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
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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