Elsevier

Neuroscience

Volume 273, 25 July 2014, Pages 39-51
Neuroscience

Activation of the ACE2/Ang-(1–7)/Mas pathway reduces oxygen–glucose deprivation-induced tissue swelling, ROS production, and cell death in mouse brain with angiotensin II overproduction

https://doi.org/10.1016/j.neuroscience.2014.04.060Get rights and content

Highlights

  • We used brain slices from transgenic mice with features of human essential hypertension.

  • Brain slices were exposed to oxygen–glucose deprivation (OGD) to model ischemic stroke.

  • Overexpression of ACE2 in neurons reduces NADPH oxidase expression, ROS production, and brain cell damage following OGD.

  • ACE2 protects brain by activating the ACE2/Ang-(1–7)/Mas signaling axis which opposes actions of the ACE/Ang II/AT1R axis.

  • Mas receptor activation can be neuroprotective for stroke in hypertensive patients.

Abstract

We previously demonstrated that mice which overexpress human renin and angiotensinogen (R+A+) show enhanced cerebral damage in both in vivo and in vitro experimental ischemia models. Angiotensin-converting enzyme 2 (ACE2) counteracts the effects of angiotensin (Ang-II) by transforming it into Ang-(1–7), thus reducing the ligand for the AT1 receptor and increasing stimulation of the Mas receptor. Triple transgenic mice, SARA, which specifically overexpress ACE2 in neurons of R+A+ mice were used to study the role of ACE2 in ischemic stroke using oxygen and glucose deprivation (OGD) of brain slices as an in vitro model. We examined tissue swelling, the production of reactive oxygen species (ROS), and cell death in the cerebral cortex (CX) and the hippocampal CA1 region during OGD. Expression levels of NADPH oxidase (Nox) isoforms, Nox2 and Nox4 were measured using western blots. Results show that SARA mice and R+A+ mice treated with the Mas receptor agonist Ang-(1–7) had less swelling, cell death, and ROS production in CX and CA1 areas compared to those in R+A+ animals. Treatment of slices from SARA mice with the Mas antagonist A779 eliminated this protection. Finally, western blots revealed less Nox2 and Nox4 expression in SARA mice compared with R+A+ mice both before and after OGD. We suggest that reduced brain swelling and cell death observed in SARA animals exposed to OGD result from diminished ROS production coupled with lower expression of Nox isoforms. Thus, the ACE2/Ang-(1–7)/Mas receptor pathway plays a protective role in brain ischemic damage by counteracting the detrimental effects of Ang-II-induced ROS production.

Introduction

Hypertension remains a prevalent condition in adults and contributes to significant mortality and morbidity (Osawa et al., 2009, Kountz, 2013, Pirkle and Freedman, 2013). Specifically, hypertension is a risk factor for hemorrhagic and ischemic stroke (Elkind, 2011, von Sarnowski et al., 2013) and is associated with increased severity of brain injury and hemorrhagic transformation in stroke patients (SHEP-Cooperative-Research-Group, 1991, Davis et al., 1998, Rashid et al., 2003, Alvarez-Sabin et al., 2013). Essential hypertension can result from increased levels of circulating vasoconstrictors and other hormones such as angiotensin (Ang-II), an active product of the renin–angiotensin system (RAS). The RAS also is present in the central nervous system (CNS) (von Bohlen and Halbach, 2005) and has been shown to have a variety of functions in physiology and pathophysiology including learning and memory, development, thirst, regulation of blood pressure and blood flow, apoptosis, and neurodegeneration (Wright et al., 2002, Xia et al., 2009).

Pharmacological and genetic manipulation of the RAS in the CNS can influence outcomes following stroke (Dai et al., 1999, Chen et al., 2009, Iwanami et al., 2009). Inhibition of angiotensin receptors confers protection in ischemic stroke beyond that due to reduced blood pressure (Padma et al., 2004). Our previous studies showed that transgenic mice overexpressing human renin and angiotensinogen (R+A+) had increased tissue swelling during oxygen–glucose deprivation (OGD) and increased subsequent cell death; both of which were reduced by contemporaneous inhibition of Ang-II/AT1 receptors (Chen et al., 2009). Another component of the RAS system, angiotensin-converting enzyme 2 (ACE2), has been identified as a negative regulator of the pro-hypertensive actions of Ang-II (Roks et al., 1999, Xu et al., 2011). ACE2 is a membrane-bound carboxymonopeptidase that metabolizes Ang-II to the heptapeptide Ang-(1–7) which then activates Mas receptors (Santos et al., 2003). Central administration of Ang-(1–7) reduces brain damage and improves neurological outcome in an animal model of ischemic stroke (Mecca et al., 2011) possibly mediated by anti-inflammatory actions (Regenhardt et al., 2013). As a negative regulator of the RAS, ACE2 over activation or expression also may have therapeutic effects on ischemic stroke (Mecca et al., 2011). While clinical studies show strong relationships between isoforms of ACE2 and the risk for hypertension (Patnaik et al., 2014) and brain ischemic stroke (Chen et al., 1895), the role of ACE2 in the mitigation of ischemic stroke injury and its mechanisms of action remain poorly understood.

There is considerable evidence that activation of the Ang-II receptor, AT1, in a variety of tissues leads to increased production of reactive oxygen species (ROS) mediated by NADPH oxidase (Nox) (Zhang et al., 1999, Lopez-Real et al., 2005, Didion et al., 2002a, Didion et al., 2002b, Franco et al., 2003, Haugen et al., 2000, Pena Silva et al., 2012, Wang et al., 2013). Nox1, Nox2 and Nox4 are the predominant isoforms in neurons of the brain (Tejada-Simon et al., 2005, Vallet et al., 2005, Ibi et al., 2006, Infanger et al., 2006), and play numerous roles in physiological cell signaling as well as for pathogenesis of brain injury (Walder et al., 1997, Atkins and Sweatt, 1999, Knapp and Klann, 2002, Kishida et al., 2005, Kishida and Klann, 2007). Nox2-deficient mice have reduced infarct volumes and in wild-type animals Nox4 is upregulated following stroke (Vallet et al., 2005). In addition, activation of the counteracting Mas receptor signaling by Ang-(1–7) can decrease ROS production and reduce tissue damage due to mechanical insult (Bodiga et al., 2011, Xia et al., 2011, Liu et al., 2012). We hypothesize that in ischemic stroke, increased ROS production and resulting cell injury mediated by the Ang-II/AT1 receptor signaling pathway is antagonized by activation of the ACE2/Ang-(1–7)/Mas receptor pathway. To test this hypothesis, we evaluated tissue swelling, ROS production, and cell death in the cerebral cortex (CX) and hippocampus in brain slices subjected to ischemic conditions induced by OGD. To evaluate the separate roles of these antagonistic pathways, we used mice which overexpress human renin and angiotensinogen in all tissues (R+A+) and SARA mice, a triple-transgenic model obtained by breeding R+A+ mice with syn-hACE2 animals overexpressing ACE2 specifically in neurons of the CNS (Xia et al., 2011).

Section snippets

Materials

Salts and other chemicals were obtained from Fischer Scientific (Pittsburgh, PA, USA). Losartan was purchased from Sigma–Aldrich (St. Louis, MO, USA). The heptapeptide Ang-(1–7) and the Mas receptor inhibitor A779 were obtained from Bachem (Torrance, CA, USA). Propidium iodide (PI) and dihydroethedium were purchased from Life Technologies (Grand Island, NY, USA). Complete Mini protease inhibitor and lysis buffer for protein extraction came from Roche Diagnostics Corporation (Indianapolis, IN,

OGD-induced tissue swelling is reduced by activation of the ACE2/Ang-(1–7)/Mas axis or by inhibition of the AT1 receptor

The intensity of light transmission through slices stabilized during the initial 30 min of perfusion with control aCSF (Fig. 1B). Similar to our previous results (Chen et al., 2009), within 10 min of the start of OGD exposure, IOS increased significantly in both brain regions with the hippocampus showing a greater increase than that observed in CX (Fig. 1A, B). The IOS of CX from SARA animals appeared to change little during the first 5 min of OGD while IOS from R+A+ animals was already

Discussion

These studies elucidate interactions between components of the brain RAS system which impact the magnitude of tissue damage during ischemic conditions in an animal model of essential hypertension. Because these studies were performed using in vitro brain preparations, the effects we observed by genetically and pharmacologically manipulating RAS pathways are independent of their effect on blood pressure or cerebrovascular tone. First, we found that inhibition of the AT1 receptor with losartan

Acknowledgments

This work was supported by the National Heart, Lung, and Blood Institute (HL-098637 to Y.C. and J.O.; HL-093178 to EL) and the National Natural Science Foundation of China (NSFC, #81271214, #81300079).

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