} A week later, baseline BP was recorded for 3 days. Mice were then randomly divided into 4 groups (n=12/group) and implanted subcutaneously either with a DOCA-silicone sheet (DOCA:silicone =1:3; DOCA 1 mg/g body weight) or an empty silicone sheet and. The mice receiving DOCA were switched to 1% NaCl in the drinking water. BP was continuously recorded for 3 additional weeks. In another set of experiments, uni-nephrectomized NT mice were infused intracerebroventricularly (i.c.v.) for 3 weeks, with COX-1 or COX-2 siRNA (GE Healthcare Dharmacon RNAi; 0.1 nmoles/day) or artificial cerebrospinal fluid (aCSF) (n=6/group) while receiving DOCA-salt. At the end of the protocol mice were euthanized, the brains were collected and stored at −80°C until used.

RESULTS

ACE2 overexpression prevents oxidative stress and improves anti-oxidant enzyme levels in the PVN

As we previously reported,² DOCA-salt treatment for 3 weeks resulted in increased MAP, systolic and diastolic BP in NT mice, which were blunted in SA mice, confirming that brain ACE2 overexpression attenuates DOCA-salt induced hypertension (Table 1). Also, DOCA-salt hypertension was associated with similar increases in fluid intake and urinary excretion in both genotypes (Table 1), suggesting that differences observed between NT and SA mice are unrelated to these parameters. Previous studies have shown that DOCA-salt hypertension is associated with increased oxidative stress.^23–25 To test whether ACE2 overexpression affects oxidative stress in neurogenic hypertension, protein expression of NADPH oxidase catalytic subunits (Nox-2 and Nox-4) and nitrotyrosine were assessed in the hypothalamic PVN using Western blotting. PVN was chosen as it is one of the pivotal brain regions integrating neuronal signals and sympathetic outflow in central BP regulation. In addition, we previously reported that ACE2 is present in this region and capable of modulating hypertension and cardiovascular function.^{7, 10} DOCA-salt treatment caused a significant increase in PVN Nox-2 (+61% ±5 %; p<0.001 vs. NT+Sham) and Nox-4 (+50% ±13 %; p<0.05 vs. NT+Sham) expression in NT mice (Figure 1A and B). This response was attenuated in SA+DOCA mice. Furthermore, nitrotyrosine, a marker of ONOO⁻ and an indicator of nitrosative stress, was significantly increased by DOCA-salt in NT mice (+89% ±32 %; p<0.001 vs. NT+Sham) compared to sham-treated mice, which was prevented by ACE2 overexpression in the brain (Figure 1C). DOCA-salt hypertension was associated with significant and similar (~30 %) decreases in catalase (Figure 1D) and total SOD (Figure 1E) activities in the hypothalamus of NT mice (p<0.05 vs. NT+Sham). However, the reduction of MnSOD activity was more pronounced with ~70 % being eliminated (p<0.05 vs. NT+Sham; Figure 1F). ACE2 overexpression had no effect on baseline catalase activity, but prevented the DOCA-salt-induced decrease in catalase activity in the hypothalamus (Figure 1D). At baseline, both SOD and MnSOD levels were significantly higher in SA mice when compared with corresponding NT control mice. ACE2 overexpression dramatically prevented the DOCA-salt mediated reduction in MnSOD activity (Figure 1F, p<0.05 for interaction between DOCA treatment and genotype).

Table 1.

Physiological measurements of mice treated with sham or DOCA-salt for 3 weeks.

Parameters	NT+Sham	NT+DOCA	SA+Sham	SA+DOCA
MAP (mmHg)	104 ±2	138 ±3*	104 ±2	122 ±3†
Systolic Pressure (mmHg)	122 ±4	161 ±5*	116 ±5	138 ±6†
Diastolic Pressure (mmHg)	88 ±4	112 ±3*	87 ±2	103 ±7
HR (bpm)	533 ±13	538 ±15	519 ±22	543 ±43
Fluid intake (1% NaCl, mL/24 hr)	3.2 ±0.1	18.8 ±2.1*	3.5 ±0.1	17.3 ±1.2*
Urine output (mL/24 hr)	2.2 ±0.1	16.1 ±1.8*	2.8 ±0.2	15.1 ±0.9*

Values are mean ±SEM. NT, Non transgenic mice; SA, Syn-ACE2 mice; MAP, Mean arterial pressure; HR, Heart rate; bpm, beats per minute.

^*p<0.05 vs. NT+Sham,

^†p<0.05 vs. NT+DOCA.

Figure 1. Brain ACE2 over-expression prevents the DOCA-salt-induced oxidative stress and improves anti-oxidant enzyme activities.

Representative western blots and densitometric analysis of group data for Nox-2 (A), Nox-4 (B) and Nitrotyrosine (C) protein expression in the hypothalamic paraventricular nucleus (PVN) of DOCA-salt-, or sham-, treated non-transgenic (NT) and syn-hACE2 (SA) mice. Data are means ±SEM (n=9 mice/group). *P<0.05 vs. sham and ^†P<0.05 vs. NT+DOCA. Antioxidants Catalase (D), Superoxide dismutase (SOD) (E), and MnSOD (F) enzyme activities in hypothalamic homogenates of sham or DOCA-salt treated non-transgenic (NT) and syn-hACE2 (SA) mice. Data are means ±SEM (n=6–8 mice/group). *P<0.05 vs. NT+Sham, ^†P<0.05 vs. NT+DOCA, and ^#P<0.05 vs. SA+Sham.

ACE2 overexpression attenuates DOCA-salt-induced decreases in NOS expression

To determine the effect of ACE2 overexpression on NO signaling we measured expression of NOS isoforms in the PVN using western blot analysis. DOCA-salt treatment significantly decreased phosphorylated eNOS and total eNOS expression in the PVN of both NT and SA mice, but this decrease was significantly lower in SA mice (Figure 2A and B, p<0.05 for interaction between DOCA treatment and genotype). At baseline, nNOS protein levels were significantly higher in SA mice when compared with corresponding NT control mice (Figure 2C). Furthermore, the importance of ACE2 for activation/reinforcement of NO-dependent pathways was illustrated by baseline up-regulation of nNOS expression (+30%; p<0.05 vs. NT+Sham) in SA mice. Three weeks of DOCA-salt treatment resulted in decreased expression of nNOS in NT mice (−26%; p<0.05 vs. NT+Sham), which was prevented by overexpression of ACE2 in SA mice (Figure 2C, p<0.01 for SA+DOCA vs. NT+DOCA). These results further confirm that ACE2 overexpression reinforces NO signaling within the brain thus attenuating neurogenic hypertension.

Figure 2. Nitric oxide synthase (NOS) expression in the paraventricular nucleus (PVN).

Representative western blots and quantification data for phosphorylated levels of eNOS (A), total eNOS (B), nNOS (C), phosphorylated and total Akt (D), and phosphorylated and total ERK1/2 (E) protein expression in the hypothalamic PVN homogenates of non-transgenic (NT) and syn-hACE2 (SA) mice treated with sham or DOCA-salt for three weeks. Data are means ±SEM (n=9 mice/group). *P<0.05 vs. NT+Sham, ^†P<0.05 vs. NT+DOCA and ^#P<0.05 vs. SA+Sham.

Brain ACE2 overexpression prevents DOCA salt induced increase in Akt and ERK1/2 phosphorylation

Since MAPK and ERK1/2 signal transduction pathways are involved in the pathogenesis of DOCA-salt hypertension²⁶ and coupled with MasR activation,²⁷ we evaluated the phosphorylation of Akt and ERK1/2 in the brain. Previous studies showed that Ang-II mediated increase in ROS production leads to activation of p38 MAPK, which regulates the phosphorylation of Akt on Ser^473.28 Therefore, we measured the phosphorylation of Akt on Ser⁴⁷³ in the PVN. As shown in Figure 2D, phosphorylation of Akt in the PVN was significantly increased in NT+DOCA mice, which was prevented in SA+DOCA animals. Phosphorylation at residues Thr²⁰²/Tyr²⁰⁴ results in activation of ERK1/2. Therefore, phosphorylation of Thr²⁰²/Tyr²⁰⁴ was used as an index of ERK1/2 activation. No differences in total ERK1/2 protein levels in the PVN were observed among the groups (Figure 2E). However, phosphorylation of ERK1/2 at Thr²⁰²/Tyr²⁰⁴ was increased in the PVN from NT+DOCA mice, but not in SA+DOCA group. These results suggest that Akt and ERK1/2 signaling pathways are targeted by ACE2 counter regulation during DOCA-salt hypertension.

ACE2 overexpression attenuates DOCA-salt-induced increase in COX expression

The COX-derived prostanoids, generated by either the largely constitutive isoform COX-1 or by the inducible form COX-2, have long been implicated in the pathogenesis of Ang-II-induced hypertension, although their precise role is unclear.^{19, 29, 30} Accordingly, we sought to determine the expression of PVN COX isoforms in DOCA-salt hypertension and whether ACE2 has any modulatory effect. In NT mice, DOCA-salt treatment resulted in a 2.5 fold increase in COX-1 and a 5 fold increase in COX-2 mRNA expression in the PVN (Figure 3A and B, p<0.05 vs. NT+Sham). However, this increase was attenuated in SA+DOCA mice. Furthermore, DOCA-salt treatment increased COX-1 and COX-2 protein expression in both NT and SA mice (Figure 3C and D, p<0.05 vs. NT+Sham), but this increase tended to be significantly lower in SA mice (p<0.05 vs. NT+DOCA, p<0.05 for interaction between DOCA treatment and genotype). Together, these data suggest that both COX-1 and COX-2 expression is increased in DOCA-salt hypertension and ACE2 exerts an inhibitory effect on COX expression.

Figure 3. ACE2 overexpression blunts DOCA-salt induced cyclooxygenase (COX) expression.

mRNA expression of COX-1 (A) and COX-2 (B) in the hypothalamic paraventricular nucleus (PVN) was measured by quantitative real time RT-PCR. Representative western blots and quantification data shows the protein expression of (C) COX-1 and (D) COX-2 in PVN homogenates of sham or DOCA-salt treated non-transgenic (NT) and syn-hACE2 (SA) mice. Data are means ±SEM (n=9 mice/group). *P<0.05 vs. NT+Sham and ^†P<0.05 vs. NT+DOCA and ^#P<0.05 vs. SA+Sham.

Brain ACE2 overexpression prevents DOCA-salt-induced changes in EP receptor expression

The COX-derived major prostanoids prostaglandin E2 via acting on 4 G-protein coupled receptors (EP1 through EP4), participates in the mechanisms of hypertension and related complications.^{16, 18, 30} To determine the specific receptors involved, mRNA expression was assessed in the PVN. DOCA-salt hypertension was associated with significant increase in EP1, EP3 and decrease in EP4 receptors mRNA expression in NT+DOCA mice (Figure S1). DOCA-salt hypertension did not affect mRNA expression of EP2 receptor in any of the groups. ACE2 overexpression had no effect on these receptors mRNA at baseline. However, ACE2 expression in the brain reversed the DOCA-salt-induced up-regulation of these receptors expression.

Brain-targeted inhibition of COX-1 or COX-2 attenuates DOCA-salt hypertension

Since our data show that, both COX1 and COX-2 mRNA and protein were significantly increased in PVN, we further investigated whether COX-1 and/or COX-2 contribute to the development of DOCA-salt hypertension using siRNA. To validate our gene silencing approach, we first validated the specificity of COX-1 and COX-2 siRNA in Neuro2A cells (Figure S2). COX-1 and COX-2 siRNA were then infused (0.1 nmoles/day, i.c.v.) in NT mice during DOCA-salt treatment for 3 weeks. In NT mice, DOCA-salt treatment significantly increased BP (Figure 4A, 144 ±2 mmHg; p<0.001 vs. NT+Sham). Knockdown of either COX-1 (122 ±4 mmHg, p<0.001 NT+DOCA+siCOX-1 vs. NT+DOCA) or COX-2 (115 ±2 mmHg, p<0.001 NT+DOCA+siCOX-2 vs. NT+DOCA) in the brain attenuated DOCA-salt-induced hypertension, although COX-2 silencing produced a more efficient reduction of BP (Figure 4A). Efficiency of the silencing was confirmed in vivo by the lack of increase in both COX-1 and COX-2 mRNA (Figure 4B, and C) and protein (Figure 4D and E) expression in the hypothalamic PVN compared to NT+DOCA mice. We further investigated whether COX-1 and COX-2 are involved in MAPK and ERK1/2 signaling mechanisms. DOCA-salt induced increases in phosphorylation of Akt on Ser⁴⁷³ and ERK1/2 at Thr²⁰²/Tyr²⁰⁴ were prevented by either COX-1 or COX-2 gene silencing using siRNA (Figure 4F and G) suggesting that COX-mediated effects on inflammation and hypertension were mediated by Akt and ERK1/2 signaling.

Figure 4. Brain-targeted inhibition of COX-1 or COX-2 attenuates DOCA-salt hypertension.

Mean arterial pressure was measured by telemetry in mice with or without DOCA-salt treatment combined with intracerebroventricular infusion of COX-1 or COX-2 siRNA (A). mRNA expression of COX-1 (B) and COX-2 (C) in the hypothalamus was measured by quantitative real time RT-PCR. Representative western blots and quantification data for COX-1 (D), COX-2 (E), phosphorylated and total Akt (F), and phosphorylated and total ERK1/2 (G) protein expression in the PVN of sham or DOCA-salt treated non-transgenic (NT), COX-1 and COX-2 siRNA infused mice. Data are means ±SEM (n=6-9 mice/group). *P<0.05 vs. NT+Sham and ^†P<0.05 vs. NT+DOCA.

Since microglia in the PVN is activated and release inflammatory cytokines during hypertension,¹² we determined PVN microglial activation using immunostaining, and inflammatory cytokine gene expression using real time RT PCR. DOCA-salt hypertension resulted in microglial activation in the PVN, as indicated by increased specific microglial marker Iba-1 immunofluorescence staining (retracted processes, enlarged perikarya, and concentrated staining; Figure 5A) and increased Iba-1 protein expression (Figure 5B) in NT+DOCA group, which was prevented by either COX-1 or COX-2 gene silencing. DOCA-salt hypertension also increased PVN mRNA expression of pro-inflammatory cytokines TNF, IL-1β and IL-6, which was attenuated by either COX-1 or COX-2 gene silencing (Figure 5C, D, and E). Furthermore, assessment of COX-1 and COX-2 immunoreactivity in the PVN revealed enhanced COX-1 (Figure S3) and COX-2 (Figure S4) protein expression in the PVN of NT+DOCA group mice. COX-1 immuno-reactivity was more prominent in neurons (co-localization with NeuN) while COX-2 was expressed in both neurons and non-neuronal cells. The size and shape of these latter cells point to astrocytes, including those located in the proximity of blood vessels. Blockade of COX using siRNA infusion resulted in the reduction of COX-1 and COX-2 immunoreactivity in all cell types within the PVN after DOCA-salt treatment. These findings along with reduced inflammation and microglial activation by COX knockdown suggest that COX expression in the hypothalamic PVN plays an important role in the induction of neuro inflammation and progression of DOCA-salt hypertension.

Figure 5. Brain-targeted inhibition of COX-1 or COX-2 attenuates DOCA-salt hypertension induced microglial activation and inflammation.

Immunofluorescence staining for Iba-1 to show microglial activation in the PVN (A). Representative western blot and quantification data for Iba-1 protein expression in the PVN of sham or DOCA-salt treated non-transgenic (NT), COX-1 and COX-2 siRNA infused mice (B). mRNA expression of TNF (C), IL-1β (D), and IL-6 (E) in the PVN was measured by quantitative real time RT-PCR. Data are means ±SEM (n=4–9 mice/group). *P<0.05 vs. NT+Sham and ^†P<0.05 vs. NT+DOCA.

DISCUSSION

Our lab previously reported that overexpression of ACE2 in the brain blunts the development of Ang-II-induced hypertension^{10, 13} and DOCA-salt hypertension.² Yet, the signaling mechanisms are still elusive. In the present study, we show that DOCA-salt hypertension was associated with increased oxidative stress and decreased NOS expression, which was attenuated by brain specific ACE2 overexpression. In addition ACE2 overexpression resulted in decreased phosphorylation of Akt and ERK1/2 which was increased by DOCA-salt hypertension. Importantly, DOCA-salt hypertension resulted in increased COX-1 and COX-2 expression in the hypothalamic PVN which was blunted by ACE2 overexpression. Furthermore, gene silencing of COX-1 and COX-2 specifically in the brain attenuated microglial activation and accompanied neuro-inflammation, blunted phosphorylation of Akt and ERK1/2, ultimately reducing DOCA-salt hypertension. Taken together, these results coupled with our previous studies suggest that brain targeted ACE2 overexpression prevents DOCA-salt hypertension by modulating NOS and ERK1/2 phosphorylation and decreased COX-mediated neuro-inflammation. There are conflicting reports about the involvement of AT₁R, inflammation and oxidative stress in DOCA-salt hypertension.^{6, 31, 32} These differences have been attributed to species, sex, duration of DOCA-treatment, and nephrectomy of the animals. Although the DOCA-salt model is associated with a suppressed systemic RAS, there is evidence for activation of brain RAS.^{1, 2, 33} Rodrigues and Granger reported that DOCA-salt hypertension was not significantly altered in mice treated with the ROS scavenger tempol or losartan systemically for 3 weeks and there was no difference in plasma cytokine levels.³¹ However, other studies have shown that treatments with tempol⁶ or apocynin,³² a NADPH oxidase inhibitor, attenuate DOCA-salt hypertension. We recently reported that DOCA-salt hypertension was associated with increased expression of pro-inflammatory cytokines in the hypothalamic PVN and ACE2 overexpression in the brain attenuated this inflammatory response.² Also, central infusion of losartan blunted the DOCA-salt-induced increase in BP.² In the present study, catalase, SOD and MnSOD activities in the hypothalamus were reduced in DOCA-salt treated mice compared with normotensive controls, suggesting that defense mechanisms against oxidative stress are decreased in hypertensive mice. This was associated with increased oxidative stress as indicated by increased Nox-2, Nox-4 and nitrotyrosine levels and decreased NOS expression and phosphorylation in the PVN. On the other hand ACE2 overexpression in the brain improved the antioxidant enzyme activities and decreased oxidative stress suggesting that the beneficial effects of ACE2 overexpression might be mediated by reduction in oxidative stress. In addition, we showed previously that overexpression of ACE2, which cleaves Ang-II to Ang-(1-7), in the brain prevents the development of hypertension induced by peripheral Ang-II infusion by, at least partly, increasing NOS and NO production.^{13, 34} Thus ACE2 overexpression not only decreases Ang-II-mediated oxidative stress but also improves NO antioxidant mechanisms likely by Ang-(1-7) formation.

Ang-II acting through AT₁R can trigger signaling cascades that stimulate MAPKs, including ERK1/2.^35–37 It has been shown that male DOCA rats displayed more severe hypertension versus females, which was associated with over activation of ERK1/2 pathway in the vasculature.³⁸ Also, Akt phosphorylation is an important phenomenon in the signal transduction pathway activated by growth factors, including Ang-II.³⁸ In vitro studies using cultured rat vascular smooth muscle cells⁸ or neuronal cells,³⁶ showed that MAP kinase and ERK1/2 pathways are involved in AT₁R mediated changes in ACE2 expression. In the present in vivo study, we show that ACE2 overexpression prevented the DOCA-salt induced phosphorylation of both Akt and ERK1/2 in the PVN suggesting that the beneficial effects of ACE2 overexpression on hypertension are mediated by Akt and ERK1/2 signaling pathways.

A major finding in the present study is that the DOCA-salt hypertension was associated with increased expression of COX-1 and COX-2 in the hypothalamic PVN of DOCA-salt treated NT mice. ACE2 overexpression attenuated this increased COX expression, suggesting that ACE2 beneficial effects on DOCA-salt hypertension are in part mediated by down-regulation of COX expression. Several studies suggest that COX products differentially modulate the pressor response in different models of hypertension.^{18–20, 39, 40} Qi and colleagues previously showed that pharmacological blockade or genetic deletion of COX-1 but not COX-2 reduced the acute pressor effects of Ang-II in mice.²⁰ Similarly, Cao and colleagues recently demonstrated that elevations in BP during slow pressor Ang-II infusions are abolished in mice with global null mutations of EP1R or COX-1 but not COX-2 and that COX-1-derived PGE2 signaling through EP1R in the SFO is required for ROS-mediated hypertension induced by systemic infusion of Ang-II.¹⁸ On the other hand, it has been shown that BP response and superoxide production were significantly blunted in COX-2 knockout mice.³⁹ Furthermore, COX-2 inhibition by rofecoxib and nimesulide attenuated Ang-II-induced oxidative stress, hypertension, and cardiac hypertrophy in rats.²⁹ In addition, in heart failure rats with chronic elevation of pro-inflammatory cytokines, COX-1 was not altered but COX-2 was increased in the PVN.⁴¹ A recent study also suggests that systemic inflammation activates microglia in RVLM to induce COX-2-dependent neuro-inflammation leading to an increase in oxidative stress that contributes to neurogenic hypertension.⁴² Together, these studies highlight the importance of COX expression in the manifestation of a variety of disease conditions including neurogenic hypertension. The reasons attributed to these variations included study design, experimental models, tissue specificity, sex of the animals, background of the transgenic mice, and selectivity of the COX inhibitors. In the present study, we used a gene silencing approach to block COX isoforms specifically in the brain thus preventing developmental compensatory mechanisms often arising in knockout animals. Both COX-1 and COX-2 gene and protein expression significantly increased in the PVN during DOCA-salt hypertension, accompanied by increased microglia activation and inflammation. Moreover, our study highlighted the contribution of multiple cell types within the brain. Indeed, our data show that COX-1 immuno-reactivity was more prominent in neurons while COX-2 was expressed in both neurons and non-neuronal cells, likely astrocytes. A study by Wang and colleagues demonstrated the co-localization of AT₁R and COX-1 in SFO neurons, and suggested the possibility of cross talk between COX-2/EP1R and NOX/ROS signaling in modulating Ang II-induced hypertension.⁴³ In our study, brain targeted COX inhibition with siRNA resulted in reduced microglial activation, decreased inflammatory cytokine production, and attenuation of hypertension suggesting a role for both COX isoforms in the regulation of DOCA-salt hypertension. However, additional studies are needed to determine the relative contribution of COX mediated signaling, and to identify the contribution of each cell type in these signaling events during the development of DOCA-salt hypertension.

It is important to note, however, that due to the infusion of COX siRNA i.c.v. in the present study, the COX inhibitory effects may have impacted additional cardiovascular regulatory regions in the brain and elicited a similar response as in the PVN. Previous publications from our group highlighted the important role of ACE2 in the PVN in the regulation of hypertension.^2,7,10–12 PVN-specific overexpression of ACE2 using bilateral injection of hACE2-adenovirus attenuated Ang-II-induced hypertensive response in rats,¹⁰ and reduced oxidative stress and normalized cardiac dysautonomia in ACE2 knockout mice.⁷ We also showed that knockdown of ACE2 in the PVN partially reversed the protective effects of brain ACE2 in DOCA-salt hypertension, suggesting the involvement of other cardiovascular centers apart from PVN in the blood pressure homeostasis.⁴⁴ Further studies will be required to determine the predominant central site or sites of action in COX-mediated inflammation in DOCA-salt hypertension.

In summary, we have provided evidence that DOCA-salt hypertension was associated with increased oxidative stress and decreased anti-oxidant enzyme activity in the brain and ACE2 overexpression prevents these changes. This beneficial effect of ACE2 seems to be mediated by reduction in activated Akt and ERK1/2-induced COX-1 and COX-2 expression in the brain. In addition, brain targeted COX inhibition reduced microglia activation, decreased neuro-inflammation, and attenuated DOCA-salt hypertension. As summarized schematically in Fig. 6, these findings collectively support important role for ACE2 and COX isoforms in the pathogenesis of DOCA-salt hypertension in the setting of increased brain oxidative stress and inflammation.

Figure 6. Working model for the role of ACE2 in the regulation of DOCA-salt hypertension and COX-mediated inflammation.

In DOCA-salt hypertension, elevated brain RAS activity results in increased oxidative stress, activation of Akt and ERK1/2 pathways, and activation of COX-mediated increased neuro-inflammation, contributing to hypertension. Overexpression of ACE2 in the brain, which converts pro-hypertensive Ang-II to anti-hypertensive Ang-(1-7), is associated with decreased oxidative stress, reduction of MAPK and ERK1/2 activation and blunting of COX-mediated neuro-inflammation, ultimately leading to the attenuation of hypertension.

PERSPECTIVES

While in the last decade ACE2 has emerged as a potential therapeutic target in a variety of cardiovascular diseases, the molecular and cellular mechanisms underlying these beneficial effects of ACE2 remain unknown. This study provides evidence that MAPK-mediated elevated COX-1 and COX-2 expression in the hypothalamic PVN region is key underlying mechanism responsible for the pathogenesis of DOCA-salt hypertension. Brain specific ACE2 expression prevented DOCA-salt induced oxidative stress, improved anti-oxidant and NOS signaling, blunted the MAPK activation and COX expression, thereby attenuating DOCA-salt hypertension. Furthermore, brain targeted gene silencing of either COX-1 or COX-2 resulted in decreased inflammation and attenuation of hypertension confirming the pivotal role of COX isoforms in the pathogenesis of hypertension.

Novelty and Significance.

What is new?

• This study demonstrates that both COX-1 and COX-2 are elevated in the brain hypothalamic paraventricular nucleus during DOCA-salt hypertension.

• Neuron-specific overexpression of ACE2 or brain targeted gene silencing of either COX-1 or COX-2 attenuated DOCA-salt hypertension.

What is relevant?

• Activation of ACE2 in the central nervous system or targeted gene silencing of COX isoforms can be used as a novel therapeutic approach for hypertension and related cardiovascular diseases.

Summary

Brain specific overexpression of ACE2 prevents oxidative stress and COX-mediated neuro-inflammation, improves anti-oxidants and nitric oxide signaling, thereby preventing the development of DOCA-salt hypertension.