Angiotensin II and Amyloid-ß Synergistically Induce Brain Vascular Smooth Muscle Cell Senescence.

BACKGROUND: Amyloid-ß (Aß) induces cerebrovascular damage and is reported to stimulate endothelial cell senescence. We previously demonstrated that angiotensin II (Ang II)-promoted vascular senescence. We examined the possible cross-talk between Ang II and Aß in regulating brain vascular smooth muscle cell (BVSMC) senescence. METHODS: BVSMCs were prepared from adult male mice and stimulated with Ang II (0, 0.1, 1, 10, and 100 nmol/l) and/or Aß 1-40 (0, 0.1, 0.3, 0.5, 1, 3, and 5 µmol/l) for the indicated times. Cellular senescence was evaluated by senescence-associated ß-galactosidase staining. RESULTS: Treatment with Ang II (100 nmol/l) or Aß (1 µmol/l) at a higher dose increased senescent cells compared with control at 6 days. Treatment with Ang II (10 nmol/l) or Aß (0.5 µmol/l) at a lower dose had no effect on senescence whereas a combined treatment with lower doses of Ang II and Aß significantly enhanced senescent cells. This senescence enhanced by lower dose combination was markedly blocked by valsartan (Ang II type 1 receptor inhibitor) or TAK-242 (Aß receptor TLR4 inhibitor) treatment. Moreover, lower dose combination caused increases in superoxide anion levels and p-ERK expression for 2 days, NF-κB activity, p-IκB, p-IKKα/ß, p16 and p53 expression for 4 days, and an obvious decrease in pRb expression. These changes by lower dose combination, except in p-IκB expression and NF-κB activity, were significantly inhibited by pretreatment with U0126 (ERK inhibitor). CONCLUSIONS: Ang II and Aß synergistically promoted BVSMC senescence at least due to enhancement of the p-ERK-p16-pRb signaling pathway, oxidative stress, and NF-κB/IκB activity.

Deterioration of cognitive function after transient cerebral ischemia with amyloid-ß infusion-possible amelioration of cognitive function by AT2 receptor activation.

BACKGROUND: To promote understanding of the pathogenesis of cognitive impairment or dementia, we explored the potential interaction between transient cerebral ischemia and amyloid-ß (Aß) infusion in mediating cognitive decline and examined the possible ameliorative effect of angiotensin II type 2 (AT2) receptor activation in vascular smooth muscle cells (VSMC) on this cognitive deficit. METHODS: Adult male wild-type mice (WT) and mice with VSMC-specific AT2 receptor overexpression (smAT2) were subjected to intracerebroventricular (ICV) injection of Aß1-40. Transient cerebral ischemia was induced by 15 min of bilateral common carotid artery occlusion (BCCAO) 24 h after Aß injection. RESULTS: Aß injection in WT induced a cognitive decline, whereas BCCAO did not cause a significant cognitive deficit. In contrast, WT with BCCAO following Aß injection exhibited more marked cognitive decline compared to Aß injection alone, in concert with increases in superoxide anion production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and expression of p22phox, p40phox, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1ß in the hippocampus, and upregulation of RAGE (receptor for advanced glycation end product), an Aß transporter. BCCAO following Aß injection further enhanced neuronal pyknosis in the hippocampus, compared with BCCAO or Aß injection alone. In contrast, smAT2 did not show a cognitive decline, increase in oxidative stress, inflammation, and RAGE level or neuronal pyknosis, which were induced by BCCAO with/without Aß injection in WT. CONCLUSIONS: Transient cerebral ischemia might worsen Aß infusion-mediated cognitive decline and vice versa, with possible involvement of amplified oxidative stress and inflammation and impairment of the RAGE-mediated Aß clearance system, contributing to exaggerated neuronal degeneration. AT2 receptor activation in VSMC could play an inhibitory role in this cognitive deficit.

AT2 receptor stimulation inhibits phosphate-induced vascular calcification.

Vascular calcification is a common finding in atherosclerosis and in patients with chronic kidney disease. The renin-angiotensin system plays a role in the pathogenesis of cardiovascular remodeling. Here, we examined the hypothesis that angiotensin II type 2 receptor (AT2) stimulation has inhibitory effects on phosphate-induced vascular calcification. In vivo, calcification of the thoracic aorta induced by an adenine and high-phosphate diet was markedly attenuated in smooth muscle cell-specific AT2-overexpressing mice (smAT2-Tg) compared with wild-type and AT2-knockout mice (AT2KO). Similarly, mRNA levels of relevant osteogenic and vascular smooth muscle cell marker genes were unchanged in smAT2-Tg mice, while their expression was significantly altered in wild-type mice in response to high dietary phosphate. Ex vivo, sections of thoracic aorta were cultured in media supplemented with inorganic phosphate. Aortic rings from smAT2-Tg mice showed less vascular calcification compared with those from wild-type mice. In vitro, calcium deposition induced by high-phosphate media was markedly attenuated in primary vascular smooth muscle cells derived from smAT2-Tg mice compared with the two other mouse groups. To assess the underlying mechanism, we investigated the effect of PPAR-γ, which we previously reported as one of the possible downstream effectors of AT2 stimulation. Treatment with a PPAR-γ antagonist attenuated the inhibitory effects on vascular calcification observed in smAT2-Tg mice fed an adenine and high-phosphate diet. Our results suggest that AT2 activation represents an endogenous protective pathway against vascular calcification. Its stimulation may efficiently reduce adverse cardiovascular events in patients with chronic kidney disease.

Deletion of interferon-regulatory factor-1 results in cognitive impairment.

Interferon-regulatory factor (IRF)-1-dependent genes in neurons play a role in ischemic neuronal death; however, the roles of IRF-1 in dementia are not well investigated. Therefore, we assessed the effect of IRF-1 on cognitive function using a vascular cognitive impairment mouse model created by chronic cerebral hypoperfusion. Male 10-week-old C57BL/6 (wild-type; WT) and IRF-1-knockout (IRF-1KO) mice were used in this study. A chronic cerebral hypoperfusion mouse model was generated by bilateral common carotid artery stenosis (BCAS) treatment. After 6 weeks of BCAS, the mice were subjected to the Morris water maze test five times a day for 5 days. In the Morris water maze task, escape latency was significantly prolonged in sham-operated IRF-1KO mice compared with sham-operated WT mice. However, BCAS treatment cancelled such difference in spatial learning between WT and IRF-1KO mice. BCAS treatment decreased CBF, but no significant difference was observed between the two strains after BCAS. Sham-operated IRF-1KO mice showed a decrease in mRNA expression of caspase-1 and an increase in IRF-2 expression in the hippocampus. Expression of angiotensin II type 2 (AT2) receptor, which induces better cognitive function, is regulated by IRF-1; however, no obvious difference in AT2 receptor expression was observed between the two strains even after BCAS. These results suggest that IRF-1 has a protective effect on cognitive decline in a normal condition; however, there was no obvious effect on cognition after chronic cerebral hypoperfusion treatment.

Attenuation of stroke damage by angiotensin II type 2 receptor stimulation via peroxisome proliferator-activated receptor-gamma activation.

The brain renin-angiotensin system plays a crucial role in ischemic stroke. It is known that stimulation of the angiotensin II type 2 (AT2) receptor protects against ischemic brain injury. We recently demonstrated that AT2 receptor stimulation by compound 21 (C21), a direct AT2 receptor agonist, inhibited vascular intimal proliferation with activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ). However, whether direct AT2 receptor stimulation protects against ischemic brain injury via PPAR-γ activation is still unknown. 8-week-old male C57BL/6 J mice were subjected to middle cerebral artery (MCA) occlusion. 2 weeks before MCA occlusion, they were administered C21 with or without GW9662, a PPAR-γ antagonist. Neurologic deficit, ischemic size, superoxide anion, superoxide dismutase (SOD) activity, expression of NADPH subunits and blood brain barrier (BBB) stabilization were assessed 24 h after MCA occlusion. Cerebral blood flow (CBF) was measured in the core and periphery of the MCA territory before, immediately after, 1 h and 24 h after MCA occlusion. Treatment with C21 markedly decreased the neurologic deficit and ischemic size with an increase in CBF, SOD activity and BBB stabilization genes compared with the non-treated group. Co-administration of GW9662 partially attenuated this protective effect of C21 on neurologic deficit and ischemic size via an increase in superoxide anion production and a decrease of SOD activity and BBB stabilization genes, while GW9662 treatment alone had no significant effect on neurologic deficit and ischemic size. These results suggest that direct AT2 receptor stimulation has a preventive effect on stroke-induced brain injury partly due to activation of PPAR-γ.

Recognition of early stage thigmotaxis in Morris water maze test with convolutional neural network.

The Morris water maze test (MWM) is a useful tool to evaluate rodents' spatial learning and memory, but the outcome is susceptible to various experimental conditions. Thigmotaxis is a commonly observed behavioral pattern which is thought to be related to anxiety or fear. This behavior is associated with prolonged escape latency, but the impact of its frequency in the early stage on the final outcome is not clearly understood. We analyzed swim path trajectories in male C57BL/6 mice with or without bilateral common carotid artery stenosis (BCAS) treatment. There was no significant difference in the frequencies of particular types of trajectories according to ischemic brain surgery. The mouse groups with thigmotaxis showed significantly prolonged escape latency and lower cognitive score on day 5 compared to those without thigmotaxis. As the next step, we made a convolutional neural network (CNN) model to recognize the swim path trajectories. Our model could distinguish thigmotaxis from other trajectories with 96% accuracy and specificity as high as 0.98. These results suggest that thigmotaxis in the early training stage is a predictive factor for impaired performance in MWM, and machine learning can detect such behavior easily and automatically.

Beneficial Effect of Mas Receptor Deficiency on Vascular Cognitive Impairment in the Presence of Angiotensin II Type 2 Receptor.

BACKGROUND: The classical renin-angiotensin system is known as the angiotensin (Ang)-converting enzyme/Ang II/Ang type 1 receptor axis, which induces various organ damage including cognitive decline. The angiotensin-converting enzyme 2/Ang-(1-7)/Mas axis is known to exert antagonistic actions against the classical renin-angiotensin system axis in the cardiovascular system. However, its roles in the brain remain unclear. We examined possible roles of the angiotensin-converting enzyme 2/Ang-(1-7)/Mas axis in cognitive function, employing vascular cognitive impairment model mice. METHODS AND RESULTS: Male 10-week-old C57BL6 (wild-type mice, Mas1 knockout mice, Ang II type 2 receptor knockout mice, and Ang II type 2 receptor/Mas1 double knockout mice were subjected to bilateral carotid artery stenosis (BCAS) surgery. Six weeks after treatment, they were subjected to cognitive tasks. Brain samples were used for histopathological analysis. Cognitive function was significantly impaired in wild-type and double knockout mice after BCAS. On the other hand, the cognitive function of Mas1 knockout mice was maintained in spite of the reduction of cerebral blood flow with BCAS. Total cell number in the dentate gyrus region was significantly reduced after BCAS in wild-type but not in Mas1 knockout mice. The number of doublecortin-positive cells in the subgranular zone was not significantly different between wild-type and Mas1 knockout mice. Ang-(1-7) administration did not improve cognitive function in all mice after BCAS surgery. CONCLUSIONS: Lack of the Mas receptor may have a protective effect against chronic brain ischemia when the Ang II type 2 receptor exists.

Predicting outcome of Morris water maze test in vascular dementia mouse model with deep learning.

The Morris water maze test (MWM) is one of the most popular and established behavioral tests to evaluate rodents' spatial learning ability. The conventional training period is around 5 days, but there is no clear evidence or guidelines about the appropriate duration. In many cases, the final outcome of the MWM seems predicable from previous data and their trend. So, we assumed that if we can predict the final result with high accuracy, the experimental period could be shortened and the burden on testers reduced. An artificial neural network (ANN) is a useful modeling method for datasets that enables us to obtain an accurate mathematical model. Therefore, we constructed an ANN system to estimate the final outcome in MWM from the previously obtained 4 days of data in both normal mice and vascular dementia model mice. Ten-week-old male C57B1/6 mice (wild type, WT) were subjected to bilateral common carotid artery stenosis (WT-BCAS) or sham-operation (WT-sham). At 6 weeks after surgery, we evaluated their cognitive function with MWM. Mean escape latency was significantly longer in WT-BCAS than in WT-sham. All data were collected and used as training data and test data for the ANN system. We defined a multiple layer perceptron (MLP) as a prediction model using an open source framework for deep learning, Chainer. After a certain number of updates, we compared the predicted values and actual measured values with test data. A significant correlation coefficient was derived form the updated ANN model in both WT-sham and WT-BCAS. Next, we analyzed the predictive capability of human testers with the same datasets. There was no significant difference in the prediction accuracy between human testers and ANN models in both WT-sham and WT-BCAS. In conclusion, deep learning method with ANN could predict the final outcome in MWM from 4 days of data with high predictive accuracy in a vascular dementia model.

Roles of angiotensin II type 2 receptor in mice with fetal growth restriction.

Our previous report indicated that vascular injury enhances vascular remodeling in fetal growth restriction (FGR) mice. The angiotensin II type 2 receptor (AT2R) is relatively highly expressed in fetal mice. Therefore, we investigated the roles of AT2R in FGR-induced cardiovascular disease using AT2R knockout (AT2KO) mice. Dams (wild-type and AT2KO mice) were fed an isocaloric diet containing 20% protein (NP) or 8% protein (LP) until delivery. Arterial blood pressure, body weight, and histological changes in organs were investigated in offspring. The birth weight of offspring from dams fed an LP diet (LPO) was significantly lower than that of offspring from dams fed an NP diet. The heart/body and kidney/body weight ratios in AT2KO-LPO at 12 weeks of age were significantly higher than those in the other groups. Greater thickness of the left ventricular wall, larger cardiomyocyte size and enhancement of perivascular fibrosis were observed in AT2KO-LPO. Interestingly, mRNA expression of collagen I and inflammatory cytokines was markedly higher in the AT2KO-LPO heart at 6 weeks of age but not at 12 weeks of age. AT2R signaling may be involved in cardiovascular disorders of adult offspring with FGR. Regulation of AT2R could contribute to preventing future cardiovascular disease in FGR offspring.

Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice.

There is a growing interest in identifying natural food ingredients that may serve to prevent dementia such as that due to Alzheimer disease (AD). Peptides derived from food proteins have been demonstrated to have various physiological activities such as a hypotensive action. Recent findings have indicated possible associations of hypertension with AD progression, and suggest that angiotensin converting enzyme (ACE) inhibitors with potential to pass through the blood brain barrier (BBB) may reduce the risk of AD. In this study, we investigated the effect of milk peptide (CH-3) on cognitive function in AD model mice. CH-3 contains a tripeptide (methionine-lysine-proline, MKP) that has been found to have a strong ACE inhibitory effect and the potential to pass through the BBB. Adult male ddY mice were used in this study, and an animal model of AD was induced by intracerebroventricular (ICV) injection of Aß1-42. CH-3 (250 mg/kg/day) or MKP (0.5 mg/kg/day) was orally administered every day starting 2 days before ICV injection. At 3 weeks after ICV injection, cognitive function was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and expression of inflammatory cytokines and NADPH oxidase subunits was measured by real-time quantitative RT-PCR. ICV injection of Aß1-42 significantly impaired cognitive function compared with that in PBS-injected mice. Daily administration of CH-3 markedly attenuated this Aß1-42-induced cognitive decline. Aß1-42 injection significantly enhanced the expression of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and p22phox in the mouse hippocampus compared with PBS injection, and showed a tendency to increase the expression of monocyte chemoattractant protein-1 (MCP-1), p47phox and gp91phox, whereas CH-3 treatment markedly reduced Aß1-42-induced TNF-α, MCP-1, iNOS, p47phox and gp91phox expression. Finally, administration of MKP also attenuated Aß1-42-induced cognitive impairment with an increase in cerebral blood flow. The present study demonstrated that repeated oral administration of CH-3 to AD model mice not only improved cognitive function but also suppressed the expression of inflammatory cytokines and production of oxidative stress, and suggests its therapeutic potential for preventing cognitive impairment in AD.

Enhancement of Adipocyte Browning by Angiotensin II Type 1 Receptor Blockade.

Browning of white adipose tissue (WAT) has been highlighted as a new possible therapeutic target for obesity, diabetes and lipid metabolic disorders, because WAT browning could increase energy expenditure and reduce adiposity. The new clusters of adipocytes that emerge with WAT browning have been named 'beige' or 'brite' adipocytes. Recent reports have indicated that the renin-angiotensin system (RAS) plays a role in various aspects of adipose tissue physiology and dysfunction. The biological effects of angiotensin II, a major component of RAS, are mediated by two receptor subtypes, angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R). However, the functional roles of angiotensin II receptor subtypes in WAT browning have not been defined. Therefore, we examined whether deletion of angiotensin II receptor subtypes (AT1aR and AT2R) may affect white-to-beige fat conversion in vivo. AT1a receptor knockout (AT1aKO) mice exhibited increased appearance of multilocular lipid droplets and upregulation of thermogenic gene expression in inguinal white adipose tissue (iWAT) compared to wild-type (WT) mice. AT2 receptor-deleted mice did not show miniaturization of lipid droplets or alteration of thermogenic gene expression levels in iWAT. An in vitro experiment using adipose tissue-derived stem cells showed that deletion of the AT1a receptor resulted in suppression of adipocyte differentiation, with reduction in expression of thermogenic genes. These results indicate that deletion of the AT1a receptor might have some effects on the process of browning of WAT and that blockade of the AT1 receptor could be a therapeutic target for the treatment of metabolic disorders.

Interferon regulatory factor 1 attenuates vascular remodeling; roles of angiotensin II type 2 receptor.

We previously reported interferon regulatory factor (IRF) 1 plays physiological roles in "growth"-regulated angiotensin II type 2 (AT2) receptor expression in fibroblasts. Here, we investigated whether IRF-1 is involved in attenuation of vascular remodeling in association with AT2 receptor upregulation. Neointimal area in injured artery after 14 days of cuff placement was significantly increased in IRF-1 knockout mice (IRF-1KO) and AT2 receptor knockout mice (AT2KO) compared with wild-type mice (WT: C57BL/6J). Treatment with compound 21 attenuated neointima formation in both WT and IRF-1KO. AT2 receptor mRNA expression after 7 days of cuff placement was significantly decreased in IRF-1KO compared with WT; however, IRF-1 expression did not differ between AT2KO and WT. Apoptotic changes in injured artery after 14 days of cuff placement were significantly attenuated in IRF-1KO, with a decrease in interleukin-1ß-converting enzyme and inducible nitric oxide synthase mRNA levels. These results indicate IRF-1 is one of the key transcriptional factors for the prevention of neointimal formation involving AT2 receptors.

Synergistic Inhibitory Effect of Rosuvastatin and Angiotensin II Type 2 Receptor Agonist on Vascular Remodeling.

We investigated the possibility that coadministration of rosuvastatin and compound 21 (C21), a selective angiotensin II type 2 (AT2) receptor agonist, could exert synergistic preventive effects on vascular injury. Vascular injury was induced by polyethylene cuff placement on the femoral artery in 9-week-old male C57BL/6J mice. Mice were treated with rosuvastatin and/or with C21 after cuff placement. Neointima formation was determined 14 days after the operation and cell proliferation, and superoxide anion production and expression of inflammatory cytokines were examined 7 days after cuff placement. Neointima formation was significantly attenuated by the treatment of rosuvastatin (5 mg kg(-1) day(-1)) or C21 (10 µg kg(-1) day(-1)), associated with the decreases in proliferating cell nuclear antigen (PCNA) labeling index, oxidative stress, and the expression of inflammatory markers. Treatment with a noneffective dose of rosuvastatin (0.5 mg kg(-1) day(-1)) plus a low dose of C21 (1 µg kg(-1) day(-1)) inhibited the PCNA labeling index, superoxide anion production, mRNA expressions of NAD(P)H subunits, and mRNA and protein expressions of inflammatory markers associated with marked inhibition of neointima formation. Angiotensin II type 1 (AT1) receptor mRNA expression did not differ the groups. By contrast, AT2 receptor mRNA expression was increased by administration of C21 at the dose of 10 µg kg(-1) day(-1) but not by C21 at the dose of 1 µg kg(-1) day(-1) or rosuvastatin. The combination of rosuvastatin and AT2 receptor agonist exerted synergistic preventive effects on vascular remodeling associated with the decreases in cell proliferation, oxidative stress, and inflammatory reaction. That could be a powerful approach to vascular disease prevention.

Angiotensin II Type 2 Receptor Inhibits Vascular Intimal Proliferation With Activation of PPARγ.

BACKGROUND: Angiotensin II type 2 (AT2) receptor stimulation could exert beneficial effects on vascular remodeling. Previously, we reported that AT2 receptor stimulation ameliorated insulin resistance in diabetic mice accompanied by PPARγ activation which also plays a variety of crucial roles in the vasculature. Therefore, this study aimed to investigate the vascular protective effect of the AT2 receptor with activation of PPARγ involving AT2 receptor-interacting protein (ATIP). METHODS AND RESULTS: Vascular injury was induced by polyethylene-cuff placement around the femoral artery in C57BL/6J mice. Treatment with compound 21 (C21), an AT2 receptor agonist, decreased neointimal formation, cell proliferation, and the mRNA levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and interleukin-1ß, and phosphorylation of nuclear factor-kappa B, and increased PPARγ DNA-binding activity in the injured artery, whereas these inhibitory effects of C21 were attenuated by co-treatment with a PPARγ antagonist, GW9662. Treatment of vascular smooth muscle cells (VSMC) with C21 prepared from smAT2 transgenic mice, which highly express the AT2 receptor in VSMC, increased both PPARγ activity and its DNA-binding activity determined by dual-luciferase assay and electrophoresis mobility shift assay (EMSA), respectively. We observed that ATIP was involved in PPARγ complex formation, and that transfection of siRNA of ATIP1 attenuated the AT2 receptor-mediated increase in PPARγ activity in VSMC. In response to AT2 receptor stimulation, ATIP was translocated from the plasma membrane to the nucleus. CONCLUSIONS: Our results suggest a new mechanism by which AT2 receptor stimulation activates PPARγ, thereby resulting in amelioration of vascular intimal proliferation, and that ATIP plays an important role in AT2 receptor-mediated PPARγ activation.

Deficiency of angiotensin-converting enzyme 2 causes deterioration of cognitive function.

The classical renin-angiotensin system (RAS), known as the angiotensin (Ang)-converting enzyme (ACE)/Ang II/Ang II type 1 (AT1) receptor axis, induces various organ damages including cognitive decline. On the other hand, the ACE2/Ang-(1-7)/Mas receptor axis has been highlighted as exerting antagonistic actions against the classical RAS axis in the cardiovascular system. However, the roles of the ACE2/Ang-(1-7)/Mas axis in cognitive function largely remain to be elucidated, and we therefore examined possible roles of ACE2 in cognitive function. Male, 10-week-old C57BL6 (wild type, WT) mice and ACE2 knockout (KO) mice were subjected to the Morris water maze task and Y maze test to evaluate cognitive function. ACE2KO mice exhibited significant impairment of cognitive function, compared with that in WT mice. Superoxide anion production increased in ACE2KO mice, with increased mRNA levels of NADPH oxidase subunit, p22phox, p40phox, p67phox, and gp91phox in the hippocampus of ACE2KO mice compared with WT mice. The protein level of SOD3 decreased in ACE2KO mice compared with WT mice. The AT1 receptor mRNA level in the hippocampus was higher in ACE2KO mice compared with WT mice. In contrast, the AT2 receptor mRNA level in the hippocampus did not differ between the two strains. Mas receptor mRNA was highly expressed in the hippocampus compared with the cortex. Brain-derived neurotrophic factor (BDNF) mRNA and protein levels were lower in the hippocampus in ACE2KO mice compared with WT mice. Taken together, ACE2 deficiency resulted in impaired cognitive function, probably at least in part because of enhanced oxidative stress and a decrease in BDNF.

Role of angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis in the hypotensive effect of azilsartan.

The possible counteracting effect of angiotensin (Ang)-converting enzyme (ACE)2/Ang-(1-7)/Mas axis against the ACE/Ang II/Ang II type 1 (AT1) receptor axis in blood pressure control has been previously described. We examined the possibility that this pathway might be involved in the anti-hypertensive effect of a newly developed AT1 receptor blocker (ARB), azilsartan, and compared azilsartan's effects with those of another ARB, olmesartan. Transgenic mice carrying the human renin and angiotensinogen genes (hRN/hANG-Tg) were given azilsartan or olmesartan. Systolic and diastolic blood pressure, as determined by radiotelemetry, were significantly higher in hRN/hANG-Tg mice than in wild-type (WT) mice. Treatment with azilsartan or olmesartan (1 or 5 mg kg(-1) per day) significantly decreased systolic and diastolic blood pressure, and the blood pressure-lowering effect of azilsartan was more marked than that of olmesartan. The urinary Na concentration decreased in an age-dependent manner in hRN/hANG-Tg mice. Administration of azilsartan or olmesartan increased urinary Na concentration, and this effect was weaker with olmesartan than with azilsartan. Azilsartan decreased ENaC-α mRNA expression in the kidney and decreased the ratio of heart to body weight. Olmesartan had a similar but less-marked effect. ACE2 mRNA expression was lower in the kidneys and hearts of hRN/hANG-Tg mice than in WT mice. This decrease in ACE2 mRNA expression was attenuated by azilsartan, but not by olmesartan. These results suggest that the hypotensive and anti-hypertrophic effects of azilsartan may involve activation of the ACE2/Ang-(1-7)/Mas axis with AT1 receptor blockade.

Direct stimulation of angiotensin II type 2 receptor initiated after stroke ameliorates ischemic brain damage.

BACKGROUND: Stroke is a leading cause of death and disability; however, meta-analysis of randomized controlled trials of blood pressure-lowering drugs in acute stroke has shown no definite evidence of a beneficial effect on functional outcome. Accumulating evidence suggests that angiotensin II type 1 receptor blockade with angiotensin II type 2 (AT2) receptor stimulation could contribute to protection against ischemic brain damage. We examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) initiated even after stroke can prevent ischemic brain damage. METHODS: Stroke was induced by middle cerebral artery (MCA) occlusion, and the area of cerebral infarction was measured by magnetic resonant imaging. C21 (10 µg/kg/day) treatment was initiated immediately after MCA occlusion by intraperitoneal injection followed by treatment with C21 once daily. RESULTS: We observed that ischemic area was enlarged in a time dependent fashion and decreased on day 5 after MCA occlusion. Treatment with C21 initiated after MCA occlusion significantly reduced the ischemic area, with improvement of neurological deficit in a time-dependent manner without affecting blood pressure. The decrease of cerebral blood flow after MCA occlusion was also ameliorated by C21 treatment. Moreover, treatment with C21 significantly attenuated superoxide anion production and expression of proinflammatory cytokines, monocyte chemoattractant protein 1, and tumor necrosis factor α. Interestingly, C21 administration significantly decreased blood-brain barrier permeability and cerebral edema on the ischemic side. CONCLUSIONS: These results provide new evidence that direct AT2 receptor stimulation with C21 is a novel therapeutic approach to prevent ischemic brain damage after acute stroke.

Possible role of angiotensin-converting enzyme 2 and activation of angiotensin II type 2 receptor by angiotensin-(1-7) in improvement of vascular remodeling by angiotensin II type 1 receptor blockade.

Cross talk between the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis and the ACE2/Ang-(1-7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Furthermore, possible stimulation of the Ang II type 2 (AT2) receptor by Ang-(1-7) has been highlighted as a new pathway. Therefore, we examined the possibility of whether the ACE2/Ang-(1-7)/Mas axis and Ang-(1-7)/AT2 receptor axis are involved in the inhibitory effects of AT1 receptor blockers on vascular remodeling. Wild-type, Mas-knockout, and AT2 receptor knockout mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. Some mice were treated with azilsartan, an AT1 receptor blocker, or Ang-(1-7). Neointimal formation 2 weeks after cuff placement was more marked in Mas-knockout mice compared with wild-type mice. Treatment with azilsartan or Ang-(1-7) attenuated neointimal area, vascular smooth muscle cell proliferation, increases in the mRNA levels of monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-1ß, and superoxide anion production in the injured artery; however, these inhibitory effects of azilsartan and Ang-(1-7) were less marked in Mas-knockout mice. Administration of azilsartan or Ang-(1-7) attenuated the decrease in ACE2 mRNA and increased AT2 receptor mRNA but did not affect AT1 receptor mRNA or the decrease in Mas mRNA. The inhibitory effect of Ang-(1-7) on neointimal formation was less marked in AT2 receptor knockout mice compared with wild-type mice. These results suggest that blockade of the AT1 receptor by azilsartan could enhance the activities of the ACE2/Ang-(1-7)/Mas axis and ACE2/Ang-(1-7)/AT2 receptor axis, thereby inhibiting neointimal formation.

Possible synergistic effect of direct angiotensin II type 2 receptor stimulation by compound 21 with memantine on prevention of cognitive decline in type 2 diabetic mice.

Type 2 diabetes mellitus (T2DM) is known to be associated with increased risk of cognitive impairment including Alzheimer disease. Recent studies have suggested an interaction between angiotensin II and N-methyl-d-aspartic acid (NMDA) glutamate receptors. We previously reported that stimulation of the angiotensin II type 2 (AT2) receptor exerts brain protective effects. A newly developed AT2 receptor agonist, compound 21 (C21), has enabled examination of the direct effect of AT2 receptor stimulation in vivo. Accordingly, we examined the possible synergistic effect of C21 and memantine on cognitive impairment in T2DM mice, KKAy. KKAy were divided into four groups; (1) control, (2) treatment with C21 (10 µg/kg/day), (3) treatment with memantine (20mg/kg/day), and (4) treatment with both for 4 weeks, and subjected to Morris water maze tasks. Treatment with C21 or memantine alone at these doses tended to shorten escape latency compared to that in the control group. C21 treatment increased cerebral blood flow (CBF), but memantine did not influence CBF. Treatment with C21 or C21 plus memantine increased hippocampal field-excitatory postsynaptic potential (f-EPSP). Moreover, treatment with memantine or C21 increased acetylcholine level, which was lower in KKAy than in wild-type mice, and C21 plus memantine treatment enhanced memantine or C21-induced acetylcholine secretion. This study provides an insight into new approaches to understand the interaction of angiotensin II and neurotransmitters. We can anticipate a new therapeutic approach against cognitive decline using C21 and memantine.

Effect of angiotensin II type 2 receptor-interacting protein on adipose tissue function via modulation of macrophage polarization.

We demonstrated that angiotensin II type 2 (AT2) receptor-interacting protein (ATIP) 1 ameliorates inflammation-mediated vascular remodeling independent of the AT2 receptor, leading us to explore the possibility of whether ATIP1 could exert anti-inflammatory effects and play a role in other pathophysiological conditions. We examined the possible anti-inflammatory effects of ATIP1 in adipose tissue associated with amelioration of insulin resistance. In mice fed a high-cholesterol diet, adipose tissue macrophage (ATM) infiltration and M1-to-M2 ratio were decreased in ATIP1 transgenic mice (ATIP1-Tg) compared with wild-type mice (WT), with decreased expression of inflammatory cytokines such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in white adipose tissue (WAT), but an increase in interleukin-10, an anti-inflammatory cytokine. Moreover, 2-[(3)H]deoxy-d-glucose (2-[(3)H]DG) uptake was significantly increased in ATIP1-Tg compared with WT. Next, we examined the roles of ATIP1 in BM-derived hematopoietic cells, employing chimeric mice produced by BM transplantation into irradiated type 2 diabetic mice with obesity, KKAy, as recipients. ATM infiltration and M1-to-M2 ratio were decreased in ATIP1 chimera (ATIP1-tg as BM donor), with improvement of insulin-mediated 2-[(3)H]DG uptake and amelioration of inflammation in WAT. Moreover, serum adiponectin concentration in ATIP1 chimera was significantly higher than that in WT chimera (WT as BM donor) and KKAy chimera (KKAy as BM donor). These results indicate that ATIP1 could exert anti-inflammatory effects in adipose tissue via macrophage polarization associated with improvement of insulin resistance, and ATIP1 in hematopoietic cells may contribute to these beneficial effects on adipose tissue functions in type 2 diabetes.