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

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 cerebral cortex (CX) and the hippocampal CA1 region during OGD. Expression levels of NADPH oxidase 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 results from diminished ROS production coupled with lower expression of NADPH oxidases. 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.

1. INTRODUCTION

Hypertension remains a prevalent condition in adults and contributes to significant mortality and morbidity ^{49, 60, 64}. Specifically, hypertension is a risk factor for hemorrhagic and ischemic stroke ^{21, 83} and is associated with increased severity of brain injury and hemorrhagic transformation in stroke patients ^{3, 16, 66, 73}. 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) ⁸² 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 ^{87, 88}.

Pharmacological and genetic manipulation of the RAS in the CNS can influence outcomes following stroke ^{13, 15, 39}. Inhibition of angiotensin receptors confers protection in ischemic stroke beyond that due to reduced blood pressure ⁶¹. 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 ¹³. 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 ^{70, 90}. ACE2 is a membrane-bound carboxymonopeptidase that metabolizes Ang-II to the heptapeptide Ang-(1-7) which then activates Mas receptors ⁷¹. Central administration of Ang-(1-7) reduces brain damage and improves neurological outcome in an animal model of ischemic stroke ⁵⁷ possibly mediated by anti-inflammatory actions ⁶⁷. As a negative regulator of the RAS, ACE2 over activation or expression also may have therapeutic effects on ischemic stroke ⁵⁷. While clinical studies show strong relationships between isoforms of ACE2 and the risk for hypertension ⁶² and brain ischemic stroke ¹², 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) ^{18, 19, 27, 33, 55, 63, 85, 94}. Nox1, Nox2 and Nox4 are the predominant isoforms in neurons of the brain ^{35, 36, 78, 81}, and play numerous roles in physiological cell signaling as well as for pathogenesis of brain injury ^{6, 44, 45, 47, 84}. Nox2-deficient mice have reduced infarct volumes and in wild type animals Nox4 is upregulated following stroke ⁸¹. 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 ^{8, 54, 89}. 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 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 ⁸⁹.

2. EXPERIMENTAL PROCEDURES

2.1. Materials

Salts and other chemicals were obtained from Fischer Scientific (Pittsburgh, PA). Losartan was purchased from Sigma-Aldrich (St. Louis, MO). The heptapeptide Ang-(1-7) and the Mas receptor inhibitor A779 were obtained from Bachem (Torrance, CA). Propidium iodide and dihydroethedium were purchased from Life Technologies (Grand Island, NY). Complete Mini protease inhibitor and lysis buffer for protein extraction came from Roche Diagnostics Corporation (Indianapolis, IN). The Bradford protein analysis kit and precast polyacrylamide gels (Cat #456-1034) were obtained from Bio-Rad Laboratories (Hercules, CA). Primary antibodies for β-actin were purchased from Sigma-Aldrich (St. Louis, MO) and rabbit polyclonal antibodies for Nox2 and Nox4 came from Abcam (Cambridge, MA). Jackson Immunoresearch Laboratories (West Grove, PA) provided HRP-conjugated donkey anti-rabbit IgG antibody. ECL reagent was purchased from Cell Signaling (Danvers, MA).

2.2. Animals

All procedures involving animals were approved by the Laboratory Animal Care and Use Committee of Wright State University and conformed to the NIH Guide for the Care and Use of Laboratory Animals. Founder mice for generating transgenic mice were obtained from Dr. Eric Lazartigues (Louisiana State University Health Sciences Center, New Orleans). Male R+A+ individuals which overexpress renin and angiotensinogen (R+A+) ⁵⁸ were bred with heterozygous transgenic females which constitutively express angiotensin converting enzyme 2 (ACE2+) in neurons of the CNS ²⁴. Their offspring were genotyped as previously described ⁴³ to determine animals that were R+A+ double transgenic or R+A+ plus ACE2+ triple transgenic (SARA). Animals were maintained on a 12 hour light/dark cycle and provided access to food and water ad libitum.

2.3. Brain Slice Preparation

Brain slices were prepared from R+A+ and SARA mice as previously described ¹³. Following rapid decapitation of the animal, brains were dissected from the cranium, mounted on a block of agar with cyanoacrylate cement, and sectioned into 400 μm-thick coronal slices using a Series 1000 Vibratome Section System (Technical Products, Inc., St. Louis, MO). Brain slices were incubated in control artificial cerebrospinal fluid (aCSF) bubbled with 95% O₂ plus 5% CO₂ at room temperature for 1–2 hr. The aCSF consisted of 124 mM NaCl, 3.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1 mM NaH₂PO₄, 26 mM NaHCO₃, and 10 mM glucose. After the room temperature incubation, slices were transferred to a Haas interface-type recording stage (Harvard Apparatus, Holliston, MA) and perfused for 30 min with aCSF at 35° C under a humidified atmosphere of 95% O₂ plus 5% CO₂.

2.4. Oxygen and Glucose Deprivation (OGD)

Some slices were exposed to OGD as an in vitro model of brain ischemia while other slices remained in control aCSF conditions. For OGD treatment, the perfusion solution was changed to 35° C aCSF which had been bubbled with 95% N₂ plus 5% CO₂ and which contained no glucose. Simultaneous with this change in perfusion solution, the humidified gas mixture flowing over the slice was changed to 95% N₂ plus 5% CO₂. Control or OGD conditions were maintained for 30 min. For some experiments, the AT1 receptor antagonist losartan (20 μM), the Mas receptor inhibitor A779 (10 μM), or the Mas receptor agonist Ang-(1-7) (10 μM) was added to the perfusing aCSF solutions at the start of OGD exposure.

2.5. Assessment of Tissue Swelling

Tissue swelling as a measure of brain edema was determined indirectly as we and others previously described ^{5, 9, 51} by measuring the intrinsic optical signal (IOS) defined as the intensity of transmitted light during OGD treatment expressed as a percent of the intensity measured prior to the start of OGD ⁵. Slices were transilluminated using a DC-regulated halogen lamp which delivered white light to the recording stage via a randomized fiber optic bundle. Images of the slice were acquired as a single standard NTSC video frame using a fixed gain video camera and 8-bit image processor board (DT2867, Data Translation, Inc., Marlboro, MA). By capturing images of dry laboratory tissue paper, this image acquisition system was found to have a linear intensity drift of −0.1% per hour with a standard deviation of 0.05% about this drift line. At the beginning of each experiment, the light source was adjusted such that the average light intensity transmitted through the slice was in the middle of the image acquisition system’s dynamic range (0–255 units). Then images were acquired at 60 sec intervals. Image analysis was subsequently performed using NIH-Image and ImageJ software. Regions of interest (ROI) were defined in stratum radiatum of the hippocampal CA1 area and in an equivalent area in the center of the adjacent cerebral cortex. The average light intensities in these ROIs were calculated for each image and then normalized to the average ROI intensity measured immediately prior to the start of OGD exposure. In addition, regional IOS was displayed by creating a pseudo-color image with red indicating increases in light intensity and blue indicating decreases in light intensity.

2.6. Measurement of Reactive Oxygen Species Production

Dihydroethidium (DHE) was used to evaluate the generation of ROS products in slices exposed to control conditions or OGD. After the initial 30 min perfusion with control aCSF at 35° C, slices in control conditions or during OGD were perfused with 10 μM DHE for 30 min. During DHE treatment the light source illuminating the slice and other room lights were turned off. Thus, no IOS measurements were made on slices used for studies of ROS production. Non-fluorescent DHE molecules freely penetrate cell membranes where they may be oxidized to ethidium by ROS ^{10, 32, 86}. Ethidium fluorescence then is greatly enhanced after binding to endogenous nucleic acids. After this treatment, slices were fixed for at least 1 hr with 4% paraformaldehyde in staining buffer (SB) consisting of 137 mM NaCl plus 10 mM Na₂HPO₄ (pH 7.4). They then were washed for 20 min with SB and mounted on glass slides under coverslips using Fluoro-Gel aqueous mounting medium (Electron Microscopy Services, Hatfield, PA). Photographs of the pyramidal cell layer in the middle of the CA1 region and in the center of the adjacent cerebral cortex were captured under epifluorescence illumination. To eliminate bias in selecting the region of interest photographed for each brain region, we first identified the general brain area in the slice using a 10× objective. Then the objective was switched to 40×, the image focused without further manipulation of the stage, and a single image acquired. The number of DHE-positive cells per microscope field was counted using ImageJ software after subtracting background fluorescence. Data representing cell density are expressed as the number of cells per microscope field.

2.7. Evaluation of Cell Death

Cell death was determined using propidium iodide (PI) staining of slices used for measurements of IOS. Following a 30 min exposure to control or OGD conditions, slices were incubated with 20 μg/ml PI in aCSF for 15 min at room temperature and then fixed overnight with 4% paraformaldehyde in SB. Slices were washed with SB and mounted under coverslips with Fluoro-Gel. PI-positive cells were visualized using laser confocal microscopy and digital images of the CA1 region and cerebral cortex captured for analysis using an unbiased selection procedure to identify regions of interest as described above. The approximate surface of the slice was found by adjusting the focus. Then, to avoid the influence of cell injury at the slice surface caused during slice preparation, we counted only PI-positive nuclei which were situated in a single plane approximately 80–90 μm below the slice surface. The cell density of PI-positive cells is expressed as the number of cells per microscope field.

2.8. Expression levels of Nox2 and Nox4

Following a 30 min exposure to control or OGD conditions, slices were removed from the perfusion chamber following measurements of IOS and homogenized on ice in lysis buffer containing Complete Mini protease inhibitors and EDTA. Total protein content of the solution was determined using the Bradford protein assay. Equal amounts (50 μg) of protein were loaded into lanes of a 10% precast polyacrylamide gel, separated by electrophoresis, and then electrotransferred onto a PVDF membrane. Membranes were blocked for 1 hour in 5% non-fat dry milk and were probed with primary monoclonal rabbit anti-mouse β-actin (1:4000), polyclonal rabbit anti-mouse Nox2 (1:1000) or polyclonal rabbit anti-mouse Nox4 (1:500) overnight at 4° C. Membranes then were washed and incubated with HRP-conjugated donkey anti-rabbit IgG (1:40,000). Blots were probed using enhanced chemiluminescence with ECL reagent and visualized using a Fuji film image analyzer (Molecular Dynamics Image Quant, Sunnyvale, CA). Bands for β-actin, Nox2, and Nox4 were visualized at mean ± SD apparent molecular weights of 47.0 ± 1.3 kDa, 61.6 ± 2.4 kDa, and 60.7 ± 2.2 kDa, respectively (N=8).

2. 9. Data Analysis

A power analysis was performed to determine the number of animals needed for these studies. Based on results from our previous report ¹³, the OGD-induced IOS in R+A+ and wild type animals differed by 34% with a pooled standard deviation of 16%. This yielded a requirement of 3 animals per group to detect a similar difference between the final IOS values from the transgenic animals used in the present study for α = 0.05 and β = 0.80. Similarly, we previously found that the number of cell counts for PI-positive cells following OGD decreased by 78% with pharmacological AT1 receptor inhibition. From this we calculated a requirement of only 2 animals to find this magnitude of effect for α = 0.05 and β = 0.80. IOS data are displayed as the mean ± SEM and were log-transformed before being analyzed using ANOVA followed by Dunnett’s test for multiple comparisons. Cell count data were analyzed with the Mann-Whitney, Kruskal-Wallis, and Student-Neuman-Keuls tests as appropriate to evaluate differences between experimental groups. Nox expression data were log-transformed and then analyzed using two-way ANOVA with animal genotype and treatment as independent variables followed by Neuman-Keuls multiple comparison tests. Percent changes in Nox expression levels were analyzed by Neuman-Keuls test. Significance was indicated for p < 0.05.

3. Results

3.1. 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 (Figure 1.B.). Similar to our previous results ¹³, 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 cerebral cortex (Figures 1.A and 1.B.). The IOS of cerebral cortex from SARA animals appeared to change little during the first 5 min of OGD while IOS from R+A+ animals was already significantly different from the initial value by this time point. IOS from both animal groups continued to increase throughout the period of OGD exposure; however, the increase was significantly greater for slices from R+A+ animals compared with slices from SARA animals. After 30 min of OGD, IOS measured in the cerebral cortex and hippocampus of slices from SARA animals was 39% and 68% of that measured in slices from R+A+ animals (Figure 1.C.). As previously reported, treating slices with 20 μM losartan significantly attenuated IOS during OGD to values not different than 100% ¹³. In addition, treating slices from R+A+ animals with the Mas receptor agonist Ang-(1-7) (10 μM) reduced the magnitude of IOS after 30 min of OGD in both brain regions. The average magnitude of IOS response was greater for SARA animals treated with the Mas receptor antagonist A779 (10 μM); however the increase in each brain region was not statistically different.

Figure 1.

Tissue swelling during oxygen and glucose deprivation (OGD) exposure. Brain slices were perfused with control aCSF until OGD exposure was initiated at time t=0 min. A. Representative images of brain slices from R+A+ and SARA animals. For each pair of images the top panel is the transmitted light image prior to OGD exposure. The hippocampal CA1 region and cerebral cortex are labeled for anatomical reference. The lower panel is a pseudocolor image of the same slice showing percent changes in light transmission after 30 min of OGD calculated on a pixel-by-pixel basis. A color calibration scale for the magnitude of the change in light transmission is shown in the figure insets. B. Mean IOS values from R+A+ and SARA animals before and during OGD exposure measured from regions of interest in cerebral cortex and hippocampal CA1 region. IOS is defined as the intensity of transmitted light during OGD treatment expressed as a percent of the value measured prior to the start of OGD. Values are the mean ± SEM for slices from 12 animals C. IOS values measured in cerebral cortex and the hippocampal CA1 region after 30 min of IOS exposure for brain slices from R+A+ and SARA animals with and without various drug treatments. The mean IOS from regions of interest in the cerebral cortex and hippocampal CA1 region for the final 5 images during OGD exposure was calculated for each slice and these results averaged to give the mean ± SEM values shown. The number of animals for each group is in the parentheses. Log-transformed data were analyzed by ANOVA with post hoc Dunnett’s test. For slices from SARA animals given no drug treatment, * indicates values that were significantly different from that measured in slices from R+A+ animals with no drug treatment. † indicates values significantly different from those measured in slices from animals of the same genotype with no drug treatment.

3.2. OGD-induced ROS production is reduced by activation of the ACE2/Ang-(1-7)/Mas axis or by inhibition of the AT1 receptor

Following perfusion with control aCSF, a small number of cells stained positive for DHE in cerebral cortex and the hippocampal CA1 region of slices from R+A+ and SARA animals (Figure 2.B.). The number of DHE-positive cells was not significantly different for each animal genotype under control perfusion conditions. Exposure to OGD significantly increased the number of DHE-positive cells in both animal groups. Nevertheless, the number of DHE-positive cells per microscope field in cerebral cortex was significantly greater in brain slices from R+A+ animals compared with the number observed in brain slices from SARA animals. Losartan treatment during OGD significantly decreased the number of DHE-positive cells in both brain regions of R+A+ and SARA animals to levels that were not significantly different from those measured in slices from animals of the same genotype without OGD exposure.

Figure 2.

ROS production during oxygen and glucose deprivation (OGD) exposure. Slices were exposed to DHE for 30 min during control or OGD conditions. Some slices also were treated with losartan during OGD exposure. A. Epifluorescence images of cerebral cortex and the hippocampal CA1 region of slices from R+A+ and SARA animals showing DHE-positive cells following OGD exposure. The entire microscope field is shown in these images with the scale bar indicating 25 μm. B. Cell counts of DHE-positive cells in cerebral cortex and hippocampal CA1 region. Boxes designate the interquartile range with the horizontal line indicating the median cell counts for the number of animals shown in parentheses. 95^th and 5^th percentiles are indicated with vertical lines where possible. Data were analyzed by Kruskal-Wallis One-Way ANOVA and Newman-Keuls test for multiple comparisons. For slices from SARA animals, * indicates values significantly different from those measured in slices from R+A+ animals exposed to the same perfusion conditions. † indicates values significantly different from those measured in slices from animals of the same genotype under control conditions.

3.3. OGD-induced cell death is reduced by activation of ACE2/Ang-(1-7)/Mas axis or by inhibition of the AT1 receptor

Slices from both R+A and SARA animals exposed to control aCSF showed low intensity fluorescence in a small number of cells (Figure 3.A.). For each genotype, a significantly greater number of cells appeared brightly PI-positive following 30 min of OGD. Compared with slices from R+A+ animals, slices from SARA mice had fewer PI-positive cells per microscope field in the cerebral cortex and CA1 region following OGD exposure (Figure 3.B.). In addition, treating slices from R+A+ animals with Ang-(1-7) reduced the PI-positive cell count in both brain regions while treating slices from SARA animals with A779 significantly increased the PI-positive cell count in cerebral cortex. Finally, adding the Ang-II receptor antagonist, losartan (20 μM) to slices from SARA animals during OGD significantly reduced the number of DHE-positive cells in cerebral cortex and hippocampal CA1 region.

Figure 3.

Cell death following oxygen and glucose deprivation (OGD) exposure. Slices were exposed to control aCSF or OGD for 30 min and then stained with PI for 15 min. Some slices had drug treatments during OGD as indicated. A. Confocal images of cerebral cortex and hippocampal CA1 region of slices from R+A+ and SARA animals following 30 min perfusion with control or OGD conditions. The entire microscope field is shown in these images with scale bar indicating 75 μm. B. Cell counts of PI-positive cells in cerebral cortex and hippocampal CA1 region. Boxes designate the interquartile range with the horizontal line indicating the median of cell counts for the number of animals shown in parentheses. 95^th and 5^th percentiles are indicated with vertical lines where possible. Data were analyzed by Kruskal-Wallis One-Way ANOVA and Newman-Keuls test for multiple comparisons. For slices given no drug treatment, * indicates values significantly different from those measured in slices from R+A+ animals. † indicates values significantly different from those measured in slices from animals of the same genotype with no drug treatment.

3.4. Nox2 and Nox4 expression levels are reduced by neuronal ACE2 expression

Both Nox2 and Nox4 were expressed in brain slices from wild type, R+A+, and SARA animals (Figure 4A and 4B). For control slices from R+A+ animals, the relative expression of Nox2 was similar while the relative expression of Nox4 was elevated compared with that measured in slices from wild type animals. We observed lower expression levels for Nox2 and Nox4 in control slices from SARA mice compared with those measured in slices from R+A+ mice. For slices from all genotypes, relative expression levels of Nox2 increased following OGD exposure; however, the percent increase relative to control slices was smaller in slices from SARA animals compared with that measured in slices from wild type and R+A+ mice (Figure 4C). Additionally, the relative expression of Nox2 in slices from SARA animals remained significantly below that measured in slices from R+A+ animals. For Nox4, OGD exposure increased expression in slices from wildtype and SARA mice, but not in slices from R+A+ animals. The percent increase in Nox4 expression in slices from SARA animals following OGD was similar to that observed for slices from wild type mice.

Figure 4.

Nox2 and Nox4 expression following oxygen and glucose deprivation (OGD) exposure. Slices were perfused with control aCSF or OGD conditions for 30 min and then prepared for western blot analysis. Expression levels of Nox2 (A) and Nox4 (B) in brain slices from wild type (WT), R+A+, and SARA animals following control or OGD exposure are shown relative to expression levels of β-actin. C shows Nox expression in brain slices following OGD as a percent of the expression measured contemporaneously in brain slices from the same animal exposed to control conditions. Values are the mean ± SEM of data from 5 R+A+ animals, 5 wild type animals, and 4 SARA animals. Representative bands from western blots for Nox2, Nox4, and β-actin are shown under each bar in A and B. Data were analyzed by ANOVA with post hoc Neuman-Keuls test for multiple comparisons. * indicates values which are significantly different from those measured slices from wild type animals given the same perfusion treatment. † indicates values which are significantly different from those measured in slices from animals of the same genotype exposed to control conditions. For slices from SARA animals, ‡ indicates values which are significantly different from those measured in slices from R+A+ animals exposed to the same perfusion treatment.

4. 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 reduces or eliminates tissue swelling during OGD in slices from SARA animals which constitutively overexpress human renin and angiotensinogen and also overexpress ACE2 selectively in neurons. These results expand upon our previous studies which showed that treatment with losartan reduced tissue swelling in slices from wild type and R+A+ animals exposed to OGD ¹³. Second, the current study further extends results from our previous investigation by demonstrating that losartan reduces cell death in brain slices from SARA animals as well as in brain slices from R+A+ mice. Third, our data demonstrate that ACE2 overexpression in neurons reduces cell damage from OGD in cerebral cortex and hippocampus. Compared with brain tissue from R+A+ animals, the brain slices of SARA animals developed less tissue swelling, as evidenced by a smaller change in IOS, and had a lower density of PI-positive cells after a 30 min OGD treatment. Finally, we observed decreased levels of ROS production and lower expression levels of Nox2 following OGD exposure in slices from SARA animals compared with those measured in R+A+ animals. Thus, we propose that the decrease in cellular injury following OGD in slices from animals with neuronal ACE2 overexpression results from decreased oxidative stress mediated by the Ang-(1-7)/Mas receptor pathway. Although we did not measure Nox expression levels separately in hippocampus and cerebral cortex in this initial study, we expect there were comparable responses of these enzymes to OGD in each region since we observed similar qualitative changes in tissue swelling, cell death, and ROS production in cerebral cortex and hippocampus.

The hypothesized role of the Ang-(1-7)/Mas receptor pathway for brain protection is strongly supported by our results showing that exposure of slices from R+A+ animals to the Mas agonist Ang-(1-7) decreases OGD-induced injury while exposing slices from SARA animals to the Mas inhibitor A779 increases oxidative stress and cell injury following OGD. Activation of Mas receptors with Ang-(1-7) and inhibition of AT1 receptors with losartan both ameliorate OGD-induced injury. However, we cannot infer a relative efficiency of these two signaling pathways as our studies were designed and statistically powered only to compare results from R+A+ animals with those from SARA animals. Interpretation of these pharmacological studies also must take into account recent reports which suggest that part of the beneficial effects of AT1 receptor blockers are mediated through enhanced activity of ACE2 or the Ang-(1-7)/Mas signaling pathway ^{37, 40, 59}. Blockade of the AT1 receptor increases Ang-II levels making it more available for conversion into Ang-(1-7) by ACE2. Accordingly it is not surprising that the combination of an AT1 receptor blocker with enhanced expression of ACE2 would provide a stronger benefit than application of Ang-(1-7).

In many cell systems, the Ang-(1-7)/Mas intracellular signaling pathway is functionally opposed to the actions of the Ang-II/AT1 receptor pathway ^{23, 25, 52, 90}. Our data are consistent with this relationship in that overexpression of ACE2 in SARA mice appears to reverse the effects of AT1 receptor activation that results from overproduction of Ang-II in the R+A+ animals. This reversal may result from ACE2-mediated conversion of Ang-I and Ang-II into Ang-(1-9) and Ang-(1-7), respectively; peptides that are inactive at the AT1 receptor. However, we conclude that the Ang-II/AT1 receptor pathway is not completely inactive in SARA animals during OGD since losartan reduces cell death and levels of oxidative stress in slices from these mice. While the data support our hypothesis that activation of the Mas receptor by Ang-(1-7) is responsible for the reduction in cell death in brain slices from SARA animals following OGD, the cellular location of the Mas receptor which mediates this neuroprotection is not clear nor is its mechanism of action. Potentially, Mas receptor activation reduces ROS production in this animal model as has been observed in cultured neuroblastoma cells, rat autonomic system, vascular smooth muscle and adipocytes from mouse ^{8, 89}.

Various isoforms of NADPH have been identified in neurons and glial cells of the CNS ^{11, 14, 65, 68, 93} and previous clinical and animal studies have shown their importance for pathogenesis of injury following ischemia ^{48, 53}. Compared with slices from wild type mice in control conditions, slices from R+A+ animals had similar expression levels of Nox2, but elevated expression levels of Nox4. A different expression pattern was seen in slices from SARA mice where Nox2 levels were decreased while Nox4 levels were similar to those measured in wild type mice. We found Nox2 and Nox 4 expression levels were increased in slices from wild type mice within 30 min of initiating the OGD treatment. A similarly rapid increase in Nox expression levels occur in human endothelial cells following atrial natriuretic peptide treatment due to increased transcription to mRNA ²⁸. Once expressed the activity of Nox4 can persist for 24 hr ⁷².

The reduction in the density of DHE-positive cells with losartan treatment following OGD indicates that the AT1 receptor is coupled to ROS production in mouse brain. Ang-II activation of ROS production has been observed in a number of tissues including brain. Previously, Griendling et al. demonstrated that activation of the AT1 receptor in cultured smooth muscle cells increases ROS production by activating NADPH oxidase to produce superoxide ³¹. Others have shown that the actions of Ang-II in the CNS are mediated by superoxide production or by increased activity of specific Nox enzymes ^{85, 95, 96}. Similarly pharmacologic inhibition of the AT1 receptor can limit oxidative stress in cardiac tissue and brain ^{42, 69, 77}. Our results suggest that increased ROS production following OGD is related to elevated expression levels of both Nox2 and Nox4 isoforms. Conversely we suggest that brain cell protection in slices from SARA mice follows from lower expression levels of Nox2 and Nox4 after OGD compared with those measured in slices from R+A+ animals and this lower expression of Nox isoforms results in decreased ROS production. OGD induces a smaller percent change in Nox2 expression in slices from SARA mice compared with the percent change measured in R+A+ animals. Thus the decreased expression of Nox isoforms in slices from SARA mice is a consequence of both lower control levels of Nox activity and a limited increase in Nox expression during OGD. In contrast, the OGD-induced percent change in Nox4 expression is greater in SARA animals while the relative expression level of Nox4 is similar to that observed in slices from R+A+ mice. These results suggest ACE2 mediates its effect on ROS production predominantly via modulation of Nox2 expression. We previously reported OGD-induced cell death is greater in brain slices from R+A+ mice compared to that found in slices from wild type mice ¹³. However, levels of Nox2 and Nox4 expression in R+A+ mice following OGD are similar to those measured in slices from wild type mice suggesting changes in Nox activity rather than expression levels are affecting OGD-induced cell death in R+A+ mice. Mechanisms by which activation of AT1 and Mas receptors may alter Nox isoform expression in this animal model of ischemic stroke are unknown.

Tissue swelling as a result of cytotoxic or vasogenic edema is commonly observed following clinical stroke and in in vivo and in vitro models of hypoxia/ischemia ^{46, 56, 74, 92}. In ischemia, the magnitude of the change in tissue water content is dependent on the degree of brain injury ⁵⁶. Changes in brain extracellular or intracellular water content as a result of exposure to aniso-osmotic, hypoxic, or ischemic conditions alter the optical properties of the tissue, measured here as the IOS ^{1, 9, 30, 41}. In the present studies, we found a consistent increase in IOS in slices from SARA mice during OGD, similar to our previous studies using brain slices from wild type and R+A+ mice ¹³. We suggest these responses represent swelling of the brain tissue, but cannot determine to what degree the edema is due to changes in volumes of extracellular or intracellular spaces. Although we find quantitative differences in IOS during OGD for slices from R+A+ and SARA animals, our measurements of PI-positive and DHE-positive cell densities cannot be corrected for tissue swelling since the relation between IOS and tissue volume are not known for this animal model. By itself, the smaller degree of tissue swelling during OGD seen for slices from SARA animals would result in a higher cell density compared with slices from R+A+ animals. Thus, the quantitative difference in PI-positive cell density between slices from SARA and R+A+ animals is likely to be greater than reported in Figure 3.

These investigations do not indicate whether neurons or glial cells are responsible for the tissue volume changes measured by the IOS, which cell type has increased ROS production, or which cells die during OGD. IOS is a general measure of tissue swelling and can result from changes in light reflection, scattering, and absorption ^{1, 22} and quantitatively may depend on slice configuration along the optical axis between the light source and imaging device ⁵⁰. IOS increases in osmotically swollen brain slices and decreases when hyperosmotic solutions are applied to cause dehydration and cellular shrinkage ^{4, 22, 51}. Thus, an increase in IOS is generally believed to represent swelling by both glial cells and neurons ⁴. In these studies IOS increased by approximately 25% to 30% during OGD, a value comparable to our previous studies of rat brain slices treated with 200 mOsm aCSF ⁵¹ but considerably higher than the 4%-6% change in response to exogenous ROS treatment (2 mM H₂O₂) ⁷⁹. However during OGD and associated spreading depression, changes in the volume of intracellular organelles and localized swelling (beading) of neuronal dendrites also may contribute significantly to changes in light transmission ^{22, 80}. These latter processes may cause the persistent IOS elevation during OGD in the presence of losartan despite complete inhibition of excess ROS production and cell death by this drug.

Cellular localization of the various RAS system components which give rise to elevated ROS production and cell death during OGD also are not defined by the current studies. Astrocytes and neuroblastoma cells express Ang-II receptors ^{26, 34} and angiotensinogen is synthesized in neurons and astrocytes in vivo ⁷⁶. Certain cortical brain structures may produce some but not all of the major peptide components of the RAS system (reviewed by von Bohlen und Halbach ⁸²); however, in the hippocampus angiotensinogen, angiotensin, renin, and ACE are all present ⁹¹. The AT1 and AT2 receptors for Ang-II are found throughout the hippocampus, but may be expressed regionally in cerebral cortex ²⁹. Previous results from our group and others show that ACE2 is distributed throughout the mouse brain and expressed in both on the cell surface and in the cytoplasm of neurons but not in glial cells ²⁰. SARA animals are genetically engineered to overexpress ACE2 in neurons of the CNS ²⁴. The differences in sensitivity we observed in cerebral cortex and hippocampus for IOS, ROS production, and cell death may be related to the abundance and localization of the sources and targets of Ang-II and Ang-(1-7) in these brain regions. Cell-specific relations between these signaling pathways, localization of Nox enzymes, ROS production, and cell injury during ROS are currently unknown.

Clinical studies have suggested that Ang-II receptor blockers can have beneficial effects in pathological conditions beyond their effects on blood pressure alone. They may lower the risk for myocardial infarction ⁷, improve the treatment of patients in end stage renal disease ⁷⁵, decrease the incidence of stroke ^{38, 61}, and improve stroke outcome ². Because of the antagonist relations between the ACE2/Ang-(1-7)/Mas axis and the Ang-II/AT1 pathway, others have suggested that augmentation of Mas signaling by pharmacological or genetic methods also may be a means of improving outcomes in these pathological situations ^{17, 52}. The results of our studies support this conjecture for the treatment of stroke in patients with essential hypertension. The ultimate utility of using activators of the Ang(1-7)/Mas signaling pathway to ameliorate brain damage following stroke must await results from future clinical investigations.

Bullet 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