Journal of the American Society of Hypertension 9(4) (2015) 250–256
Research Article
Direct angiotensin II type 2 receptor stimulation by compound 21 prevents vascular dementia Jun Iwanami, PhD, Masaki Mogi, MD, PhD, Kana Tsukuda, BS, Xiao-Li Wang, MD, Hirotomo Nakaoka, MS, Harumi Kan-no, MS, Toshiyuki Chisaka, MD, Hui-Yu Bai, MD, Bao-Shuai Shan, MD, Masayoshi Kukida, MD, and Masatsugu Horiuchi, MD, PhD* Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan Manuscript received August 19, 2014 and accepted January 16, 2015
Abstract Angiotensin II type 2 (AT2) receptor activation has been reported to play a role in cognitive function, although its detailed mechanisms and pathologic significance are not fully understood. We examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) could prevent cognitive decline associated with hypoperfusion in the brain.We employed a bilateral common carotid artery stenosis (BCAS) model in mice as a model of vascular dementia. The Morris water maze task was performed 6 weeks after BCAS operation. Azilsartan (0.1 mg/kg/day) or C21 (10 mg/kg/day) was administered from 1 week before BCAS. Cerebral blood flow (CBF) and inflammatory cytokine levels were also determined. Wild– type (WT) mice showed significant prolongation of escape latency after BCAS, and this cognitive impairment was attenuated by pretreatment with azilsartan. Cognitive impairment was more marked in AT2 receptor knockout (AT2KO) mice, and the preventive effect of azilsartan on cognitive decline was weaker in AT2KO mice than in WT mice, suggesting that the improvement of cognitive decline by azilsartan may involve stimulation of the AT2 receptor. The significant impairment of spatial learning after BCAS in WT mice was attenuated by C21 treatment. The decrease in CBF in the BCAS–treated group was blunted by C21 treatment, and the increase in TNF–a and MCP–1 mRNA expression after BCAS was attenuated by C21 treatment. These findings indicate that direct AT2 receptor stimulation attenuates ischemic vascular dementia induced by hypoperfusion at least in part through an increase in CBF, and a reduction of inflammation. J Am Soc Hypertens 2015;9(4):250–256. Ó 2015 American Society of Hypertension. All rights reserved. Keywords: Cerebral blood flow; cognitive function; inflammation; renin-angiotensin system.
Introduction Cardiovascular risk factors such as hypertension, diabetes, and hypercholesterolemia are known to be associated with the onset and progression of dementia, including Alzheimer disease. Vascular cognitive impairment is related to aging and doubles every 5.3 years.1 Vascular dementia is classified into six groups.2 Multi–infarction dementia, Dr Horiuchi received research support and lecturing fees from Takeda Pharmaceutical Company Ltd. This study was supported by JSPS KAKENHI Grant Numbers 25293310 (MH), 25462220 (MM), and 24791506 (JI), and research grants from pharmaceutical companies: Astellas Pharma Inc, Bayer Yakuhin, Ltd, Daiichi-Sankyo Pharmaceutical Co, Ltd, Nippon Boehringer lngelheim Co, Ltd, Novartis Pharma KK, Shionogi & Co., Ltd., and Takeda Pharmaceutical Co, Ltd.
strategic infarction dementia and hemorrhagic dementia, which develop relatively suddenly, mixed subcortical ischemic vascular dementia, and other forms of vascular dementia. Chronic cerebral hypoperfusion is known to increase inflammation and oxidative stress, disrupt the blood–brain barrier (BBB), and induce the development and progression of white matter lesion and disruption.
*Corresponding author: Masatsugu Horiuchi, MD, PhD, Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. Tel: þ 81-89-960-5248; Fax: þ 81-89-960-5251. E-mail: [email protected]
1933-1711/$ - see front matter Ó 2015 American Society of Hypertension. All rights reserved. http://dx.doi.org/10.1016/j.jash.2015.01.010
J. Iwanami et al. / Journal of the American Society of Hypertension 9(4) (2015) 250–256
Pharmacologic targeting against the renin–angiotensin system (RAS) is one of the most effective ways to intervene in the pathogenesis of hypertension and diabetes. Li et al reported that angiotensin II type 1 (AT1) receptor blockers (ARBs) were associated with a significant reduction in the incidence and progression of Alzheimer disease and dementia compared with angiotensin–converting enzyme (ACE) inhibitors and other cardiovascular drugs in a population of 819,491 predominantly male participants aged 65 years or more with cardiovascular disease.3 Similar results were reported by Davies et al, who conducted a nested case–control analysis within the UK general practice research database.4 Fuentes et al reported that patients taking antihypertensive drugs at stroke onset had a lower rate of poor outcome than those not on antihypertensive treatment, and those taking an ARB had better outcomes than those without an ARB, with no difference in the analysis of other antihypertensive drugs including ACE inhibitors.5 Angiotensin II type 2 (AT2) receptor stimulation by unbound angiotensin II could also be expected during treatment with ARBs, and there is recent accumulating evidence suggesting that the AT2 receptor not only opposes the AT1 receptor, but also has unique effects beyond interaction with AT1 receptor signaling.6 These results suggest the idea that both AT1 receptor blockade and AT2 receptor stimulation by unbound angiotensin II are important in the neuroprotective effect of ARBs. AT2 receptor agonists such as compound 21 (C21) have been newly developed and are expected to be useful agents for improving pathologic disorders. This compound also enables us to examine AT2 receptor actions in vitro and in vivo by direct receptor stimulation, and principally offers the possibility to use AT2 receptor stimulation as a therapeutic tool.7,8 Our recent study also pointed out that direct AT2 receptor stimulation with C21 enhanced cognitive function in both normal mice and an Alzheimer disease mouse model through increases in cerebral blood flow and neurite outgrowth.9 Therefore, we investigated the effect of direct AT2 receptor stimulation by C21 on cognitive function using a mouse chronic cerebral hypoperfusion model.
Methods This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All animal studies were reviewed and approved by the Animal Studies Committee of Ehime University.
Animals Adult male C57BL/6 mice (Clea Japan Inc, Tokyo, Japan) as wild–type (WT) mice and AT2 receptor knockout mice
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(AT2KO mice; based on C57BL/6J strain)10 (23–25 g; 10 weeks old) were used. Mice were housed in a room in which lighting was controlled (12 hours on, 12 hours off), and the temperature was kept at 25 C. They were given a standard diet (MF, Oriental Yeast, Tokyo, Japan) and water ad libitum. They were given control chow or chow containing azilsartan (0.1 mg/kg/day) from 1 week before induction of bilateral common carotid artery stenosis (BCAS) until the cognitive task. An AT2 receptor agonist, C21 (10 mg/kg/day; provided by Vicore Pharma, Gothenburg, Sweden), was administered daily by intraperitoneal injection from 1 week before BCAS operation, and C21 administration was kept until the brain sample was finally taken. The cognitive task was performed 1 hour after injection of C21. BCAS was induced by a microcoil technique.11 We used microcoil with inner diameters of 0.18 mm and total length of 2.5 mm (Sawane Spring Co, Hamamatsu, Japan). Mice were anesthesized with 65 mg/kg nembutal in saline. A middle incision was made in the neck, and the bilateral common carotid artery was isolated. Silk suture was placed around the right common carotid artery (CCA). Then, the CCA was gently lifted by suture and placed between the loops of the microcoil. The microcoil was trinned by rotating it around the CCA. After that, another microcoil was twined around the left CCA in the same way. The microcoil’s inside diameter of 0.18 mm resulted in a stenosis of about 50%, because the outside diameter of the common carotid artery was 0.35-0.40 mm under anesthesia. We checked the cerebral blood flow before and just after BCAS operation. Cerebral blood flow just after was decreased to 60%–70%. Systolic blood pressure was measured in conscious mice by the tail-cuff method (MK-1030; Muromachi Co, Tokyo, Japan) 6 weeks after the BCAS operation.
Morris Water Maze Test The Morris water maze task was performed in mice 6 weeks after the BCAS operation as previously described.10 A white circular tank (120 cm diameter) was filled with water (23 2 C). A transparent platform (a 6 cm 6 cm acrylic board) was placed 1.5 cm below the surface of the water. Four objects in the corners of the pool helped mice to know their position. After they were placed on the platform for 10 seconds, they were put into the water. After reaching the platform, they were returned to their cages. If they did not reach the platform within 120 seconds, they were placed on it, kept for 10 seconds, and returned to their cages. Mice were trained five times a day at 20–minute intervals for five consecutive days. In each trial, mice were given 120 seconds to find the platform. Swimming was video–tracked (AnyMaze, Wood Dale, IL), and latency, path length, swim speed, and cumulative distance from the platform were recorded. Mean swim latency each day was evaluated and compared between groups. The area under the curve of days 1–5 was quantified using
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computer–imaging software (Densitograph; ATTO Corporation, Tokyo, Japan). All analyses were performed by an investigator blinded to the experimental conditions.
Measurement of Cerebral Blood Flow After the Morris water maze task, cerebral blood flow (CBF) was measured by laser speckle flowmetry (Omegazone laser speckle blood flow imager; Omegawave, Tokyo, Japan), which obtains high–resolution 2D images in a matter of seconds as previously described.9 Mice were anesthetized with nembutal, and a midline incision was made in the scalp. Anesthesia did not significantly affect blood pressure. The skull was exposed and wet with saline. A 780–nm laser semiconductor laser illuminated the whole skull surface. Mean CBF on the skull surface was measured. Light intensity was accumulated in a charge–coupled device camera and transferred to a computer for analysis. Image pixels were analyzed to produce average perfusion values.
Real–time Reverse Transcription Polymerase Chain Reaction (RT–PCR) Method Samples of cerebral cortex were frozen in liquid nitrogen and stored at 80 C until analysis. Total mRNA was extracted from brain samples after homogenization in Sepazol (Nacalai Tesque Inc, Kyoto, Japan). Quantitative real– time RT–PCR was performed with a SYBR green kit (MJ Research, Inc, Waltham, MA). PCR primers were as follows: 50 -CGAGTGACAAGCCTGTAGCC-30 (forward) and 50 -GGTGAGGAGCACGATGTCG-30 (reverse) for tumor necrosis factor (TNF)–a, 50 -TTAACGCCCCACT CACCTGCTG-30 (forward) and 50 -GCTTCTTTGGGA CACCTGCTGC-30 (reverse) for monocyte chemoattractant protein-1 (MCP-1), and 50 -ATGTAGGCCATGAGGTCCA C-30 (forward) and 50 -TGCGACTTCAACAGCAACTC-30 (reverse) for glyceraldehyde–3–phosphate dehydrogenase (GAPDH).
Statistical Analysis All values are expressed as mean standard error of the mean in the text and figures. Data were evaluated by analysis of variance followed by post hoc analysis for multiple comparisons. A difference with P < .05 was considered significant.
Results Preventive Effect of ARB on Decrease in Spatial Learning after Bilateral Common Carotid Artery Stenosis and Possible Involvement of AT2 Receptor To examine the possible protective effects of ARBs on cognitive decline in vascular dementia, we employed a
BCAS mouse model. The Morris water maze task was performed 6 weeks after the BCAS operation. WT mice showed significant prolongation of escape latency assessed by Morris water maze test compared with mice without BCAS (Figure 1A). This cognitive impairment was attenuated by pretreatment with azilsartan at a dose of 0.1 mg/kg/ day, without a change in systolic blood pressure. To investigate the possible involvement of AT2 receptor stimulation in the preventive effect of azilsartan on the cognitive decline associated with BCAS, we employed AT2 receptor–null mice. Body weight and systolic blood pressure did not differ between both strains before or after BCAS (data not shown). There was no significant difference in spatial learning memory between and AT2KO and WT mice without BCAS, although spacial learning in AT2KO mice tended to be impaired. Prolongation of escape latency after BCAS was more marked in AT2KO mice (Figure 1A and B). Interestingly, the preventive effect of azilsartan on cognitive decline was weaker in AT2 KO mice than in WT mice (Figure 1C). These results suggested that the improvement of cognitive decline by azilsartan may involve stimulation of the AT2 receptor. Therefore, we examined the effects of direct AT2 receptor stimulation on cognitive decline after BCAS and its mechanism.
Preventive Effect of Direct AT2 Receptor Stimulation by Compound 21 on Decrease in Spatial Learning Treatment with C21 was started 1 week before BCAS. There were no significant differences in body weight and systolic blood pressure among all groups before start of treatments. Administration of C21 did not influence body and brain weights or systolic blood pressure (Table 1). Mice showed significant prolongation of escape latency after BCAS, as shown in Figure 1A, and this prolongation of escape latency was significantly attenuated by C21 treatment (Figure 2A). Path length and cumulative distance were increased in BCAS–treated mice, and this increase was attenuated by treatment with C21 (Figures S1 and S2). There was no significant differences in swim speed among three groups (Figure S3). In the probe trials, treatment with C21 prevented the decrease in the number of platform area crossings induced by the BCAS operation (Figure 2B).
Increase in Cerebral Blood Flow and Decrease in Inflammation by Direct AT2 Receptor Stimulation by Compound 21 Cerebral blood flow in the whole brain in the BCAS– treated group was significantly decreased compared with that in the sham group at 6 weeks after BCAS operation (Figure 3). This decrease was increased by C21 treatment. Next, we assessed expression of inflammatory cytokines such as TNF–a and MCP–1 in the cerebral cortex
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Figure 1. Effect of administration of azilsartan on escape latency in wild type (WT; A) and Angiotensin II type 2 knockout (AT2KO; B) mice. Effect of azilsartan on learning ability using the area under the curve of days 1 to 5 (C). Escape latency decreased in bilateral common carotid artery stenosis (BCAS)–operated mice, and this decrease was improved by azilsartan treatment. n ¼ 5–6 for each group. *P < .05 vs. sham–operated mice.
(Figure 4A and B). TNF–a and MCP–1 mRNA expression were significantly increased after BCAS operation, but significantly attenuated by C21 treatment. We observed that expression of TNF–a in the brain was decreased by treatment with azilsartan, whereas this decrease was attenuated by co–treatment with PD123319 (data not shown).
Discussion Vascular dementia is induced by a reduction of cerebral blood flow and is characterized by white matter changes. A
chronic cerebral hypoperfusion mouse model induced by BCAS is widely used as a model of vascular dementia.12 Previous studies demonstrated that chronic cerebral hypoperfusion induced white matter lesions and cognitive impairment through a decrease in cerebral blood flow and increases in inflammatory cytokines and apoptosis.11,13 In this study, we showed that an ARB, azilsartan, prevented cognitive decline derived from chronic cerebral hypoperfusion. This preventive effect of ARB was attenuated in AT2KO mice, suggesting that this effect of azilsartan may involve AT2 receptor stimulation with blockade of the
Table 1 Effects of bilateral common carotid artery stenosis (BCAS) or compound 21 (C21) treatment on body and brain weights, and systolic blood pressure in before and after treatment Treatment
Sham BCAS BCAS þ C21
Body Weight, g
Systolic Blood Pressure, mm Hg
Before
After
Before
After
24.0 0.2 23.8 0.3 23.9 0.3
27.3 0.4 26.3 0.8 26.2 0.3
93.7 2.0 93.7 1.5 94.1 1.2
94.7 1.1 97.4 2.0 94.0 0.9
Brain Weight, g
Brain/Body Weight Ratio, %
0.45 0.003 0.44 0.008 0.43 0.006
1.64 0.03 1.69 0.04 1.65 0.03
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Figure 2. Effects of administration of compound 21 (C21) on escape latency (A) and platform area crossings (B) determined by Morris water maze test. Escape latency and number of platform area crossings were decreased after bilateral common carotid artery stenosis (BCAS), and these decreases were attenuated by C21 administration. n ¼ 7–10 for each group. *P < .05 vs. sham–operated mice.
AT1 receptor. Moreover, we observed that co–treatment with PD123319 attenuated the effect of azilsartan on improving cognitive decline in the BCAS–treated mice (data not shown). Accordingly, we examined the effects of direct AT2 receptor stimulation in a chronic cerebral hypoperfusion mouse model. We demonstrated that direct AT2 receptor stimulation by C21 improved cognitive decline in the BCAS mouse model, possibly through multiple pathways at least due to prevention of CBF reduction, and a
Figure 3. Effect of administration of compound 21 (C21) on cerebral blood flow (CBF). CBF was measured with a two– dimensional blood flow meter. Representative images of each groups are indicated (A). CBF was decreased after bilateral common carotid artery stenosis (BCAS), and this decrease was attenuated by C21 administration (B). n ¼ 7–10 for each group.
decrease in inflammatory cytokine expression. Treatment with C21 did not influence the cognitive decline in BCAS–treated AT2KO mice (Figure S4). Since protection from neuronal apoptosis is a potential link between inflammation and cognitive function. Therefore, we examined the effect of C21 on the possible apoptosis in hippocampus. TUNEL staining showed there were no significant differences in the number of apoptotic cells in CA1 region of hippocampus 6 weeks after BCAS among three groups (data not shown). We also examined the cell number by staining the samples with hematoxylin–eosin. We observed that in CA1 region of hippocampus, cell number did not differ among three groups. Moreover, we did not observe karyopyknosis in BCAS–treated mice. Consistent with this, we recently reported that treatment with C21 enhanced cognitive function in C57BL6 mice and improved cognitive decline in a mouse Alzheimer disease model, with increased CBF and neurite outgrowth.9 Recently, we and others reported that C21 promotes neurite elongation in primary cultured hippocampal neurons.9,14 Shraim et al suggested minimal passage of C21 to the striatum.15 However, it is reported that the BBB is disrupted in vascular cognitive impairment.16,17 In this mouse model, it is reported that chronic cerebral hypoperfusion induces disruption of the BBB.18 Therefore, we can expect that C21 protects against neural damage and promotes neuron development. NO in the hippocampus is known to play a role in synaptic plasticity.19,20 We reported previously that the increases in cerebral blood flow and neurite outgrowth by C21 treatment were inhibited by co–administration of icatibant, a bradykinin B2 receptor antagonist.9 Moreover, it is reported that AT2 receptor stimulation increases angiogenesis in hypoxia condition through AT2 receptor–bradykinin–NO pathway.21,22 Recently, Alhusban et al reported that treatment with C21 increased vascular density after stroke.23 We speculate that the increase in cerebral blood flow in
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Figure 4. Effect of administration of compound 21 (C21) on inflammatory cytokines, tumor necrosis factor–a (TNF–a; A) and monocyte chemoattractant protein-1 (MCP–1; B). mRNA expression of TNF–a and MCP–1 in the brain were measured by real–time reverse transcription polymerase chain reaction (RT–PCR). mRNA expression of TNF–a and MCP–1 were increased after bilateral common carotid artery stenosis (BCAS), and these increases were attenuated by C21 administration. n ¼ 5–6.
C21–treated mice after BCAS was at least partly due to an increase in vasodilation and angiogenesis. Taken together, our results indicate that direct AT2 stimulation by C21 prevents ischemic vascular dementia induced by hypoperfusion at least in part through an increase in CBF and a reduction of inflammation. We expect that direct AT2 receptor stimulation could be a new therapeutic strategy for preventing and treating vascular dementia.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jash.2015.01.010.
References 1. Gorelick PB, Scuteri A, Black SE, Decarli C, Greenberg SM, Iadecola C, et al; American Heart Association Stroke Council, Council on Epidemiology and Prevention, Council on Cardiovascular Nursing, Council on Cardiovascular Radiology and Intervention, and Council on Cardiovascular Surgery and Anesthesia. Vascular Contributions to Cognitive Impairment and Dementia: A Statement for Healthcare Professionals from the American Heart Association/American Stroke Association. Stroke 2011;42:2672–713. 2. Roh JH, Lee JH. Recent updates on subcortical ischemic vascular dementia. J Stroke 2014;16:18–26. 3. Li NC, Lee A, Whitmer RA, Kivipelto M, Lawler E, Kazis LE, et al. Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis. BMJ 2010;340:b5465.
4. Davies NM, Kehoe PG, Ben-Shlomo Y, Martin RM. Associations of anti-hypertensive treatments with Alzheimer’s disease, vascular dementia, and other dementias. J Alzheimers Dis 2011;26:699–708. 5. Fuentes B, Fernandez-Domınguez J, OrtegaCasarrubios MA, SanJose B, Martınez-Sanchez P, Dıez-Tejedor E. Treatment with angiotensin receptor blockers before stroke could exert a favourable effect in acute cerebral infarction. J Hypertens 2010;28:575–81. 6. Horiuchi M, Mogi M. Role of angiotensin II receptor subtype activation in cognitive function and ischaemic brain damage. Br J Pharmacol 2011;163:1122–30. 7. Wan Y, Wallinder C, Plouffe B, Beaudry H, Mahalingam AK, Wu X, et al. Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist. J Med Chem 2004;47:5995–6008. 8. Unger T, Dahlof B. Compound 21, the first orally active, selective agonist of the angiotensin type 2 receptor (AT2): implications for AT2 receptor research and therapeutic potential. J Renin Angiotensin Aldosterone Syst 2010;11:75–7. 9. Jing F, Mogi M, Sakata A, Iwanami J, Tsukuda K, Ohshima K, et al. Direct stimulation of angiotensin II type 2 receptor enhances spatial memory. J Cereb Blood Flow Metab 2012;32:248–55. 10. Sakata A, Mogi M, Iwanami J, Tsukuda K, Min LJ, Fujita T, et al. Sex different effect of angiotensin II type 2 receptor on ischemic brain injury and cognitive function. Brain Res 2009;1300:14–23. 11. Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke 2004; 35:2598–603. 12. Ihara M, Tomimoto H. Lessons from a mouse model characterizing features of vascular cognitive impairment
256
13.
14.
15.
16.
17.
J. Iwanami et al. / Journal of the American Society of Hypertension 9(4) (2015) 250–256
with white matter changes. J Aging Res 2011;2011: 978761. Washida K, Ihara M, Nishio K, Fujita Y, Maki T, Yamada M, et al. Nonhypotensive dose of telmisartan attenuates cognitive impairment partially due to peroxisome proliferator activated receptor gamma activation in mice with chronic cerebral hypoperfusion. Stroke 2010;41:1798–806. Namsolleck P, Boato F, Schwengel K, Paulis L, Matho KS, Geurts N, et al. AT2 receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating BDNF expression. Neurobiol Dis 2013; 51:177–91. Shraim N, Mertens B, Clinckers R, Sarre S, Michotte Y, Van Eeckhaut A. Microbore liquid chromatography with UV detection to study the in vivo passage of compound 21, a non-peptidergic AT2 receptor agonist, to the striatum in rats. J Neurosci Methods 2011;202:137–42. Yang Y, Rosengerg G. Blood-brain barrier breakdown in acute and chronic cerebrovascular disease. Stroke 2011;42:3323–8. Rosenberg G. Neurological diseases in relation to the blood brain barrier. J Cereb Blood Flow Metab 2012; 32:1139–51.
18. Nakaji K, Ihara M, Takahashi C, Itohara S, Moda M, Takahashi R, et al. Matrix metalloproteinase-2 plays a critical role in the pathogenesis of white matter lesions after chronic cerebral hypoperfusion in rodents. Stroke 2006;37:2816–23. 19. Bon CL, Garthwaite J. On the role of nitric oxide in hippocampal long term potentiation. J Neurosci 2003; 23:1941–8. 20. Hopper RA, Garthwaite J. Tonic and phasic nitric oxide signals in hippocampal long term potentiation. J Neurosci 2006;26:11513–21. 21. Munk VC, Sanchez de Miguel L, Petrimpol M, Butz N, Banfi A, Eriksson U, et al. Angiotensin II induces angiogenesis in the hypoxic adult mouse heart in vitro through an AT2-B2 receptor pathway. Hypertension 2007;49: 1178–85. 22. Li P, Kondo T, Numaguchi Y, Kobayashi K, Aoki M, Inoue N, et al. Role of bradykinin, nitric oxide, and angiotensin II type 2 receptor in imidapril-induced angiogenesis. Hypertension 2008;51:252–8. 23. Alhusban A, Fouda AY, Bindu Pillai, Ishrat T, Soliman S, Fagan SC. Compound 21 is pro-angiogenic in the brain and results in sustained recovery after ischemic stroke. J Hypertens 2015;33:170–80.
Research Article
Direct angiotensin II type 2 receptor stimulation by compound 21 prevents vascular dementia Jun Iwanami, PhD, Masaki Mogi, MD, PhD, Kana Tsukuda, BS, Xiao-Li Wang, MD, Hirotomo Nakaoka, MS, Harumi Kan-no, MS, Toshiyuki Chisaka, MD, Hui-Yu Bai, MD, Bao-Shuai Shan, MD, Masayoshi Kukida, MD, and Masatsugu Horiuchi, MD, PhD* Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Tohon, Ehime, Japan Manuscript received August 19, 2014 and accepted January 16, 2015
Abstract Angiotensin II type 2 (AT2) receptor activation has been reported to play a role in cognitive function, although its detailed mechanisms and pathologic significance are not fully understood. We examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) could prevent cognitive decline associated with hypoperfusion in the brain.We employed a bilateral common carotid artery stenosis (BCAS) model in mice as a model of vascular dementia. The Morris water maze task was performed 6 weeks after BCAS operation. Azilsartan (0.1 mg/kg/day) or C21 (10 mg/kg/day) was administered from 1 week before BCAS. Cerebral blood flow (CBF) and inflammatory cytokine levels were also determined. Wild– type (WT) mice showed significant prolongation of escape latency after BCAS, and this cognitive impairment was attenuated by pretreatment with azilsartan. Cognitive impairment was more marked in AT2 receptor knockout (AT2KO) mice, and the preventive effect of azilsartan on cognitive decline was weaker in AT2KO mice than in WT mice, suggesting that the improvement of cognitive decline by azilsartan may involve stimulation of the AT2 receptor. The significant impairment of spatial learning after BCAS in WT mice was attenuated by C21 treatment. The decrease in CBF in the BCAS–treated group was blunted by C21 treatment, and the increase in TNF–a and MCP–1 mRNA expression after BCAS was attenuated by C21 treatment. These findings indicate that direct AT2 receptor stimulation attenuates ischemic vascular dementia induced by hypoperfusion at least in part through an increase in CBF, and a reduction of inflammation. J Am Soc Hypertens 2015;9(4):250–256. Ó 2015 American Society of Hypertension. All rights reserved. Keywords: Cerebral blood flow; cognitive function; inflammation; renin-angiotensin system.
Introduction Cardiovascular risk factors such as hypertension, diabetes, and hypercholesterolemia are known to be associated with the onset and progression of dementia, including Alzheimer disease. Vascular cognitive impairment is related to aging and doubles every 5.3 years.1 Vascular dementia is classified into six groups.2 Multi–infarction dementia, Dr Horiuchi received research support and lecturing fees from Takeda Pharmaceutical Company Ltd. This study was supported by JSPS KAKENHI Grant Numbers 25293310 (MH), 25462220 (MM), and 24791506 (JI), and research grants from pharmaceutical companies: Astellas Pharma Inc, Bayer Yakuhin, Ltd, Daiichi-Sankyo Pharmaceutical Co, Ltd, Nippon Boehringer lngelheim Co, Ltd, Novartis Pharma KK, Shionogi & Co., Ltd., and Takeda Pharmaceutical Co, Ltd.
strategic infarction dementia and hemorrhagic dementia, which develop relatively suddenly, mixed subcortical ischemic vascular dementia, and other forms of vascular dementia. Chronic cerebral hypoperfusion is known to increase inflammation and oxidative stress, disrupt the blood–brain barrier (BBB), and induce the development and progression of white matter lesion and disruption.
*Corresponding author: Masatsugu Horiuchi, MD, PhD, Department of Molecular Cardiovascular Biology and Pharmacology, Ehime University, Graduate School of Medicine, Shitsukawa, Tohon, Ehime 791-0295, Japan. Tel: þ 81-89-960-5248; Fax: þ 81-89-960-5251. E-mail: [email protected]
1933-1711/$ - see front matter Ó 2015 American Society of Hypertension. All rights reserved. http://dx.doi.org/10.1016/j.jash.2015.01.010
J. Iwanami et al. / Journal of the American Society of Hypertension 9(4) (2015) 250–256
Pharmacologic targeting against the renin–angiotensin system (RAS) is one of the most effective ways to intervene in the pathogenesis of hypertension and diabetes. Li et al reported that angiotensin II type 1 (AT1) receptor blockers (ARBs) were associated with a significant reduction in the incidence and progression of Alzheimer disease and dementia compared with angiotensin–converting enzyme (ACE) inhibitors and other cardiovascular drugs in a population of 819,491 predominantly male participants aged 65 years or more with cardiovascular disease.3 Similar results were reported by Davies et al, who conducted a nested case–control analysis within the UK general practice research database.4 Fuentes et al reported that patients taking antihypertensive drugs at stroke onset had a lower rate of poor outcome than those not on antihypertensive treatment, and those taking an ARB had better outcomes than those without an ARB, with no difference in the analysis of other antihypertensive drugs including ACE inhibitors.5 Angiotensin II type 2 (AT2) receptor stimulation by unbound angiotensin II could also be expected during treatment with ARBs, and there is recent accumulating evidence suggesting that the AT2 receptor not only opposes the AT1 receptor, but also has unique effects beyond interaction with AT1 receptor signaling.6 These results suggest the idea that both AT1 receptor blockade and AT2 receptor stimulation by unbound angiotensin II are important in the neuroprotective effect of ARBs. AT2 receptor agonists such as compound 21 (C21) have been newly developed and are expected to be useful agents for improving pathologic disorders. This compound also enables us to examine AT2 receptor actions in vitro and in vivo by direct receptor stimulation, and principally offers the possibility to use AT2 receptor stimulation as a therapeutic tool.7,8 Our recent study also pointed out that direct AT2 receptor stimulation with C21 enhanced cognitive function in both normal mice and an Alzheimer disease mouse model through increases in cerebral blood flow and neurite outgrowth.9 Therefore, we investigated the effect of direct AT2 receptor stimulation by C21 on cognitive function using a mouse chronic cerebral hypoperfusion model.
Methods This study was performed in accordance with the National Institutes of Health guidelines for the use of experimental animals. All animal studies were reviewed and approved by the Animal Studies Committee of Ehime University.
Animals Adult male C57BL/6 mice (Clea Japan Inc, Tokyo, Japan) as wild–type (WT) mice and AT2 receptor knockout mice
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(AT2KO mice; based on C57BL/6J strain)10 (23–25 g; 10 weeks old) were used. Mice were housed in a room in which lighting was controlled (12 hours on, 12 hours off), and the temperature was kept at 25 C. They were given a standard diet (MF, Oriental Yeast, Tokyo, Japan) and water ad libitum. They were given control chow or chow containing azilsartan (0.1 mg/kg/day) from 1 week before induction of bilateral common carotid artery stenosis (BCAS) until the cognitive task. An AT2 receptor agonist, C21 (10 mg/kg/day; provided by Vicore Pharma, Gothenburg, Sweden), was administered daily by intraperitoneal injection from 1 week before BCAS operation, and C21 administration was kept until the brain sample was finally taken. The cognitive task was performed 1 hour after injection of C21. BCAS was induced by a microcoil technique.11 We used microcoil with inner diameters of 0.18 mm and total length of 2.5 mm (Sawane Spring Co, Hamamatsu, Japan). Mice were anesthesized with 65 mg/kg nembutal in saline. A middle incision was made in the neck, and the bilateral common carotid artery was isolated. Silk suture was placed around the right common carotid artery (CCA). Then, the CCA was gently lifted by suture and placed between the loops of the microcoil. The microcoil was trinned by rotating it around the CCA. After that, another microcoil was twined around the left CCA in the same way. The microcoil’s inside diameter of 0.18 mm resulted in a stenosis of about 50%, because the outside diameter of the common carotid artery was 0.35-0.40 mm under anesthesia. We checked the cerebral blood flow before and just after BCAS operation. Cerebral blood flow just after was decreased to 60%–70%. Systolic blood pressure was measured in conscious mice by the tail-cuff method (MK-1030; Muromachi Co, Tokyo, Japan) 6 weeks after the BCAS operation.
Morris Water Maze Test The Morris water maze task was performed in mice 6 weeks after the BCAS operation as previously described.10 A white circular tank (120 cm diameter) was filled with water (23 2 C). A transparent platform (a 6 cm 6 cm acrylic board) was placed 1.5 cm below the surface of the water. Four objects in the corners of the pool helped mice to know their position. After they were placed on the platform for 10 seconds, they were put into the water. After reaching the platform, they were returned to their cages. If they did not reach the platform within 120 seconds, they were placed on it, kept for 10 seconds, and returned to their cages. Mice were trained five times a day at 20–minute intervals for five consecutive days. In each trial, mice were given 120 seconds to find the platform. Swimming was video–tracked (AnyMaze, Wood Dale, IL), and latency, path length, swim speed, and cumulative distance from the platform were recorded. Mean swim latency each day was evaluated and compared between groups. The area under the curve of days 1–5 was quantified using
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computer–imaging software (Densitograph; ATTO Corporation, Tokyo, Japan). All analyses were performed by an investigator blinded to the experimental conditions.
Measurement of Cerebral Blood Flow After the Morris water maze task, cerebral blood flow (CBF) was measured by laser speckle flowmetry (Omegazone laser speckle blood flow imager; Omegawave, Tokyo, Japan), which obtains high–resolution 2D images in a matter of seconds as previously described.9 Mice were anesthetized with nembutal, and a midline incision was made in the scalp. Anesthesia did not significantly affect blood pressure. The skull was exposed and wet with saline. A 780–nm laser semiconductor laser illuminated the whole skull surface. Mean CBF on the skull surface was measured. Light intensity was accumulated in a charge–coupled device camera and transferred to a computer for analysis. Image pixels were analyzed to produce average perfusion values.
Real–time Reverse Transcription Polymerase Chain Reaction (RT–PCR) Method Samples of cerebral cortex were frozen in liquid nitrogen and stored at 80 C until analysis. Total mRNA was extracted from brain samples after homogenization in Sepazol (Nacalai Tesque Inc, Kyoto, Japan). Quantitative real– time RT–PCR was performed with a SYBR green kit (MJ Research, Inc, Waltham, MA). PCR primers were as follows: 50 -CGAGTGACAAGCCTGTAGCC-30 (forward) and 50 -GGTGAGGAGCACGATGTCG-30 (reverse) for tumor necrosis factor (TNF)–a, 50 -TTAACGCCCCACT CACCTGCTG-30 (forward) and 50 -GCTTCTTTGGGA CACCTGCTGC-30 (reverse) for monocyte chemoattractant protein-1 (MCP-1), and 50 -ATGTAGGCCATGAGGTCCA C-30 (forward) and 50 -TGCGACTTCAACAGCAACTC-30 (reverse) for glyceraldehyde–3–phosphate dehydrogenase (GAPDH).
Statistical Analysis All values are expressed as mean standard error of the mean in the text and figures. Data were evaluated by analysis of variance followed by post hoc analysis for multiple comparisons. A difference with P < .05 was considered significant.
Results Preventive Effect of ARB on Decrease in Spatial Learning after Bilateral Common Carotid Artery Stenosis and Possible Involvement of AT2 Receptor To examine the possible protective effects of ARBs on cognitive decline in vascular dementia, we employed a
BCAS mouse model. The Morris water maze task was performed 6 weeks after the BCAS operation. WT mice showed significant prolongation of escape latency assessed by Morris water maze test compared with mice without BCAS (Figure 1A). This cognitive impairment was attenuated by pretreatment with azilsartan at a dose of 0.1 mg/kg/ day, without a change in systolic blood pressure. To investigate the possible involvement of AT2 receptor stimulation in the preventive effect of azilsartan on the cognitive decline associated with BCAS, we employed AT2 receptor–null mice. Body weight and systolic blood pressure did not differ between both strains before or after BCAS (data not shown). There was no significant difference in spatial learning memory between and AT2KO and WT mice without BCAS, although spacial learning in AT2KO mice tended to be impaired. Prolongation of escape latency after BCAS was more marked in AT2KO mice (Figure 1A and B). Interestingly, the preventive effect of azilsartan on cognitive decline was weaker in AT2 KO mice than in WT mice (Figure 1C). These results suggested that the improvement of cognitive decline by azilsartan may involve stimulation of the AT2 receptor. Therefore, we examined the effects of direct AT2 receptor stimulation on cognitive decline after BCAS and its mechanism.
Preventive Effect of Direct AT2 Receptor Stimulation by Compound 21 on Decrease in Spatial Learning Treatment with C21 was started 1 week before BCAS. There were no significant differences in body weight and systolic blood pressure among all groups before start of treatments. Administration of C21 did not influence body and brain weights or systolic blood pressure (Table 1). Mice showed significant prolongation of escape latency after BCAS, as shown in Figure 1A, and this prolongation of escape latency was significantly attenuated by C21 treatment (Figure 2A). Path length and cumulative distance were increased in BCAS–treated mice, and this increase was attenuated by treatment with C21 (Figures S1 and S2). There was no significant differences in swim speed among three groups (Figure S3). In the probe trials, treatment with C21 prevented the decrease in the number of platform area crossings induced by the BCAS operation (Figure 2B).
Increase in Cerebral Blood Flow and Decrease in Inflammation by Direct AT2 Receptor Stimulation by Compound 21 Cerebral blood flow in the whole brain in the BCAS– treated group was significantly decreased compared with that in the sham group at 6 weeks after BCAS operation (Figure 3). This decrease was increased by C21 treatment. Next, we assessed expression of inflammatory cytokines such as TNF–a and MCP–1 in the cerebral cortex
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Figure 1. Effect of administration of azilsartan on escape latency in wild type (WT; A) and Angiotensin II type 2 knockout (AT2KO; B) mice. Effect of azilsartan on learning ability using the area under the curve of days 1 to 5 (C). Escape latency decreased in bilateral common carotid artery stenosis (BCAS)–operated mice, and this decrease was improved by azilsartan treatment. n ¼ 5–6 for each group. *P < .05 vs. sham–operated mice.
(Figure 4A and B). TNF–a and MCP–1 mRNA expression were significantly increased after BCAS operation, but significantly attenuated by C21 treatment. We observed that expression of TNF–a in the brain was decreased by treatment with azilsartan, whereas this decrease was attenuated by co–treatment with PD123319 (data not shown).
Discussion Vascular dementia is induced by a reduction of cerebral blood flow and is characterized by white matter changes. A
chronic cerebral hypoperfusion mouse model induced by BCAS is widely used as a model of vascular dementia.12 Previous studies demonstrated that chronic cerebral hypoperfusion induced white matter lesions and cognitive impairment through a decrease in cerebral blood flow and increases in inflammatory cytokines and apoptosis.11,13 In this study, we showed that an ARB, azilsartan, prevented cognitive decline derived from chronic cerebral hypoperfusion. This preventive effect of ARB was attenuated in AT2KO mice, suggesting that this effect of azilsartan may involve AT2 receptor stimulation with blockade of the
Table 1 Effects of bilateral common carotid artery stenosis (BCAS) or compound 21 (C21) treatment on body and brain weights, and systolic blood pressure in before and after treatment Treatment
Sham BCAS BCAS þ C21
Body Weight, g
Systolic Blood Pressure, mm Hg
Before
After
Before
After
24.0 0.2 23.8 0.3 23.9 0.3
27.3 0.4 26.3 0.8 26.2 0.3
93.7 2.0 93.7 1.5 94.1 1.2
94.7 1.1 97.4 2.0 94.0 0.9
Brain Weight, g
Brain/Body Weight Ratio, %
0.45 0.003 0.44 0.008 0.43 0.006
1.64 0.03 1.69 0.04 1.65 0.03
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Figure 2. Effects of administration of compound 21 (C21) on escape latency (A) and platform area crossings (B) determined by Morris water maze test. Escape latency and number of platform area crossings were decreased after bilateral common carotid artery stenosis (BCAS), and these decreases were attenuated by C21 administration. n ¼ 7–10 for each group. *P < .05 vs. sham–operated mice.
AT1 receptor. Moreover, we observed that co–treatment with PD123319 attenuated the effect of azilsartan on improving cognitive decline in the BCAS–treated mice (data not shown). Accordingly, we examined the effects of direct AT2 receptor stimulation in a chronic cerebral hypoperfusion mouse model. We demonstrated that direct AT2 receptor stimulation by C21 improved cognitive decline in the BCAS mouse model, possibly through multiple pathways at least due to prevention of CBF reduction, and a
Figure 3. Effect of administration of compound 21 (C21) on cerebral blood flow (CBF). CBF was measured with a two– dimensional blood flow meter. Representative images of each groups are indicated (A). CBF was decreased after bilateral common carotid artery stenosis (BCAS), and this decrease was attenuated by C21 administration (B). n ¼ 7–10 for each group.
decrease in inflammatory cytokine expression. Treatment with C21 did not influence the cognitive decline in BCAS–treated AT2KO mice (Figure S4). Since protection from neuronal apoptosis is a potential link between inflammation and cognitive function. Therefore, we examined the effect of C21 on the possible apoptosis in hippocampus. TUNEL staining showed there were no significant differences in the number of apoptotic cells in CA1 region of hippocampus 6 weeks after BCAS among three groups (data not shown). We also examined the cell number by staining the samples with hematoxylin–eosin. We observed that in CA1 region of hippocampus, cell number did not differ among three groups. Moreover, we did not observe karyopyknosis in BCAS–treated mice. Consistent with this, we recently reported that treatment with C21 enhanced cognitive function in C57BL6 mice and improved cognitive decline in a mouse Alzheimer disease model, with increased CBF and neurite outgrowth.9 Recently, we and others reported that C21 promotes neurite elongation in primary cultured hippocampal neurons.9,14 Shraim et al suggested minimal passage of C21 to the striatum.15 However, it is reported that the BBB is disrupted in vascular cognitive impairment.16,17 In this mouse model, it is reported that chronic cerebral hypoperfusion induces disruption of the BBB.18 Therefore, we can expect that C21 protects against neural damage and promotes neuron development. NO in the hippocampus is known to play a role in synaptic plasticity.19,20 We reported previously that the increases in cerebral blood flow and neurite outgrowth by C21 treatment were inhibited by co–administration of icatibant, a bradykinin B2 receptor antagonist.9 Moreover, it is reported that AT2 receptor stimulation increases angiogenesis in hypoxia condition through AT2 receptor–bradykinin–NO pathway.21,22 Recently, Alhusban et al reported that treatment with C21 increased vascular density after stroke.23 We speculate that the increase in cerebral blood flow in
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Figure 4. Effect of administration of compound 21 (C21) on inflammatory cytokines, tumor necrosis factor–a (TNF–a; A) and monocyte chemoattractant protein-1 (MCP–1; B). mRNA expression of TNF–a and MCP–1 in the brain were measured by real–time reverse transcription polymerase chain reaction (RT–PCR). mRNA expression of TNF–a and MCP–1 were increased after bilateral common carotid artery stenosis (BCAS), and these increases were attenuated by C21 administration. n ¼ 5–6.
C21–treated mice after BCAS was at least partly due to an increase in vasodilation and angiogenesis. Taken together, our results indicate that direct AT2 stimulation by C21 prevents ischemic vascular dementia induced by hypoperfusion at least in part through an increase in CBF and a reduction of inflammation. We expect that direct AT2 receptor stimulation could be a new therapeutic strategy for preventing and treating vascular dementia.
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jash.2015.01.010.
References 1. Gorelick PB, Scuteri A, Black SE, Decarli C, Greenberg SM, Iadecola C, et al; American Heart Association Stroke Council, Council on Epidemiology and Prevention, Council on Cardiovascular Nursing, Council on Cardiovascular Radiology and Intervention, and Council on Cardiovascular Surgery and Anesthesia. Vascular Contributions to Cognitive Impairment and Dementia: A Statement for Healthcare Professionals from the American Heart Association/American Stroke Association. Stroke 2011;42:2672–713. 2. Roh JH, Lee JH. Recent updates on subcortical ischemic vascular dementia. J Stroke 2014;16:18–26. 3. Li NC, Lee A, Whitmer RA, Kivipelto M, Lawler E, Kazis LE, et al. Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis. BMJ 2010;340:b5465.
4. Davies NM, Kehoe PG, Ben-Shlomo Y, Martin RM. Associations of anti-hypertensive treatments with Alzheimer’s disease, vascular dementia, and other dementias. J Alzheimers Dis 2011;26:699–708. 5. Fuentes B, Fernandez-Domınguez J, OrtegaCasarrubios MA, SanJose B, Martınez-Sanchez P, Dıez-Tejedor E. Treatment with angiotensin receptor blockers before stroke could exert a favourable effect in acute cerebral infarction. J Hypertens 2010;28:575–81. 6. Horiuchi M, Mogi M. Role of angiotensin II receptor subtype activation in cognitive function and ischaemic brain damage. Br J Pharmacol 2011;163:1122–30. 7. Wan Y, Wallinder C, Plouffe B, Beaudry H, Mahalingam AK, Wu X, et al. Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist. J Med Chem 2004;47:5995–6008. 8. Unger T, Dahlof B. Compound 21, the first orally active, selective agonist of the angiotensin type 2 receptor (AT2): implications for AT2 receptor research and therapeutic potential. J Renin Angiotensin Aldosterone Syst 2010;11:75–7. 9. Jing F, Mogi M, Sakata A, Iwanami J, Tsukuda K, Ohshima K, et al. Direct stimulation of angiotensin II type 2 receptor enhances spatial memory. J Cereb Blood Flow Metab 2012;32:248–55. 10. Sakata A, Mogi M, Iwanami J, Tsukuda K, Min LJ, Fujita T, et al. Sex different effect of angiotensin II type 2 receptor on ischemic brain injury and cognitive function. Brain Res 2009;1300:14–23. 11. Shibata M, Ohtani R, Ihara M, Tomimoto H. White matter lesions and glial activation in a novel mouse model of chronic cerebral hypoperfusion. Stroke 2004; 35:2598–603. 12. Ihara M, Tomimoto H. Lessons from a mouse model characterizing features of vascular cognitive impairment
256
13.
14.
15.
16.
17.
J. Iwanami et al. / Journal of the American Society of Hypertension 9(4) (2015) 250–256
with white matter changes. J Aging Res 2011;2011: 978761. Washida K, Ihara M, Nishio K, Fujita Y, Maki T, Yamada M, et al. Nonhypotensive dose of telmisartan attenuates cognitive impairment partially due to peroxisome proliferator activated receptor gamma activation in mice with chronic cerebral hypoperfusion. Stroke 2010;41:1798–806. Namsolleck P, Boato F, Schwengel K, Paulis L, Matho KS, Geurts N, et al. AT2 receptor stimulation enhances axonal plasticity after spinal cord injury by upregulating BDNF expression. Neurobiol Dis 2013; 51:177–91. Shraim N, Mertens B, Clinckers R, Sarre S, Michotte Y, Van Eeckhaut A. Microbore liquid chromatography with UV detection to study the in vivo passage of compound 21, a non-peptidergic AT2 receptor agonist, to the striatum in rats. J Neurosci Methods 2011;202:137–42. Yang Y, Rosengerg G. Blood-brain barrier breakdown in acute and chronic cerebrovascular disease. Stroke 2011;42:3323–8. Rosenberg G. Neurological diseases in relation to the blood brain barrier. J Cereb Blood Flow Metab 2012; 32:1139–51.
18. Nakaji K, Ihara M, Takahashi C, Itohara S, Moda M, Takahashi R, et al. Matrix metalloproteinase-2 plays a critical role in the pathogenesis of white matter lesions after chronic cerebral hypoperfusion in rodents. Stroke 2006;37:2816–23. 19. Bon CL, Garthwaite J. On the role of nitric oxide in hippocampal long term potentiation. J Neurosci 2003; 23:1941–8. 20. Hopper RA, Garthwaite J. Tonic and phasic nitric oxide signals in hippocampal long term potentiation. J Neurosci 2006;26:11513–21. 21. Munk VC, Sanchez de Miguel L, Petrimpol M, Butz N, Banfi A, Eriksson U, et al. Angiotensin II induces angiogenesis in the hypoxic adult mouse heart in vitro through an AT2-B2 receptor pathway. Hypertension 2007;49: 1178–85. 22. Li P, Kondo T, Numaguchi Y, Kobayashi K, Aoki M, Inoue N, et al. Role of bradykinin, nitric oxide, and angiotensin II type 2 receptor in imidapril-induced angiogenesis. Hypertension 2008;51:252–8. 23. Alhusban A, Fouda AY, Bindu Pillai, Ishrat T, Soliman S, Fagan SC. Compound 21 is pro-angiogenic in the brain and results in sustained recovery after ischemic stroke. J Hypertens 2015;33:170–80.
