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.

Diabetic mice exhibited a peculiar alteration in body composition with exaggerated ectopic fat deposition after muscle injury due to anomalous cell differentiation.

BACKGROUND: Sarcopenic obesity, age-related muscle loss, which is compensated by an increase in fat mass, impairs quality of life in elderly people. Although the increase in intramuscular fat is associated with decreased insulin sensitivity and increased metabolic risk factors, the origin of diabetes-associated intramuscular fat has not been elucidated. Here, we investigated intramuscular fat deposition using a muscle injury model in type 2 diabetic mice. METHODS: Male 8-week-old C57BL/6 and 8-week-old and 26-week-old KKAy underwent intramuscular injection of cardiotoxin (Ctx) (100 µL/10 µM) into the tibialis anterior (TA) muscles. After 2 weeks, the muscles were removed and evaluated. RESULTS: KKAy exhibited impaired muscle regeneration and ectopic fat deposition. Such impairment was more marked in older KKAy. These changes were also observed in another diabetic mouse model, db/db and diet-induced obese mice but not in streptozocin-induced diabetic mice. Deposited fat was platelet-derived growth factor (PDGF) receptor alpha positive and its cytoskeleton was stained with Masson's trichrome, indicating it to be of fibro-adipocyte progenitor cell origin. Expression of a myogenic marker, myoD, was lower and that of PDGF receptor alpha and CCAAT/enhancer binding protein (CEBP) alpha was higher in Ctx-injured TA of KKAy compared with that of C57BL/6. Peroxisome proliferator-activated receptor γ (PPARγ) was highly expressed in fat-forming lesions in older KKAy. Treatment with all-trans retinoic acid prevented the formation of intramuscular fat; however, treatment with GW9662, a PPARγ antagonist, increased the fibrotic change in muscle. CONCLUSIONS: Diabetic mice showed impaired muscle regeneration with fat deposition, suggesting that diabetes may enhance sarcopenic obesity through a mechanism involving anomalous fibro-adipocyte progenitor cell differentiation.

Low-Protein Diet-Induced Fetal Growth Restriction Leads to Exaggerated Proliferative Response to Vascular Injury in Postnatal Life.

BACKGROUND: We investigated the effects of fetal growth restriction (FGR) induced by maternal protein restriction on inflammatory vascular remodeling using a cuff-induced vascular injury mouse model. METHODS: Dams (C57BL/6J strain mice) were fed an isocaloric diet containing 20% protein (normal protein; NP) or 8% protein (low protein; LP) from 10 weeks of age until delivery. On the day of delivery, all dams were returned to the NP diet. After weaning, offspring were fed the NP diet. When offspring were 10 weeks of age, vascular injury was induced by polyethylene cuff placement around the femoral artery. RESULTS: Birth weight in offspring from dams fed LP until delivery (LPO) was significantly lower, but body weight was the same at 2 weeks after birth compared with that in NP offspring (NPO). Arterial blood pressure at 12 weeks of age did not differ between LPO and NPO. Neointima formation was exaggerated in LPO compared with NPO and associated with an increase in cell proliferation assessed by proliferating cell nuclear antigen (PCNA) staining index. Moreover, LPO showed enhanced expression of monocyte chemotactic protein-1, interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, and production of superoxide anion in the injured artery. Moreover, mRNA expression of isoforms of NAD(P)H oxidase subunits such as p22phox, p40phox, p47phox, p67phox, gp91phpx, and Rac1 in the injured arteries were enhanced in LPO. Furthermore, HIF-1α expression was increased in LPO compared with that in NPO. CONCLUSIONS: These results suggest that maternal low-protein diet-induced FGR increases susceptibility of the vasculature to postnatal injury.

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.

Pre-treatment with LCZ696, an orally active angiotensin receptor neprilysin inhibitor, prevents ischemic brain damage.

Angiotensin II receptor blockers (ARBs) are known to prevent ischemic brain damage after stroke. Natriuretic peptides, which are increased by a neprilysin inhibitor, are also reported to protect against brain damage. Therefore, we investigated the possible protective effect of valsartan (VAL) compared with LCZ696 (VAL+ neprilysin inhibitor; 1:1) after middle cerebral artery (MCA) occlusion. Eight-week-old male C57BL/6J mice were treated with VAL (3mg/kg per day) or LCZ696 (6mg/kg per day) for 2 weeks before MCA occlusion. Blood pressure and heart rate were measured by telemetry. Cerebral blood flow (CBF) was determined by laser-Doppler flowmetry. Ischemic area was evaluated by triphenytetrasodium chloride staining, and oxidative stress was determined by dihydroethidium staining. Blood pressure and heart rate were not significantly different before and after treatment. Pre-treatment with LCZ696 or VAL reduced the ischemic area, and this effect of LCZ696 was more marked than that of VAL pre-treatment. The decrease in CBF in the peripheral region of the ischemic area was significantly attenuated by pre-treatment with LCZ696 or VAL, without any significant effect on CBF in the core region. VAL or LCZ696 pre-treatment significantly decreased the increase of superoxide anion production in the cortex on the ischemic side. However, no significant difference in CBF and superoxide anion production was observed between VAL and LCZ696 pre-treatment. The preventive effect of LCZ696 on ischemic brain damage after stroke was more marked than that of VAL. LCZ696 could be used as a new approach to prevent brain damage after stroke. (246 words).

Direct angiotensin II type 2 receptor stimulation by compound 21 prevents vascular dementia.

Angiotensin II type 2 (AT(2)) 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 AT(2) 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 µg/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 AT(2) receptor knockout (AT(2)KO) mice, and the preventive effect of azilsartan on cognitive decline was weaker in AT(2)KO mice than in WT mice, suggesting that the improvement of cognitive decline by azilsartan may involve stimulation of the AT(2) 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-α and MCP-1 mRNA expression after BCAS was attenuated by C21 treatment. These findings indicate that direct AT(2) receptor stimulation attenuates ischemic vascular dementia induced by hypoperfusion at least in part through an increase in CBF, and a reduction of inflammation.

Drinking citrus fruit juice inhibits vascular remodeling in cuff-induced vascular injury mouse model.

Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuff-induced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.

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.

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.

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.

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.