Toshifumi Yamauchi

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.

Procalcitonin as a marker of respiratory disorder in neonates

Serum procalcitonin (PCT) increases in various respiratory disorders such as acute respiratory distress syndrome. Elevated PCT is also observed in healthy neonates. In this study, we investigated whether PCT is a good marker of respiratory disorder in neonates.

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.

Arrhythmia risk and β-blocker therapy in pregnant women with long QT syndrome

Background Pregnancy is one of the biggest concerns for women with long QT syndrome (LQTS). Objectives This study investigated pregnancy-related arrhythmic risk and the efficacy and safety of β-blocker therapy for lethal ventricular arrhythmias in pregnant women with LQTS (LQT-P) and their babies. Methods 136 pregnancies in 76 LQT-P (29±5 years old; 22 LQT1, 36 LQT2, one LQT3, and 17 genotype-unknown) were enrolled. We retrospectively analysed their clinical and electrophysiological characteristics and pregnancy outcomes in the presence (BB group: n=42) or absence of β-blocker therapy (non-BB group: n=94). Results All of the BB group had been diagnosed with LQTS with previous events, whereas 65% of the non-BB group had not been diagnosed at pregnancy. Pregnancy increased heart rate in the non-BB group; however, no significant difference was observed in QT and Tpeak–Tend intervals between the two groups. In the BB group, only two events occurred at postpartum, whereas 12 events occurred in the non-BB group during pregnancy (n=6) or postpartum period (n=6). The frequency of spontaneous abortion did not differ between the two groups. Fetal growth rate and proportion of infants with congenital malformation were similar between the two groups, but premature delivery and low birthweight infants were more common in those taking BB (OR 4.79, 95% CI 1.51 to 15.21 and OR 3.25, 95% CI 1.17 to 9.09, respectively). Conclusions Early diagnosis and β-blocker therapy for high-risk patients with LQTS are important for prevention of cardiac events during pregnancy and the postpartum period, and β-blocker therapy may be tolerated for babies in LQT-P cases.

Manifestation of recessive combined D-2-, L-2-hydroxyglutaric aciduria in combination with 22q11.2 deletion syndrome

22q11.2 deletion syndrome is one of the most common human microdeletion syndromes. The clinical phenotype of 22q11.2 deletion syndrome is variable, ranging from mild to life‐threatening symptoms, depending mainly on the extent of the deleted region. Brain malformations described in association with 22q11.2 deletion syndrome include polymicrogyria, cerebellar hypoplasia, megacisterna magna, and agenesis of the corpus callosum (ACC), although these are rare. We report here for the first time a patient who manifested combined D‐2‐ and L‐2‐hydroxyglutaric aciduria as a result of a hemizygous mutation in SLC25A1 in combination with 22q11.2 deletion. The girl was diagnosed to have ACC shortly after birth and a deletion of 22q11.2 was identified by genetic analysis. Although the patient showed cardiac anomalies, which is one of the typical symptoms of 22q11.2 deletion syndrome, her rather severe phenotype and atypical face prompted us to search for additional pathogenic mutations. Three genes present in the deleted 22q11.2 region, SLC25A1, TUBA8, and SNAP29, which have been reported to be associated with brain malformation, were analyzed for the presence of pathogenic mutations. A frameshift mutation, c.18_24dup (p.Ala9Profs*82), was identified in the first exon of the remaining SLC25A1 allele, resulting in the complete loss of normal SLC25A1 function in the patients cells. Our results support the notion that the existence of another genetic abnormality involving the retained allele on 22q11.2 should be considered when atypical or rare phenotypes are observed.

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.

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.

[PP.35.08] CEREBRAL AT2 RECEPTOR-INTERACTING PROTEIN ENHANCES PROTECTIVE EFFECTS OF AT2 RECEPTOR STIMULATION ON ISCHEMIC BRAIN DAMAGE

Objective: We previously reported that the ischemic brain area was significantly larger in angiotensin II type 2 (AT2) receptor-deficient mice after middle cerebral artery (MCA) occlusion compared to wild-type (WT) mice. 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 might play key roles in diverse mechanisms of AT2 receptor signaling. We investigated the possibility that ATIP could enhance the protective effects compound 21 (C21), a selective non-peptidic AT2 receptor agonist, on focal cerebral ischemia. Design and method: Ten week-old male ATIP1-transgenic (ATIP1-Tg) and littermate (WT) mice were subjected to (MCA) occlusion with silicon-coated micro-filament. C21 (10 &mgr;g/kg/day) was administered 2 weeks before MCA occlusion. Twenty-four hours after MCA occlusion, ischemic area was assessed by triphenylterazolium chloride staining. Neurological deficit after MCA occlusion was evaluated using a neurological score. Cerebral blood flow (CBF) was measured by laser speckle flowmetry. Expression of inflammatory cytokines and methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor were measured by real-time RT-PCR. Results: Administration of C21 did not influence the blood pressure measured by tail-cuff method. There was no significant difference in ischemic size without C21 treatment between WT and ATIP-Tg mice. 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. Administration 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 tended to increase in ATIP1-Tg mice compared with WT mice. Expression of inflammatory cytokines such as TNF-&agr; and MCP-1 was increased in ipsilateral hemisphere compared with in contralateral hemisphere. Treatment of C21 tended to reduce inflammatory cytokine expression in ipsilateral hemisphere. Conclusions: These results suggested that ATIP1 could enhance the cerebral protective effects of AT2 receptor stimulation at least in part due to the improvement of CBF.

OS 17-04 DEFICIENCY OF ANGIOTENSIN CONVERTING ENZYME 2 CAUSES COGNITIVE IMPAIRMENT

Objective: The activity of angiotensin (Ang) converting enzyme (ACE)/Ang II/Ang II type 1 (AT1) receptor axis which is the classical renin-angiotensin system (RAS), causes 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 are unknown. Here, we explored possible roles of ACE2 in cognitive function. Design and Method: Male 10-week-old C57BL6 (wild-type: WT) mice and ACE2 knockout (KO) mice were used. Vascular dementia model was induced by bilateral common carotid artery stenosis (BCAS). Morris water maze task was performed 6 weeks after BCAS operation to evaluate spatial cognitive function. Results: There were no significant differences in body weight, brain weight and systolic blood pressure between ACE2KO and WT mice. ACE2KO mice exhibited significant impairment of cognitive function, compared with that in WT mice, with no significant difference in cerebral blood flow. Mas receptor mRNA was highly expressed in the hippocampus compared with the cortex, with no significant difference between ACE2KO and WT mice. AT1 receptor mRNA in the hippocampus was higher in ACE2KO mice compared with WT mice, whereas AT2 receptor mRNA in the hippocampus did not differ between the two strains. Superoxide anion production increased in ACE2KO mice, with increased NADPH oxidase subunits mRNAs in the hippocampus. Protein level of SOD3 decreased in ACE2KO mice compared with WT mice. Brain-derived neurotrophic factor (BDNF) mRNA and protein levels were lower in the hippocampus in ACE2KO mice compared with WT mice. Administration of Ang-(1–7) (0.5 mg/kg/day) attenuated the cognitive decline in ACE2KO mice without changes in systolic blood pressure. Conclusions: Taken together, ACE2 deficiency caused impaired cognitive function with enhancement of oxidative stress and decrease in BDNF level.