Bao-Shuai Shan

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-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.

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.

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-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.

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-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.

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

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

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

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.

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

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

Angiotensin II type 2 receptor signaling affects dopamine levels in the brain and prevents binge eating disorder

Introduction: Binge eating disorder (BED) is associated with dopaminergic activation as food reward, resulting in metabolism-related disorders. Stimulation of angiotensin type 2 (AT2) receptor is reported to inhibit dopamine synthesis. We investigated the possible roles of AT2 receptor-mediated dopamine regulation in the pathogenesis of BED. Materials and methods: Male C57BL/6 mice, type 2 diabetic (KKAy) mice and AT2 receptor-null (AT2KO) mice at eight weeks old were treated with AT2 receptor agonist, compound 21 (C21) or saline for two weeks. Mice were subjected to fasting for two days followed by re-feeding for seven days. Results: Treatment with C21 attenuated the rebound proportion of body weight, food intake and water intake in KKAy mice, but not in C57BL/6 and AT2KO mice. Dopamine concentration in the striatum was further increased by fasting in KKAy and AT2KO mice. Administration of C21 significantly attenuated this fasting-induced increase in dopamine level only in KKAy mice. Dopamine receptor D1, D2 expression in the substantia nigra were markedly lower in KKAy mice compared with C57BL/6 mice, while administration of C21 increased their expression in KKAy mice. Conclusions: Our study suggests that AT2 receptor stimulation may be a new therapeutic approach to improve eating disorder associated with dopamine resistance.

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

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

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

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