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Molecular hydrogen is thought to have an inhibitory effect on oxidative stress, thereby attenuating the onset and progression of various diseases including cardiovascular disease; however, few reports have assessed the preventive effect of constitutive inhalation of hydrogen gas on of vascular remodeling. Here, we investigated the effect of constitutive inhalation of hydrogen gas on vascular neointima formation using a cuff-induced vascular injury mouse model. After constitutive inhalation of compressed hydrogen gas (O2 21%, N2 77.7%, hydrogen 1.3%) or compressed air only (O2 21%, N2 79%) by C57BL/6 mice for 2 weeks from 8 weeks of age in a closed chamber, inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia, and hydrogen gas administration was continued until sampling of the femoral artery. Neointima formation, accompanied by an increase in cell proliferation, was significantly attenuated in the hydrogen group compared with the control group. NADPH oxidase NOX1 downregulation in response to cuff injury was shown in the hydrogen group, but the expression levels of NADPH oxidase subunits, p40phox and p47phox, did not differ significantly between the hydrogen and control groups. Although the increase in superoxide anion production did not significantly differ between the hydrogen and control groups, DNA damage was decreased as a result of reduction of reactive oxygen species such as hydroxyl radical (⋅OH) and peroxynitrite (ONOO-) in the hydrogen group. These results demonstrate that constitutive inhalation of hydrogen gas attenuates vascular remodeling partly via reduction of oxidative stress, suggesting that constitutive inhalation of hydrogen gas at a safe concentration in the living environment could be an effective strategy for prevention of vascular diseases such as atherosclerosis.

Background Lung injury is a life-threatening complication in patients with liver dysfunction. We recently provided an experimental lung injury model in mouse with common bile duct ligation. In this study, we aimed to characterize the pathologic and biochemical features of lung tissues in common bile duct ligation mice using a proteomic approach. Methods Common bile ducts of BALB/c mice, 8 weeks of age, were ligated operatively. CD31-expressing pulmonary cells were sorted with immunomagnetic microbeads, and protein profiles were examined by 2-dimensional gel electrophoresis. Based on the results of protein identification, immunohistochemistry and quantitative reverse transcription polymerase chain reaction were carried out in pulmonary and hepatic tissues. Results Two-dimensional gel electrophoresis revealed 3 major inflammation-associated proteins exhibiting considerable increases in the number of CD31-positive pulmonary cells after common bile duct ligation. Mass spectrometry analysis identified these proteins as SerpinB1a (48 kDa), ANXA1 (46 kDa), and S100A9 (16 kDa). Furthermore, the 3 proteins were more highly expressed in dilated pulmonary blood vessels of common bile duct ligation mice, in which neutrophils and monocytes were prominent, as shown by immunohistochemistry. More importantly, SerpinB1a mRNA and protein were significantly upregulated in the liver, whereas S100A9 and ANXA1 mRNA and protein were upregulated in the lungs, as shown by quantitative reverse transcription polymerase chain reaction and Western blotting. Conclusion We identified 3 proteins that were highly expressed in the lung after common bile duct ligation using a proteomics-based approach.

Introduction: Distant organ dysfunction has been highlighted whereby acute or chronic damage in one organ may induce dysfunctions of the other organs such as cardiorenal syndrome. Vascular injury is an important process in various vascular diseases. However, the relationship cerebral ischemic damage and peripheral vascular disorder is still unclear. Therefore, we have investigated the possible pathophysiological interactions between brain ischemia and vascular remodeling. Methods: Eight-week-old male C57BL/6 mice underwent permanent cerebral brain ischemia by permanent occlusion of the left middle cerebral artery occlusion (MCAO) by electrocoagulation using a subtemporal approach. Four weeks after MCAO, vascular injury was induced by polyethylene cuff placement on the femoral artery. Neointima formation was determined 14 days after cuff placement by evaluating intima/media ratio. Mice were administrated 0.5% tempol, antioxidant agent, in drinking water from after MCAO. Macrophage fractions in spleen two weeks after MCAO were examined and M1 and M2-polarized macrophages were separated by flow cytometry and analyzed. Results: Cerebral ischemia did not influence the intima/media ratio 6 weeks after MCAO compared with sham-operated mice. Neointima formation in the injured artery was significantly increased 2 weeks after cuff placement. Interestingly, the neointima formation 2 weeks after cuff placement was more marked in MCAO-operated mice compared with sham-operated mice. We observed that treatment with tempol did not improve increased neointima formation in MCAO-operated mice. Next, we examined the macrophage fractions in spleen 2 weeks after MCAO. The macrophage fraction in SVF evaluated by F4/80 staining did not differ between sham- and MCAO-operated mice. There was no significant difference in M1 and M2 fraction with/without MCAO. Conclusion: These results suggest that cerebral ischemic injury aggravate peripheral vascular disorders, and we are addressing the possible roles of renin-angiotensin system and inflammation etc. involved in these interesting results, which could provide us with the discovery of new pathological mechanisms concerning the distant organ dysfunctions between brain and peripheral vasculature.

We previously reported interferon regulatory factor (IRF) 1 plays physiological roles in “growth”-regulated angiotensin II type 2 (AT 2 ) receptor expression in fibroblasts. Here, we investigated whether IRF-1 is involved in attenuation of vascular remodeling in association with AT 2 receptor upregulation. Neointimal area in injured artery after 14 days of cuff placement was significantly increased in IRF-1 knockout mice (IRF-1KO) and AT 2 receptor knockout mice (AT 2 KO) compared with wild-type mice (WT: C57BL/6J). Treatment with compound 21 attenuated neointima formation in both WT and IRF-1KO. AT 2 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 AT 2 KO and WT. Apoptotic changes in injured artery after 14 days of cuff placement were significantly attenuated in IRF-1KO, with a decrease in interleukin-1β–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 AT 2 receptors.

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.

Introduction: Distant organ dysfunction has been highlighted whereby acute or chronic damage in one organ may induce acute or chronic dysfunctions of the other organs such as cardiorenal syndrome. Vascular remodeling is an important process in various vascular diseases. However, the relationship cerebral ischemic damage and peripheral vascular disorder is still unclear. Therefore, we have investigated the possible pathophysiological interactions between brain ischemia and vascular remodeling. Methods: Eight-week-old male C57BL/6 mice underwent permanent cerebral brain ischemia by permanent occlusion of the left middle cerebral artery occlusion (MCAO) by electrocoagulation using a subtemporal approach. Four weeks after MCAO, vascular injury was induced by polyethylene cuff placement on the femoral artery. Neointima formation was determined 14 days after cuff placement by evaluating intima/media ratio. Macrophage fractions in spleen two weeks after MCAO were examined and M1 and M2-polarized macrophages were separated by flow cytometry and analyzed. Results: Body weight after MCAO and cuff placement did not differ among all groups. Cerebral ischemia did not influence the intima/media ratio 6 weeks after MCAO (MCAO-Cuff (-), 0.123) compared with sham-operated mice (sham-Cuff (-), 0.120). Neointima formation in the injured artery was significantly increased 2 weeks after cuff placement (sham-Cuff (+), 0.258). Interestingly, the neointima formation 2 weeks after cuff placement was more marked in MCAO-operated mice (MCAO-Cuff (+), 0.369) compared with sham-Cuff (+). Next, we examined the macrophage fractions in spleen 2 weeks after MCAO. The macrophage fraction in SVF evaluated by F4/80 staining did not differ between sham- (7.5%) and MCAO-operated (7.6%) mice. There was no significant difference in M1 and M2 fraction with/without MCAO. Conclusion: These results suggest that cerebral ischemic injury aggravate peripheral vascular disorders, and we are addressing the possible roles of renin-angiotensin system and oxidative stress etc. involved in these interesting results, which could provide us with the discovery of new pathological mechanisms concerning the distant organ dysfunctions between brain and peripheral vasculature.

Calcification of bioprosthetic valves (BVs) implanted in aortic position can result in gradual deterioration and necessitate aortic valve replacement. The molecular mechanism of calcium deposition on BV leaflets has been investigated, but remains to be fully elucidated. The present study aimed to identify explanted bioprosthetic valve (eBV)-specific proteins using a proteomics approach and to unveil their biochemical and histological involvements in calcium deposition on BV leaflets. Calcification, fibrosis, and glycosylation of the valves were histologically assessed using Von Kossa, Masson's Trichrome and Alcian Blue staining, as well as immunostaining. Protein expression in the explanted biological valves was analysed using proteomics and western blotting. In a histological evaluation, αSMA-positive myofibroblasts were not observed in eBV, whereas severe fibrosis occurred around calcified areas. SDS-PAGE revealed three major bands with considerably increased intensity in BV leaflets that were identified as plasminogen and fibrinogen gamma chain (100 kDa), and fibrinogen beta chain (50 and 37 kDa) by mass analysis. Immunohistochemistry showed that fibrinogen β-chain was distributed throughout the valve tissue. On the contrary, plasminogen was strongly stained in CD68-positive macrophages, as evidenced by immunofluorescence. The results suggest that two important blood coagulation-related proteins, plasminogen and fibrinogen, might affect the progression of BV degeneration., Summary: Fibrinogen was specifically deposited on whole deteriorated tissue valve leaflets, and plasminogen-positive macrophages strongly invaded the areas around calcified bioprosthetic and native tissues.

Objective: Accumulating evidences and previous our research suggest that angiotensin II type 2 (AT 2 ) receptor stimulation could contribute to protection against ischemic brain damage. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and reported that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on ischemic brain damage is still unclear. Therefore, we investigated the effects of the ATIP and compound 21 (C21), a selective non-peptidic AT2 receptor agonist, on focal cerebral ischemia. Method: Ten-week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to middle cerebral artery occlusion (MCAO) with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCAO. Twenty-four hours after MCAO, ischemic area was determined. Cerebral blood flow (CBF) before and after MCAO was measured by laser speckle flowmetry. Collateral circulation was evaluated by the perfusion of India ink. Expression of mRNA was determined by real-time RT-PCR. Results: There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size in both strains. Interestingly, this protective effect of C21 was more marked in ATIP-Tg compared with WT mice. In CBF of core region of ischemic area, there were no significant differences among all groups. However, the reduction of CBF in penumbra region just after MCA occlusion was attenuated in ATIP-Tg mice with C21 administration. Treatment with C21 tended to increase the cerebral collateral number before MCA occlusion in ATIP-Tg mice. Expression of vascular endothelial growth factor (VEGF) mRNA in the cortex before MCA occlusion did not differ among all groups. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the attenuation of CBF reduction and increase of MMS2 expression after ischemia.

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.

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.

Objectives: We reported the preventive effects of angiotensin II type 2 (AT2) receptor stimulation on ischemic brain damage. Moreover, we have cloned ATIP (AT2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT2 receptor, and suggest that ATIP should play key roles in diverse mechanisms of AT2 receptor actions. However, the effect of ATIP on ischemic brain damage is unclear. Therefore, we investigated the effects of the ATIP and compound 21 (C21), a selective non-peptide AT2 receptor agonist, on focal cerebral ischemia. Method: Ten-week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to middle cerebral artery occlusion (MCAO) with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCAO. Twenty-four hours after MCAO, ischemic volume was determined and depicted as mm 3 . Expression of methyl methanesulfonate sensitive 2 (MMS2) mRNA as a neuroprotective factor was determined by real-time RT-PCR. Cerebral blood flow (CBF) before and after MCA occlusion were measured by laser speckle flowmetry. Collateral circulation was evaluated by the perfusion of India ink. Results: Systolic blood pressure did not differ among all groups. There was no significant difference in ischemic size without C21 treatment between two strains (WT, 80 mm 3 ; ATIP-Tg, 75 mm 3 ). Treatment with C21 significantly decreased ischemic size in both strains (WT, 66 mm 3 ; ATIP-Tg, 50 mm 3 ). Interestingly, this protective effect of C21 was more marked in ATIP-Tg compared with WT mice (WT, 16% (66 of 80); ATIP-Tg, 27% (50 of 75) reduction, respectively). MMS2 expression increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. There were no significant differences in CBF of core region of ischemic area, among all groups. However, the reduction of CBF in penumbra region after MCA occlusion was attenuated in ATIP-Tg mice with C21 administration. Treatment with C21 tended to increase the cerebral collateral number before MCA occlusion in ATIP-Tg mice not in WT. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the increase of CBF and MMS2 expression after ischemia.

Objective: We previously reported that the ischemic brain area was significantly larger in angiotensin II type 2 (AT 2 ) receptor-deficient mice after middle cerebral artery (MCA) occlusion compared to wild-type mice. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and suggest that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on brain damage has not been clarified. We investigated the possibility that ATIP could enhance the protective effects of compound 21 (C21), a selective direct non-peptidic AT 2 receptor agonist, on focal cerebral ischemia. Method: Ten week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to permanent MCA occlusion with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCA occlusion. Twenty-four hours after MCA occlusion, ischemic area and neurological deficit was assessed. Cerebral blood flow (CBF) was measured by laser speckle flowmetry. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor were measured by real-time RT-PCR. Collateral circulation was evaluated by the perfusion of India ink. Results: Systolic blood pressure did not differ between ATIP-Tg and WT mice with or without C21. There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size and improved neurological deficit in both strains. These protective effects by C21 were more marked in ATIP-Tg mice compared with WT mice. Treatment of C21 did not affect CBF in the core region of ischemic area after MCA occlusion in both strains; however, the reduction of CBF in penumbra region was markedly attenuated in ATIP-Tg mice treated with C21. MMS2 expression increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. C21 treatment tended to increase collateral number in ATIP-Tg mice. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the improvement of CBF and increase of MMS2 expression.

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.

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.

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.

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.

Introduction: 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 damage 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. Methods: Male 10-week-old C57BL6 (wild-type: WT) mice and ACE2 knockout (KO) mice were subjected to the Morris water maze task to evaluate spatial cognitive function. Vascular dementia model were induced by bilateral common carotid artery stenosis (BCAS). The Morris water maze task was performed 6 weeks after BCAS operation. Results: ACE2KO mice exhibited significant impairment of spatial cognitive function, compared with that in WT mice, without significant difference in cerebral blood flow determined by laser speckle flowmetry and morphological changes in the hippocampus between both strains. Superoxide anion production in the hippocampus tended to be increased in ACE2KO mice, with increased mRNA levels of NADPH oxidase subunit in the hippocampus of ACE2KO compared with WT mice. Protein level of superoxide dismutase (SOD) 3 tended to be decreased in ACE2KO mice compared with WT mice. AT1 receptor mRNA level in the hippocampus was higher in ACE2KO mice compared with WT mice. In contrast, 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, with no significant difference between ACE2KO and WT mice. Brain-derived neurotrophic factor (BDNF) mRNA was lower in the hippocampus in ACE2KO mice compared with WT mice. Escape latency after BCAS was prolonged in WT mice compared with sham operated mice, whereas impaired cognitive function in ACE2 KO mice was not further exaggerated after BCAS. Conclusion: Taken together ACE2 deficiency resulted in impaired cognitive function probably due to enhanced oxidative stress and a decrease in BDNF.

Introduction: It is well known that activation of angiotensin type 1 (AT1) receptor results in cognitive impairment. In contrast, accumulating evidences suggest that angiotensin II type 2 (AT2) receptor stimulation might prevent cognitive impairment in mice. We recently reported that administration of compound 21, newly developed direct AT2 receptor agonist, improved cognitive decline in the mouse model of vascular dementia induced by bilateral common carotid artery stenosis (BCAS). Cerebral histone deacetylase (HDAC) 2 has been highlighted in terms of epigenetic modulation of cognitive function. These results led us to examine the effects of HDAC on AT2 receptor-mediated improvement of cognitive decline. Methods: Ten-week-old male wild type (WT) (C57BL/6) or AT2 receptor knockout (AT2KO) mice were subjected to BCAS. HDAC inhibitor (SAHA; 25 mg/kg/day) was administrated from 4 weeks after BCAS operation. The Morris water maze task was performed 6 weeks after BCAS operation. HDAC2 and inflammatory cytokine levels in the hippocampus were determined. Cerebral blood flow (CBF) were assessed by laser speckle flowmetry. Results: HDAC2 protein level in the hippocampus did not differ between WT and AT2KO mice at 10 weeks of age, whereas HDAC2 of AT2KO mice at 20 weeks of age was higher compared with that of WT mice. Cognitive impairment after BCAS was more marked in AT2KO mice. Treatment with SAHA attenuated BCAS-induced prolongation of escape latency in the Morris water maze test both in WT and AT2KO mice; however this effect of SAHA was more marked in AT2KO mice. Decrease in CBF after BCAS was not affected by SAHA treatment. Expression of inflammatory cytokine mRNA was increased by BCAS and this increase was attenuated by SAHA treatment. Conclusions: We speculate that modulation of cerebral AT2 receptor could be new therapeutic tool to prevent cognitive impairment via epigenetic abnormalities.

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.

Research highlights: {yields} Atherosclerotic apolipoprotein E-deficient (ApoEKO) mice were treated with irbesartan. {yields} Irbesartan decreased white adipose tissue weight without affecting body weight. {yields} DNA-binding for PPAR{gamma} was increased in white adipose tissue in vivo by irbesartan. {yields} Irbesartan increased adipocyte number in white adipose tissue. {yields} Irbesatan increased the expression of adiponectin and leptin in white adipose tissue. -- Abstract: The effect of the PPAR{gamma} agonistic action of an AT{sub 1} receptor blocker, irbesartan, on adipose tissue dysfunction was explored using atherosclerotic model mice. Adult male apolipoprotein E-deficient (ApoEKO) mice at 9 weeks of age were treated with a high-cholesterol diet (HCD) with or without irbesartan at a dose of 50 mg/kg/day for 4 weeks. The weight of epididymal and retroperitoneal adipose tissue was decreased by irbesartan without changing food intake or body weight. Treatment with irbesartan increased the expression of PPAR{gamma} in white adipose tissue and the DNA-binding activity of PPAR{gamma} in nuclear extract prepared from adipose tissue. The expression of adiponectin, leptin and insulin receptor was also increased by irbesartan. These results suggest that irbesartan induced activation of PPAR{gamma} and improved adipose tissue dysfunction including insulin resistance.

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-1β, 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.

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).

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-1β, 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.

Objectives: Recent clinical evidence demonstrated that angiotensin II type 1 receptor blockers (ARBs) were associated with a significant reduction in the incidence and progression of dementia compared with angiotensin converting enzyme inhibitor. Therefore, we examined the possibility that direct angiotensin II type 2 (AT2) receptor stimulation by AT2 receptor agonist, compound 21 (C21), could prevent cognitive decline associated with hypoperfusion in the brain. Methods: 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. ARB, azilsartan (0.1 mg/kg/day) or C21 (10 μg/kg/day) was administered from 1 week before BCAS. Cerebral blood flow (CBF) was measured by laser speckle flowmetry and inflammatory cytokine levels were determined by real-time RT-PCR. Results: Wild-type (WT) mice showed the significant prolongation of escape latency after BCAS and this cognitive impairment was attenuated by the pretreatment of azilsartan. Cognitive impairment was more marked in AT2 receptor knockout (AT2KO) mice, and preventive effect of azilsartan on cognitive decline was weaker in AT2KO mice than in WT mice, suggesting that improvement of cognitive decline by azilsartan may involve the stimulation of AT2 receptor. The significant impairment of spatial learning after BCAS in WT mice was attenuated by C21 treatment. CBF in the whole brain in the BCAS-treated group was significantly decreased compared with that in the sham group at 6 weeks after BCAS operation. This decrease was increased by treatment with C21. We assessed expression of inflammatory cytokines such as TNF-[[Unable to Display Character: ]]α and MCP-1 in the cerebral cortex. TNF-α and MCP-1 mRNA expression were significantly increased after BCAS operation, but significantly attenuated by treatment with C21. Azilsartan or C21 at these doses did not affect systolic blood pressure. Conclusions: These findings indicate that direct AT2 stimulation prevents ischemic vascular dementia induced by hypoperfusion at least in part through an increase in CBF, and reduction of inflammation. We expect that direct AT2 receptor stimulation could be a new therapeutic strategy for preventing and treating vascular dementia.

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-1β, 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.

The dispersibility of aqueous BaTiO 3 slurries with poly ammonium acrylate-based dispersant has been investigated. BaTiO 3 particles were well-dispersed in the high pH region and the adsorption behavior of dispersant strongly depended on the slurry pH. The dispersion of BaTiO 3 particles at various pH was also elucidated by characterizing the sedimentation density, viscosity of slurries, amount of adsorbed dispersant and tape-casted green sheets. The microstructures of green sheets fabricated using aqueous BaTiO 3 slurries were found to depend greatly upon the dispersion state of BaTiO 3 particles in the slurries.

Objective: It is well known that excessive salt intake and/or activation of renin-angiotensin system (RAS) increases blood pressure in part via an increase in sympathetic nerve activity. To further examine the interaction of these factors, we employed transgenic mice carrying both the human renin and angiotensinogen genes (Tsukuba hypertensive mice) and studied the effects of high salt loading and telmisartan on mortality, blood pressure and kidney functions. Design and Method: Ten-week-old male Tsukuba hypertensive mice (TH) were administrated control chow or the chow containing 8% NaCl with or without telmisartan (1 mg/kg/day) and PPAR-γ antagonist, GW9662, in drinking water for 8 weeks. Blood pressure was assessed by radio telemetry method. Urine samples were obtained before and after salt-loading. Concentrations of urinary adrenalin and noradrenalin were measured by ELISA. Expressions of ENaC-α, P47 and p67-phox mRNA in the kidney were measured by RT-PCR. Results: Survival rate 8 weeks after treatment was decreased in salt-loading TH (sTH) mice compared with control TH (cTH). This decrease was improved by the treatment with telmisartan. Blood pressure in sTH mice was significantly higher compared with cTH mice and the treatment with telmisartan mice decreased blood pressure. Urinary sodium concentration was higher in sTH mice compared with cTH mice and treatment with telmisartan further increased urinary sodium concentration in sTH mice. Expression of ENaC-α in did not between sTH mice and cTH mice. Treatment with telmisartan decreased expression of ENaC-α. Urinary adrenalin and noradrenalin concentrations were higher in sTH mice compared with cTH mice. These increases were attenuated by treatment with telmisartan. Expressions of p47 and p67-phox in sTH mice were increased more markedly compared with cTH mice. These increase were also attenuated by treatment with telmisartan. However, these possible protective effects of telmisartan were not influenced by the co-treatment with GW9662. Conclusion: These results suggested that salt-loading enhanced an increase in blood pressure, superoxide anion production, sympathetic activity and mortality in concert with activated RAS.

Background: Sarcopenic obesity, a loss of muscle and a concomitant increase in ectopic fat, is reported to increase in life-style related diseases. Especially the prevalence of sarcopenia is greater in patients with type 2 diabetes mellitus (T2DM) than in non-diabetic subjects. Increase in fat filtration enhances insulin resistance and is associated with metabolic risk factors. However, the detailed mechanism of such ectopic fat filtration in muscle of T2DM patients has not been well investigated. Here, we assessed muscle regeneration using muscle-injured models in obese diabetic mice. Methods: Male eight-week-old wild-type mice (C57BL6) and T2DM mice (KKAy) were undergone intramuscular injection of cardiotoxin (Ctx) (100uL/10uM) into tibia. After two weeks, muscle were removed and stained with hematoxylin and eosin. KKAy replaced by bone marrow prepared from GFP-transgenic mice were generated by 8 Gy whole body X-ray irradiation. Cell-differentiation was evaluated by immunofluorescent analysis. Some mice were treated with all trans-retinoic acid (ATRA) and PPAR-gamma antagonist, GW9662. Results: In 26 week-old KKAy, magnetic resonance imaging analysis showed that intramuscular fat deposition was estimated as higher intensity compared with C57BL6 mice. Treatment with Ctx showed a significant increase in honey comb structure which was perilipin positive. KKAy mice exhibited impaired muscle regeneration. Tibial muscle weight was approximately a half in Ctx-injured KKAy. Such change was also observed in another diabetic mouse model, db/db, but not in streptozocin-induced diabetic mice. Fat formation was remarkably increased in aged KKAy compared with younger mice. Several fat formation was GFP-positive in GFP-chimeric mice indicating that fat tissue was partially generated from marrow cells. Fat formation was desmin-negative and vimentin-positive indicating that these are mesenchymal origin. Treatment with ATRA reduced fat filtration. On the other hand, treatment with GW9662 enhanced fat filtration. Conclusion: Obese diabetic mice showed significantly impaired muscle regeneration with replacement of fat deposition, suggesting that diabetes enhanced sarcopenic obesity possibly due to inhibitory effect of cell differentiation.

Background: We have previously reported that treatment with angiotensin II type-2 (AT 2 ) receptor agonist (compound 21; C21) ameliorated insulin resistance with the improvement of adipocyte dysfunction in obese type 2 diabetic mouse model, KKAy. Until quite recently, adipose tissue has been divided into two groups that have directory-opposed capacities; white adipose tissue is best known for its role in energy store, while brown adipose tissue functions for energy expenditure (thermogenesis). In addition, new clusters of adipocyte with thermogenic capability have been discovered in white adipose tissue, which are distinct origins from brown adipose tissue. These adipocytes have been named ‘beige’. However, the role of AT 2 receptor signaling in white-to-beige fat conversion is not known. Methods: KKAy mice were subjected to i.p. injection of C21 (10ug/kg/day) for 2 weeks. After the treatment, the expression of thermogenic genes (Ucp1, Cidea, Pgc1α) in adipose tissue were determined with real-time RT-PCR. Next, we examined the difference in conversion from white to beige fat between wild-type (WT) mice and AT 2 receptor knockout (AT 2 KO) mice. Eight-week-old mice were fed a high-fat and high-cholesterol diet (HFCD) (10% coconut oil + 1.25% cholesterol) for 2 weeks. To examine the effect of exercise on fat conversion, 9-week-old mice were forced swim test (2 minх3 times/day) for 7days. After the swim test, mice were sacrificed for examinations. Results: In KKAy mice, treatment of C21 increased the expression of thermogenic genes in subcutaneous fat, but not in visceral fat. Adipose tissue weight was not changed by treatment of C21. In AT 2 KO mice fed HFCD for 2 weeks, visceral fat was greater than that in WT mice, but subcutaneous fat was not. The expression of thermogenic genes in subcutaneous fat was lower in AT 2 KO mice than in WT mice. In both mice, swim exercise increased the expression of thermogenic genes; however, the up-regulation of these genes in AT 2 KO mice did not reach the level of that in WT mice. Conclusion: These results suggest that AT 2 receptor signaling has an important role in white-to-beige fat conversion, and therefore AT 2 receptor signaling might be a potential therapeutic target for lipid metabolic disorders.

Objective: Transcriptional control of angiotensin II type 2 (AT 2 ) receptor expression is not well known and we previously reported that interferon regulatory factor (IRF)-1 plays physiological roles in “growth”-regulated AT 2 receptor expression in fibroblast. We studied whether IRF-1 is involved in the attenuation of vascular remodeling in association with AT 2 receptor up-regulation. Methods: Inflammatory vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery in male wild-type mice (WT: C57BL/6J strain), IRF-1 knockout mice (IRF-1KO) and AT 2 receptor-null mice (AT 2 KO) at 10 weeks of age. After cuff placement, each mice were treated with an intraperitoneal injection of compound 21 (C21), AT 2 receptor agonist, at the dose of 10 μg/kg/day or saline. Formalin-fixed, paraffin-embedded sections were prepared using femoral arteries 14 days after cuff placement and subjected to Elastica van Gieson staining for the evaluation of neointima formation. Superoxide anion production and mRNA expressions 7 days after cuff placement were evaluated by dihydroethidium staining and real-time quantitative RT-PCR respectively. Results: Neointima areas in the injured artery induced by cuff placement were significantly increased in vehicle-treated group of IRF-1KO and AT 2 KO compared with those in WT. Moreover, we observed that treatment with C21 attenuated neointima formation 76% in WT, but 45% in IRF-1KO. Oxidative stress was more enhanced in vehicle-treated group of IRF-1KO compared with WT. Treatment with C21 markedly inhibited oxidative stress in WT, but not in IRF-1KO. AT 2 receptor mRNA expression was significantly decreased in IRF-1KO compared with that in WT; however, IRF-1 mRNA expression did not differ between AT 2 KO and WT. Conclusion: These results indicate that IRF-1 up-regulates AT 2 receptor expression and thereby plays an important role in the inhibition of vascular remodeling, supporting the notion that IRF-1 is one of the key transcriptional factor for AT 2 receptor expression and that targeting the immune system is pivotal in the treatment of vascular diseases.

Objective: Recent accumulating evidence suggests that an orally active angiotensin II receptor-neprilysin inhibitor, LCZ696, was supposed to be superior compared to angiotensin II receptor blocker (ARB), valsartan alone in treating cardiovascular disease. We previously reported that ARBs prevent ischemic brain damage using middle cerebral artery occlusion (MCAO) mouse model. Moreover, natriuretic peptides such as atrial natriuretic peptide (ANP) or brain natriuretic peptide (BNP) increased by neprilysin inhibitor are also reported to protect brain damage. Therefore, we investigate the possible protective effects of valsartan (VAL) compared with LCZ696 (LCZ) (VAL+ neprilysin inhibitor; 1:1) on the prevention of ischemic damage after stroke. Methods: Focal brain ischemia was induced by MCAO with the intraluminal filament technique in 10-week male C57BL/6J mice. Mice were treated with VAL (3 mg/kg) or LCZ (6 mg/kg) orally as powder in gelatin mini-capsules daily for 2 weeks before MCAO. Ischemic area was evaluated by triphenyl tetrazolium chloride staining, cerebral blood flow (CBF) by laser-Doppler flowmetry, oxidative stress by dihydroethidium staining. Results: Telemetry method showed that systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) did not change before and after treatments. Protective effect of LCZ treatment on ischemic brain damage in each brain section seemed to be more marked than that of VAL treatment. The average ischemic area ratio showed a significant reduction in VAL and LCZ groups compared with CON group. CBF in the peripheral region around ischemic core was significantly improved after treatments. Moreover, VAL and LCZ treatments significantly decreased the increase of superoxide anion production in the cortex of ischemic side. However, significant differences of CBF and superoxide anion production were not observed in VAL and LCZ. Conclusions: Both valsartan and LCZ696 treatments exerted preventive effects on ischemic stroke. The preventive effect of LCZ696 seemed more prominent than VAL. LCZ696 could be a novel powerful approach to protect brain after stroke. Further investigation has been performed to clarify the protective effect of LCZ696 on ischemic brain damage.

Cross talk between the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT 1 ) 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 (AT 2 ) 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)/AT 2 receptor axis are involved in the inhibitory effects of AT 1 receptor blockers on vascular remodeling. Wild-type, Mas-knockout, and AT 2 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 AT 1 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-1β, 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 AT 2 receptor mRNA but did not affect AT 1 receptor mRNA or the decrease in Mas mRNA. The inhibitory effect of Ang-(1–7) on neointimal formation was less marked in AT 2 receptor knockout mice compared with wild-type mice. These results suggest that blockade of the AT 1 receptor by azilsartan could enhance the activities of the ACE2/Ang-(1–7)/Mas axis and ACE2/Ang-(1–7)/AT 2 receptor axis, thereby inhibiting neointimal formation.

Objectives: Crosstalk between the angiotensin (Ang)-converting enzyme (ACE)/Ang II/ Ang II type 1 (AT 1 ) receptor axis and ACE2/Ang (1-7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Moreover, possible stimulation of Ang II type 2 (AT 2 ) receptor by Ang-(1-7) has been highlighted as new axis; however, pathophysiological significance of this possibility has to be elucidated in more detail. Therefore, we examined the possibility whether ACE2/Ang (1-7)/Mas axis and Ang (1-7)/AT 2 receptor axis is involved the inhibitory effects of AT 1 receptor blocker (ARB) on vascular remodeling. Methods: Ten-week-old male C57BL/6J (wild type: WT), Mas knockout (MasKO), and AT 2 receptor knockout (AT 2 KO) mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. After cuff placement, some mice were treated with azilsartan (0.3 mg/kg/day), an ARB, or Ang (1-7) (0.5 mg/kg per day). Two weeks after operation, femoral arteries were removed, fixed and stained by Elastica van Gieson staining for evaluating neointimal formation. Results: Neointima formation induced by cuff placement was further increased in MasKO mice compared with WT mice. Treatment with azilsartan or Ang-(1-7) decreased neointima area and vascular smooth muscle cell proliferation, and attenuated the increases in the mRNA level of MCP-1, TNF-α and IL-1β in the injured artery in WT mice without influencing blood pressure. These inhibitory effects of azilsartan or Ang-(1-7) were less in Mas KO mice. One week after cuff placement, the mRNAs of ACE2 and Mas in the injured artery were markedly decreased in WT mice. Administration of azilsartan or Ang-(1-7) attenuated the decrease in ACE2 mRNA, but not affect the decrease in Mas mRNA. Neointima formation in AT 2 KO mice was more exaggerated compared with that in WT mice. Interestingly, we observed that inhibitory effect of Ang-(1-7) on neointima formation was less in AT 2 KO mice compared with WT mice. Conclusion: These results suggest that blockade of AT 1 receptor by azilsartan could enhance the activities of the ACE2/Ang (1-7)/Mas axis and ACE2/Ang-(1-7)/AT 2 receptor axis, thereby inhibiting the neointima formation.

Background Sarcopenia is the degenerative loss of skeletal muscle mass which impaired quality of life in the elderly. On the other hand, sarcopenic obesity which is a loss of muscle and a concomitant increase in fat is reported to increase in life-style related diseases. Especially the prevalence of sarcopenia is greater in patients with type-2 diabetes mellitus (T2DM) than in non-diabetic subjects. However, the detailed mechanism of such muscle degeneration has not been well established. Here, we investigated muscle regeneration using muscle-injured models in life-style related disease mouse model such as angiotensin II overexpression mice for hypertension and obese diabetic mice for diabetes. Methods Male eight-week-old wild-type mice (C57BL6), Tsukuba hypertensive mice (transgenic mice carrying human renin and angiotensinogen genes) and T2DM mice (KKAy) were undergone intramuscular injection of cardiotoxin (Ctx) (100uL/10uM) into tibia. After two weeks, muscle were removed and stained with hematoxylin and eosin. KKAy replaced by bone marrow prepared from GFP-transgenic mice were generated by 8 Gy whole body X-ray irradiation. Results Treatment with Ctx showed increased muscle satellite cells where nucleuses were observed in the center of cells. In Tsukuba hypertensive mice, inflammatory cells were significantly accumulated in muscle compared with C57BL6, but muscle regeneration was not significantly impaired. KKAy mice exhibited impaired muscle regeneration. Tibia weight was a half in Ctx-injected KKAy compared with Ctx non-injected KKAy, and Ctx-injected and non-injected C57BL6 tibia. Moreover, remarkable fat deposition was observed in Ctx-treated muscle of KKAy. Such change was also observed in another diabetic mouse model, db/db, but not in streptozocin-induced diabetic mice. Fat deposition was remarkably increased in aged KKAy (6-month-old) compared with younger mice (8-week-old). In GFP-chimeric mice, fat tissue showed highly GFP-positive indicating that fat tissue was generated from marrow cells. Conclusion Diabetic mice showed significantly impaired muscle regeneration with fat deposition, suggesting that diabetes enhanced sarcopenic obesity possibly due to inhibitory effect of marrow cell differentiation.

Objective: Diabetes is associated with abnormal regulation of appetite. Dopaminergic activation in food reward induces binge eating disorder (BED) and leads to increase in food intake. On the other hand, recent studies have demonstrated that stimulation of angiotensin II type 2 (AT 2 ) receptor inhibits dopamine (DA) synthesis. In this study, we investigated the relationship between DA level and BED after fasting condition in diabetic mice and the inhibitory effect of AT 2 receptor stimulation on BED and body weight. Methods: Adult male type 2 diabetic mice, KKAy, at 10 weeks of age were treated with an intraperitoneal injection of AT 2 receptor agonist, compound 21 (C21), at the dose of 10 μg/kg/day or saline for 2 weeks. DA level in the striatum was measured by microdialysis. Short-term spatial memory and activity were assessed by Y-maze test. After these tests, brain samples were obtained and the expressions of DA receptor D1 (DRD1), DA receptor D2 (DRD2) and DA transporter (DAT) in the substantia nigra were evaluated by immunohistochemical staining. Results: Food and water intake and DA level in the striatum were significantly increased 48 hours after fasting compared with non-fasting mice. Administration with C21 significantly attenuated these increase without affecting systolic blood pressure. Treatment with C21 remarkably improved short-term cognitive function and increased explore activity. Moreover, C21 treatment did not affect fasting-induced weight loss, but significantly inhibited rebound weight gain after refeeding compared with vehicle-treated group. In KKAy, the expressions of DRD1, DRD2 and DAT in the substantia nigra were markedly decreased compared with wild-type (C57BL/6J strain) mice, whereas these reductions were improved by administration of C21. Interestingly, we demonstrated that fasting decreased DRD1 and DRD2 expression and increased DAT; however, administration of C21 inhibited these reductions of DRD1 and DRD2. Conclusion: Activation of AT 2 receptor could contribute to inhibition of BED and rebound weight gain with modulation of dopamine signaling. These results indicate that stimulation of AT 2 receptor may be a new therapeutic approach to improve the eating disorder associated with dopamine resistance.

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.

Objectives: Previous our studies showed that angiotensin II type-2 (AT 2 ) receptor stimulation protects neurons after brain ischemic damage using AT 2 receptor-deficient mice. Recently, Compound 21 (C21) is available to use as a direct AT 2 receptor agonist. We reported that administration of C21 induces vascular dilatation via bradykinin-nitric oxide pathway and maintains cerebral blood flow, resulting in prevention of cognitive impairment. These results inspired us the possibility that administration of C21 could protect ischemic brain damage; therefore, we assessed the effect of C21 on stroke expansion by not only pre-treatment, but also a treatment immediately after stroke. Methods: 10 week-old wild-type male C57BL6 mice were subjected to the middle cerebral artery occlusion (MCAO) by electrocoagulation using a subtemporal approach. Ischemic area after stroke was evaluated by time-course analysis by magnetic resonance imaging (MRI) of the brain. C21 was administrated to mice 2 weeks before MCAO or immediately after MCAO treatment by intraperitoneal injection. Cerebral blood flow was evaluated by using 2D-laser speckle blood flow imaging system. Results: No significant remarkable change was observed in blood pressure in mice with or without C21 treatment. Pretreatment of C21 prevented ischemic brain damage 1 day after MCAO. In contrast, such preventive effect by C21 was not observed in AT 2 receptor-deficient mice. On the other hand, C21 treatment immediately after MCAO did not reduce ischemic area 1 day after MCAO, but remarkably reduced this 3 days after MCAO. Treatment with C21 prevented the reduced cerebral blood flow after MCAO. Conclusions: These findings indicate that AT 2 receptor stimulation by C21 prevents ischemic brain damage at least in part via maintaining cerebral blood flow even after stroke. Therefore, administration of C21 could work as a new therapeutical option in patients with stroke even in acute phase.

Objective: Classical renin-angiotensin system (RAS) is mainly known as angiotensin (Ang) converting enzyme (ACE)/Ang II/Ang type 1 (AT1) receptor axis which induces various organ damages including cognitive decline. On the other hand, ACE2/Ang-(1–7)/Mas axis has been known to exert antagonistic actions against the classical RAS axis in cardiovascular system. However, the roles of ACE2/Ang-(1–7)/Mas axis in cognitive function remain to be elucidated. Here, we examined possible roles of ACE2/Ang-(1–7)/Mas axis in cognitive function in vascular dementia model. Design and Method: Male 10-week-old C57BL6 (wild-type: WT) mice, Mas1 knockout (MasKO) mice, Angiotensin type2 receptor knockout (AT2KO) mice and AT2/Mas1 double KO (WKO) mice were all conducted bilateral carotid artery stenosis (BCAS) surgery. After 6 week of surgery they were subjected to the Morris water maze task for cognitive function. Cerebral blood flow (CBF) was analyzed by laser speckle flowmetry after the cognitive task. Receptor expressions and inflammatory cytokine levels were determined by real-time RT-PCR. Results: Spatial cognitive function was significantly impaired in AT2KO mice and WKO mice compared with WT mice after BCAS (As shown in Figure 1). On the other hand, MasKO mice showed no deterioration in cognitive function after BCAS surgery even though their CBF were significantly lower than sham-operated mice. Receptor expression of AT1, AT2 receptors and MAS showed no difference between groups. There were no significant differences between groups in inflammatory cytokine expressions. Conclusions: These results indicate that AT2 receptor signal plays more important role in maintenance of cognitive function than Mas receptor signal does. In ischemic brain conditions, whether ACE2/Ang-(1–7)/Mas axis has protective effect or not should be assessed carefully. Further investigation is necessary to understand the detailed mechanism.