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.

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.

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.

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.

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.

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.

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.

Direct angiotensin II type 2 receptor stimulation ameliorates insulin resistance in type 2 diabetes mice with PPARγ activation.

OBJECTIVES: The role of angiotensin II type 2 (AT(2)) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT(2) receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue. METHODS: T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[(3)H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined. RESULTS: Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored ß cell damage in diabetic pancreatic tissue. CONCLUSION: The present study demonstrated that direct AT(2) receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic ß cells.

Angiotensin II type 2 receptor-interacting protein prevents vascular senescence.

Angiotensin II type 2 (AT(2)) receptor-interacting protein (ATIP), which interacts with the C-terminal tail of the AT(2) receptor, regulates the functions of the AT(2) receptor. We have reported that AT(2) receptor stimulation attenuated vascular senescence. Therefore, we examined the possible negative role of ATIP in regulating vascular senescence. We generated ATIP-transgenic (Tg) mice, and cultured vascular smooth muscle cells (VSMCs). Persistent angiotensin II stimulation induced increases in SA-ß-gal-positive cells and the level of a DNA damage marker, 8-OHdG in VSMC, whereas these effects of angiotensin II were attenuated in VSMC prepared from ATIP-Tg mice. Angiotensin II treatment also upregulated the expression of methyl methanesulfonate-sensitive 2 (MMS2), a DNA repair factor, and Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1) activity, whereas these effects of angiotensin II were further enhanced in ATIP-Tg VSMC. In vivo, x-ray irradiation to mice caused increases in SA-ß-gal-positive area and 8-OHdG level in the thoracic aorta; however, these effects were reduced in ATIP-Tg mice, with a significant increase in MMS2 expression. These results suggest that ATIP could inhibit VSMC senescence, involving MMS2 expression and SHP-1 activity. ATIP might be a new therapeutic molecule to treat vascular aging and age-related vascular diseases.

Improvement of cognitive impairment in female type 2 diabetes mellitus mice by spironolactone.

Patients with type 2 diabetes mellitus (T2DM) exhibit more severe cognitive decline in females compared with males; however, the preventive approach to this gender-specific cognitive decline is still an enigma. Spironolactone is a potassium-sparing diuretic that also acts as an androgen receptor antagonist. Here, we investigated whether spironolactone attenuates cognitive impairment observed in female T2DM mice. Adult wild-type (WT) mice and an obese T2DM model, KKAy mice, were employed in this study. Cognitive function was evaluated by the shuttle avoidance test and Morris water maze test. Administration of spironolactone (50 mg/kg per day in chow) had no significant effect on blood pressure, glucose tolerance or insulin resistance. In WT mice, no significant sex difference in cognitive function was observed; however, treatment with spironolactone improved spatial memory in the water maze, especially in female WT mice. Administration of spironolactone markedly improved the cognitive decline in female KKAy mice up to the level in male KKAy mice. Spironolactone treatment also improved cognitive function in ovariectomized-KKAy mice, but failed to improve it in those with administration of estradiol (200 µg/kg per day). In diabetic mice, spironolactone improved impaired cognitive function observed in female mice, suggesting that spironolactone may prevent cognitive impairment associated with diabetes in females clinically.

Effect of angiotensin II type 2 receptor deletion in hematopoietic cells on brain ischemia-reperfusion injury.

The angiotensin II type 2 (AT(2)) receptor is expressed in bone marrow cells and may affect cell differentiation. We previously reported a beneficial role of the AT(2) receptor in ischemic brain damage. Here, we investigated the effect of AT(2) receptor stimulation in hematopoietic cells on ischemic brain injury using chimeric mice. Chimeric mice were generated by bone marrow transplantation into wild-type mice after irradiation. Bone marrow cells were prepared from wild-type (Agtr2(+)) or AT(2) receptor-deficient mice (Agtr2(-)). Six weeks after bone marrow transplantation, these chimeric mice were subjected to ischemia/reperfusion injury. Both Agtr2(+) and Agtr2(-) chimeric mice did not show a significant change in systolic and diastolic blood pressures, whereas body weight decreased in Agtr2(-) chimera. Twenty-four hours after ischemia/reperfusion injury, ischemic brain damage in Agtr2(-) chimera was exaggerated compared with that in Agtr2(+) chimera. Moreover, cerebral blood flow in the peripheral region before and after ischemia/reperfusion injury was decreased in Agtr2(-) chimera. The inflammatory response in the ipsilateral hemisphere was not significantly different, whereas tumor necrosis factor-α and monocyte chemoattractant protein 1 expressions tended to increase in the Agtr2(-) chimeric brain. Expression of methylmethane sulfonate 2, which has a neuroprotective effect, was lower in the brain of Agtr2(-) chimera. These results indicate that deletion of AT(2) receptor in blood cells has a harmful effect on ischemic brain injury.

Female exhibited severe cognitive impairment in type 2 diabetes mellitus mice.

AIMS: Sex-specific medicine has been highlighted as a different approach to the diagnosis and treatment of diseases between men and women. Type 2 diabetes has been reported to be a risk factor for cognitive impairment. Here, we investigated the sex difference in cognitive function associated with diabetes using KKAy mice. MAIN METHODS: Cognitive function was evaluated by shuttle avoidance test and Morris water maze test. Changes in gene expression in the brain were evaluated by PCR array and confirmed by quantitative RT-PCR. To evaluate the effect of estradiol, some female KKAy were ovariectomized and treated with or without estradiol. KEY FINDINGS: In KKAy mice, female significantly exhibited impaired cognitive function compared with male, while there was no sex difference in these cognitive functions in C57BL6, wild-type mice. Female KKAy mice showed hyperinsulinemia, impaired glucose tolerance and increased oxidative stress compared with male KKAy mice. Female KKAy also showed a significant decrease in peroxisome proliferators-activated receptor (PPAR)-gamma expression in the brain compared with male KKAy. Estradiol treatment improved the insulin resistance and higher superoxide production, but failed to improve the cognitive task performance, serum insulin level and lower expression of PPAR-gamma. SIGNIFICANCE: In diabetic mice, female showed significantly impaired cognitive function, with greater insulin resistance, lower expression of PPAR-gamma and higher superoxide production compared with male. Estrogen had little effect on cognitive function. These results indicate that a sex-specific approach to cognitive impairment is necessary for diabetic patients, especially for women.

Angiotensin II type 1 receptor-associated protein prevents vascular smooth muscle cell senescence via inactivation of calcineurin/nuclear factor of activated T cells pathway.

Emerging new research suggests that the functions of the angiotensin (Ang) II type 1 (AT(1)) receptor are regulated in a complex manner. AT(1) receptor-associated protein (ATRAP) has been reported to reduce AT(1) receptor signaling with enhancement of AT(1) receptor internalization and to regulate the calcineurin/nuclear factor of activated T cells (NFAT) pathway. We examined the possibility that ATRAP could attenuate AT(1) receptor-mediated vascular senescence via inactivation with the calcineurin/NFAT pathway. Ang II stimulation significantly increased senescence-associated beta-galactosidase (SA-beta-gal)-stained cells, oxidative stress, and expression of p53 and p21 in wild-type (WT) vascular smooth muscle cells (VSMC). Moreover, in WT VSMC, Ang II stimulation enhanced NFAT transcriptional activity, which was prevented by CAML-siRNA treatment. NFAT-siRNA treatment attenuated Ang-II-increased SA-beta-gal activity and p53 and p21 expression. Treatment with a calcineurin activity inhibitor, cyclosporin A, reduced Ang-II-induced NFAT transcriptional activity and senescent VSMC. In contrast, VSMC prepared from ATRAP transgenic (ATRAP-Tg) mice exhibited attenuation of Ang-II-induced SA-beta-gal activity, oxidative stress, NFAT transcriptional activity, and expression of p53 and p21. Moreover, ATRAP-Tg VSMC showed a more reduction of Ang-II-induced NFAT transcriptional activity by CAML-siRNA treatment than WT VSMC. Furthermore, we demonstrated that in ATRAP-Tg VSMC, NFAT activity and senescent cells induced by ultraviolet irradiation were decreased compared with those in WT VSMC. Treatment with an AT(1) receptor blocker, valsartan, blocked these senescent cells but did not change NFAT activity in both cells. These results suggest that ATRAP negatively regulates VSMC senescence by reducing AT(1) receptor signaling, and that ATRAP-mediated inactivation of the calcineurin/NFAT pathway could be at least partly involved in prevention of VSMC senescence, irrespective of AT(1) receptor blockade in some conditions.

Sex-different effect of angiotensin II type 2 receptor on ischemic brain injury and cognitive function.

We previously reported that angiotensin II type 2 (AT(2)) receptor signaling prevents neural damage and cognitive impairment after focal cerebral ischemia. We investigated the possible roles of the AT(2) receptor in the sex difference, focusing on cognitive function and ischemic brain damage using AT(2) receptor-deficient mice (Agtr2(-)). In Agtr2(-), spatial memory evaluated by the Morris water maze test was impaired in female compared with that in male Agtr2(-) and female wild-type (Agtr2(+)), while no significant sex-different change was observed in Agtr2(+). Interestingly, bromodeoxyuridine incorporation assay showed a significant decrease of hippocampal neurogenesis in female Agtr2(-) compared with that in female Agtr2(+). In contrast, ischemic area after middle cerebral artery (MCA) occlusion was significantly increased in male compared with female mice in Agtr2(-), while no significant sex-different change was observed in Agtr2(+). Male Agtr2(-) mice showed higher AT(1) receptor expression and significantly impaired cerebral blood flow (CBF) in the ipsilateral side 24 hours after MCA occlusion compared with female Agtr2(-) mice. In conclusion, deletion of the AT(2) receptor showed a sex-different effect such as a severe cognitive impairment with a decrease of hippocampal neurogenesis in females and a larger ischemic brain damage with a decrease of CBF in males.

Cognitive deficit in amyloid-beta-injected mice was improved by pretreatment with a low dose of telmisartan partly because of peroxisome proliferator-activated receptor-gamma activation.

The pathological hallmark of Alzheimer disease is deposition of amyloid-beta protein (Abeta) in the brain. Telmisartan is a unique angiotensin II receptor blocker with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-stimulating activity. Activation of PPAR-gamma is expected to prevent inflammation and Abeta accumulation in the brain. We investigated the possible preventive effect of telmisartan on cognitive decline in an Alzheimer disease mouse model via PPAR-gamma activation. Here, male ddY mice underwent ICV injection of Abeta 1-40. Cognitive function was evaluated by the Morris water maze test. A low dose of telmisartan (0.35 mg/kg per day) was administered in drinking water with or without GW9662, a PPAR-gamma antagonist. Cerebral blood flow was evaluated by laser speckle flowmetry. Inflammatory cytokine levels were measured by quantitative RT-PCR. Abeta 1-40 ICV injection significantly impaired cognitive function. Pretreatment with telmisartan improved this cognitive decline to a similar level to that in control mice. Cotreatment with GW9662, a PPAR-gamma antagonist, attenuated this telmisartan-mediated improvement of cognition. Treatment with telmisartan enhanced cerebral blood flow and attenuated the Abeta-induced increase in expression of cytokines, such as tumor necrosis factor-alpha and inducible NO synthase in the brain. Interestingly, coadministration of GW9662 cancelled these beneficial effects of telmisartan. Abeta 1-40 concentration in the brain was significantly decreased by treatment with telmisartan, whereas administration of GW9662 attenuated the decrease in telmisartan-mediated Abeta 1-40 concentration. Taken together, our findings suggest that even a low dose of telmisartan had a preventive effect on cognitive decline in an Alzheimer disease mouse model, partly because of PPAR-gamma activation.

Attenuation of cuff-induced neointimal formation by overexpression of angiotensin II type 2 receptor-interacting protein 1.

Recently we have cloned angiotensin II type 2 receptor-interacting protein 1 (ATIP1) as a novel protein that interacts specifically with the C-terminal tail of the angiotensin II type 2 receptor; however, the pathophysiological roles of ATIP1 in vascular remodeling are still unknown. Here, we generated ATIP1-transgenic (ATIP1-Tg) mice expressing mouse ATIP1 and investigated the role of ATIP1 in vascular remodeling using these transgenic mice. ATIP1-Tg mice exhibited no significant difference in blood pressure compared with wild-type (WT) mice. Angiotensin II type 2 receptor mRNA expression in the femoral artery was increased in injured femoral arteries, reaching a peak at 7 days after operation in WT mice, and a similar result of angiotensin II type 2 receptor expression was observed in ATIP1-Tg mice. In ATIP1-Tg mice, neointimal formation of the femoral artery 14 days after cuff placement was significantly smaller than that in WT mice. 5-Bromo-2'-deoxyuridine incorporation was significantly reduced in the injured arteries of ATIP1-Tg mice compared with WT mice. In ATIP1-Tg mice, superoxide anion production and the expression of a proinflammatory cytokine, tumor necrosis factor-alpha, were markedly attenuated. Moreover, cell proliferative signaling, such as extracellular signal-regulated kinase phosphorylation, was significantly attenuated in ATIP1-Tg mice compared with WT mice. Taken together, these results suggest that ATIP1 plays an important role in cuff-induced vascular remodeling in mice.

Deletion of angiotensin II type 2 receptor attenuates protective effects of bone marrow stromal cell treatment on ischemia-reperfusion brain injury in mice.

BACKGROUND AND PURPOSE: Protective effects of bone marrow stromal cells (MSCs) on ischemic brain damage have been highlighted. We examined the possibility that deletion of AT(2) receptor could attenuate the cerebroprotective effects of MSC using AT(2) receptor-deficient mice (Agtr2 (-)) and the effect of selective AT(1) receptor blocker. METHODS: Wild-type mice (Agtr2 (+)) were subjected to 3 hours of focal brain ischemia followed by reperfusion (ischemia-reperfusion injury). Simultaneously, Agtr2 (+)-MSC, Agtr2 (-)-MSC, or saline was injected through the tail vein. RESULTS: Survival rates at 6 days after ischemia-reperfusion injury were as follows: approximately 50% in saline-injected mice, 80% in Agtr2 (+)-MSC-injected mice, and 20% in Agtr2 (-)-MSC-injected mice. Neurological deficit after ischemia-reperfusion injury was improved in Agtr2 (+)-MSC-injected mice, but not in Agtr2 (-)-MSC-injected mice. After 48 hours of ischemia-reperfusion injury, brain infarct size was reduced in Agtr2 (+)-MSC-injected mice, but not in Agtr2 (-)-MSC-injected mice. Moreover, brain edema was significantly ameliorated in Agtr2 (+)-MSC-treated mice but not in Agtr2 (-)-MSC-treated mice. Furthermore, the increase in mRNA expression of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 in the ischemic brain was less in Agtr2 (+)-MSC-treated mice in the ipsilateral site, but was similar in the contralateral hemisphere. Tumor necrosis factor-alpha level was increased in both the contralateral hemisphere and ipsilateral hemisphere of Agtr2 (-)-MSC-treated mice. In contrast, monocyte chemoattractant protein-1 levels tended to increase Agtr2 (-)-MSC-treated mice without a significant difference. Treatment of MSC with an AT(1) receptor blocker, valsartan, significantly improved survival rates in Agtr2 (-)-MSC-injected mice. CONCLUSIONS: These results suggest that AT(2) receptor signaling in MSC attenuated brain damage and neurological deficit (deleted).

Temporary pretreatment with the angiotensin II type 1 receptor blocker, valsartan, prevents ischemic brain damage through an increase in capillary density.

BACKGROUND AND PURPOSE: We investigated the effect of temporary treatment with a nonhypotensive dose of valsartan on ischemic brain damage in C57BL/6 mice. METHODS: We separated the mice into 3 groups of valsartan treatment before middle cerebral artery (MCA) occlusion: (1) for 4 weeks: Val (2W, 2W); (2) for 2 weeks followed by its cessation for 2 weeks: Val (2W, -); and (3) no treatment for 4 weeks: Val (-, -). RESULTS: Ischemic volume, DNA damage, superoxide production, and mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha on the ipsilateral side after 24 hours of MCA occlusion were significantly reduced in both Val (2W, 2W) and Val (2W, -) mice compared with those in Val (-, -) mice, whereas these parameters were larger in Val (2W, -) mice than in Val (2W, 2W) mice. Moreover, mice in both the Val (2W, 2W) and Val (2W, -) groups exhibited an increase in cerebral blood flow in the peripheral territory of the MCA 1 hour after MCA occlusion, with increases in endothelial nitric oxide synthase activation and nitric oxide production. Before MCA occlusion, treatment with valsartan did not influence superoxide production or mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in the brain. However, the capillary density in the brain in both Val (2W, 2W) and Val (2W, -) mice was increased before MCA occlusion. CONCLUSIONS: Our results suggest that temporary valsartan treatment could protect against ischemic brain damage even after its cessation, at least in part due to an increase in capillary density.