Akinori Higaki

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.

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.

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.

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.

MPS 13-09 AT2 RECEPTOR STIMULATION INHIBITS PHOSPHATE-INDUCED VASCULAR CALCIFICATION

Objective: A variety of evidence suggests that angiotensin II type 2 (AT2) receptor stimulation exerts beneficial counter-regulatory roles against angiotensin II type 1 (AT1) receptor action in cardiovascular remodeling. Whereas, vascular calcification is a common finding in atherosclerosis and a serious problem in diabetic and chronic kidney disease. Moreover, some recent reports suggest that the renin-angiotensin system plays a role in the pathogenesis of cardiovascular remodeling. Here, we examined the possibility of whether AT2 receptor stimulation is involved in the inhibitory effects on phosphate-induced vascular calcification. Design and Method: Ex vivo study, thoracic aorta sections were prepared from C57BL/6 (WT), AT2 receptor-knockout (AT2KO) and smooth muscle cell-specific AT2 receptor overexpressed (smAT2Tg) mice and then cultured in DMEM supplemented with inorganic phosphate (Pi) to induce vascular calcification. Morphometric assessment of medial calcium deposition was quantitatively performed, and the amount of dissolved calcium was measured. In vitro study, primary vascular smooth muscle cells were prepared from WT, AT2KO, smAT2Tg mice and cultured like ex vivo study. Results: Ex vivo, Vascular calcification in thoracic aortas of smAT2Tg mice was significantly attenuated compared with AT2KO and WT mice. In vitro, VSMC cultured in DMEM containing high-phosphate showed dose- and time-dependent increase in mineral deposition. Vascular calcification induced by phosphate-stimulation markedly attenuated in smAT2Tg mice compared with other mice. Conclusions: Our results suggest that AT2 receptor stimulation is an efficient endogenous vasoprotective factor in vascular calcification. The selective stimulation of AT2 receptor may decrease the risk of vascular calcification and subsequent adverse cardiovascular events during chronic kidney disease.

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

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

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

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

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

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

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

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