Kumiko Takemori

Role of angiotensin II type 1 receptor in the leucocytes and endothelial cells of brain microvessels in the pathogenesis of hypertensive cerebral injury.

Objectives To elucidate the mechanisms of activation of polymorphonuclear neutrophils (PMNs) and their adhesion to endothelial cells in hypertensive cerebral injury, and to determine the effects of angiotensin II type 1 (AT1) receptor antagonism on PMNs and endothelial cells. Design We examined expression of AT1 receptor in PMNs in relation to that in endothelial cells of brain microvessels, using mature stroke-prone spontaneously hypertensive rats (SHRSP). Methods To investigate the expression of AT1 receptor, we used 23-week-old male spSHRs and age-matched Wistar–Kyoto (WKY) rats. For the effects of AT1 receptor blockade, the AT1 receptor antagonist, TCV-116, was orally administered at a dosage of 0.5 mg/kg per day for 4 weeks in rats from age 19 weeks. A PMN-rich fraction was obtained by density gradient using Ficol-hypaque. AT1 receptor expression in PMNs was investigated by immunohistochemistry (avidin–biotinylated peroxidase complex method) and reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of macrophage-1 (Mac-1) in PMNs was examined by flow cytometry. Expression of intercellular adhesion molecule-1, glucose transporter-1 and fibrinogen in the cerebral cortex (occipital region) was investigated by immunohistochemistry. Results AT1 receptor was identified in PMNs by both immunohistochemistry and RT-PCR. It was also detected in the cerebral cortex. Expression in both types of cells was much more intense in spSHRs than in WKY rats. AT1 receptor antagonism ameliorated the enhanced expression of Mac-1 in PMNs. In addition, it was confirmed that enhanced expression of adhesion molecules and increased permeability of brain microvessels were decreased by AT1 receptor antagonism. Conclusions The results indicate that both PMNs and brain microvessel endothelial cells possess AT1 receptor, that AT1 receptor antagonism ameliorates endothelial injury via inhibition of PMNs and endothelial cell adhesion, and that angiotensin II must be a key factor in hypertensive endothelial injury.

Effects of the AT1 receptor antagonist on adhesion molecule expression in leukocytes and brain microvessels of stroke-prone spontaneously hypertensive rats

To elucidate the possible involvement of angiotensin II (AII) in the pathogenesis of microvascular changes in severe hypertension, we investigated the effects of angiotensin II type 1 (AT1) receptor antagonist and angiotensin-converting enzyme inhibitor (ACEI) on the expression of adhesion molecules of leukocytes and brain microvessels. Male stroke-prone spontaneously hypertensive rats (SHRSP) at 19 weeks of age were divided into three groups and age-matched Wistar-Kyoto rats (WKY) were used as the control group. AT1 receptor antagonist (TCV-116, 0.5 mg/kg/day) and ACEI (captopril, 20 mg/kg/day) were administered to SHRSP for 4 weeks. Mac-1 expression in leukocytes was investigated by flow cytometric analysis. For endothelial cells, we examined the expression of intercellular adhesion molecule-1 (ICAM-1), the AT1 receptor, and glucose transporter-1 (GLUT-1, a marker of the blood-brain barrier) using reverse transcription-polymerase chain reaction (RT-PCR). The blood pressure of AT1 receptor antagonist and ACEI-treated groups was slightly lower than that of the control, but was still greater than 220 mm Hg. Mac-1 expression, as well as ICAM-1 expression, was higher in control SHRSP than in WKY. Such enhanced expression of adhesion molecules in SHRSP was ameliorated by the administration of AT1 receptor antagonist or ACEI, the former being more effective. AT1 receptor expression was higher in control SHRSP than in WKY, and was lower in the AT1 receptor antagonist group, whereas no difference was found in the ACEI group. No significant differences were found in GLUT-1 expression among all groups. In the case of hypertensive cerebral injuries in SHRSP, leukocytes may have an important role for initiation via adhesion to endothelial cells. AT1 receptor antagonist showed a beneficial effect for the amelioration of enhanced expression of adhesion molecule in both leukocytes and endothelial cells. Thus, AII seems to be an important mediator for the hypertensive microvascular injuries.

Food restriction improves glucose and lipid metabolism through Sirt1 expression: a study using a new rat model with obesity and severe hypertension.

AIMS To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome. MAIN METHODS Male, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPARα, γ and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle. KEY FINDINGS FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPARα and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUT4 expression in skeletal muscle. SIGNIFICANCE These results indicate that FR corrects adipokine dysfunction by activating PPARγ via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPARα in the liver.

AT1 Receptor Antagonist Prevents Brain Edema Without Lowering Blood Pressure

In order to investigate the role of Angiotensin II (AII) for the vasogenic cerebral edema, the AT1 receptor antagonist (TCV-116) was administered to 19-week-old stroke-prone spontaneously hypertensive rats (SHRSP) for 2 weeks at a dosage which did not decrease the blood pressure. Although no remarkable changes were found in blood pressure after treatment, the average brain weight of the treated group was relatively lower as compared to that of control SHRSP and no edematous changes were found in any brains. The immunohistochemical expression of intercellular adhesion molecule-1 (ICAM-1) was less and the glucose transporter-1 (GLUT-1) expression was much more intense in the endothelial cells of the micro vessels in the cerebral cortex of the treated group. Fibrinogen expression around micro-vessels was also remarkably reduced in the treated group. A decreased expression of ICAM-1 in the treated group was confirmed by RT-PCR analysis. These results indicate that the AT1 receptor blockade ameliorates hypertensive cerebral injury in a blood pressure-independent manner and suggest that AII may have an important role for endothelial injury in severe hypertension.

A newly established strain of spontaneously hypertensive rat with a defect of ascorbic acid biosynthesis

To investigate the effects of ascorbic acid deficiency on the pathogenesis of hypertension and/or its complications, we established a rat strain with both genetic hypertension and a defect of ascorbic acid biosynthesis. The od gene (L-gulono-gamma-lactone oxidase gene) of the ODS (Osteogenic Disorder Shionogi) rat, which is a rat mutant unable to synthesize ascorbic acid, was introduced into spontaneously hypertensive rats (SHR), and a novel congenic strain, SHR-od, was established. SHR-od showed scurvy when fed an ascorbic acid-free diet. Systolic blood pressure of male SHR-od began to increase at 9 weeks of age and reached 190-200 mmHg at 20 weeks of age. In 25-week-old SHR-od, ascorbic acid deficiency when fed an ascorbic acid-free diet for 6 weeks caused a remarkable reduction of blood pressure to lower than 110 mmHg. The wall to lumen ratio of the testicular artery in ascorbic acid-deficient SHR-od was lower than that of the control rats. When rats were fed a diet supplemented with ascorbic acid (300 mg/kg), ascorbic acid concentration in SHR-od was lower in the serum and liver than that in ODS rats. These results indicate that ascorbic acid could be closely related to the development of hypertension in SHR-od. We believe that SHR-od will be a useful model for experimental studies on hypertension and its complications, since all of them suffer from hypertension spontaneously and the level of ascorbic acid deficiency in these rats could be controlled at will both in concentration and duration.

Short-term treatment of stroke-prone spontaneously hypertensive rats with an AT1 receptor blocker protects against hypertensive end-organ damage by prolonged inhibition of the renin-angiotensin system.

1 The aim of the present study was to investigate the effects of short‐term treatment with an AT1 receptor blocker (ARB) on amelioration of hypertensive end‐organ damage in stroke‐prone spontaneously hypertensive rats (SHRSP). 2 Male SHRSP were divided into two groups: (i) an ARB‐treated group; and (ii) a control group. Candesartan (1 mg/kg per day) was administered orally from 6 to 11 weeks of age. At 20 weeks of age, plasma renin activity (PRA), angiotensin II concentrations, angiotensin‐converting enzyme (ACE) activity and hydroperoxide content were measured. Expression of intercellular adhesion molecule (ICAM)‐1, renin, AT1 and AT2 receptors was investigated by reverse transcription–polymerase chain reaction. 3 Blood pressure in the ARB group was slightly lower at 7, 8, 11, 13–15 and 18 weeks of age, but no significant difference in blood pressure was found between the ARB and control groups at 20 weeks of age. All rats in the control group had cerebral oedema, whereas no lesions were found in the ARB group. In the ARB group, PRA, AII and hydroperoxide content were lower than in the control group. In the ARB‐treated group, lower ICAM‐1 expression was found in the cerebral cortex and slightly, albeit not significantly, lower expression of renin was found in the kidney. In contrast, AT1 receptor expression in the cerebrum and kidney was higher in the ARB group compared with the control group. 4 These results indicate that short‐term treatment of SHRSP with ARB at a young age is effective in preventing cerebral oedema after maturation. Such beneficial effects of ARB may be due, in part, to decreased blood pressure and is likely mainly due to inhibition of total circulating and local renin–angiotensin systems.

Effects of Inducible Nitric Oxide Synthase Inhibition on Cerebral Edema in Severe Hypertension

In order to clarify the causative role of cytotoxic nitric oxide (NO) in hypertensive cerebral injury, the effects of inducible nitric oxide synthase (iNOS) inhibition on leukocytes and endothelial function were examined using stroke-prone spontaneously hypertensive rats (SHRSP). For the iNOS inhibition, S-methylisothiourea (SMT) was administered to 12-week-old male SHRSP for 3 weeks. Immunohistochemical examination were carried out for the expression of intercellular adhesion molecule-1 (ICAM-1), glucose transporter-1 (GLUT-1), fibrinogen and glial fibrillary acidic protein (GFAP) in cerebral cortex. The effects of iNOS inhibition was also examined for Mac-1 expression by flow cytometric analysis. Plasma NO metabolites level was significantly lower in the SMT group than in the control group. Mac-1 expression was inhibited by SMT. In the SMT group, brain weight was significantly lower than in the control. By SMT administration, ICAM-1 expression was suppressed, GLUT-1 was enhanced, fibrinogen was decreased and GFAP was decreased as compared to those in control group. In hypertensive cerebral injury in SHRSP, iNOS-derived NO, mainly in activated leukocytes, could be an important causative factor for endothelial injury.

Ternatin, a cyclic peptide isolated from mushroom, and its derivative suppress hyperglycemia and hepatic fatty acid synthesis in spontaneously diabetic KK-Ay mice

(-)-Ternatin is a highly methylated cyclic heptapeptide isolated from mushroom Coriolus versicolor. Ternatin has an inhibitory effect on fat accumulation in 3T3-L1 adipocytes. [D-Leu(7)]ternatin, a ternatin derivative, also inhibited fat accumulation in 3T3-L1 cells, although the effectiveness of [D-Leu(7)]ternatin was lower than that of ternatin. In this study, we investigated the effects of ternatin and [D-Leu(7)]ternatin on obesity and type 2 diabetes in KK-A(y) mice, an animal model for spontaneously developed type 2 diabetes. We continuously administered ternatin (8.5 or 17 nmol/day) or [D-Leu(7)]ternatin (68 nmol/day) to mice via a subcutaneous osmotic pump. Unexpectedly, neither ternatin nor [D-Leu(7)]ternatin affected body weight or adipose tissue weight in KK-A(y) mice. In contrast, it was demonstrated that both ternatin and [D-Leu(7)]ternatin suppress the development of hyperglycemia. In liver, the SREBP-1c mRNA level tended to be lower or significantly decreased in mice treated with ternatin or [D-Leu(7)]ternatin, respectively. Moreover, we found that ternatin directly lowered the SREBP-1c mRNA level in Hepa1-6 hepatocyte cells. This study showed that ternatin and [D-Leu(7)]ternatin each had a preventive effect on hyperglycemia and a suppressive effect on fatty acid synthesis in KK-A(y) mice.

Changes in catecholamine metabolism by ascorbic acid deficiency in spontaneously hypertensive rats unable to synthesize ascorbic acid.

We have previously reported the establishment of a novel rat strain, SHR-od, with both spontaneous hypertension and a defect of ascorbic acid biosynthesis. Blood pressure in mature SHR-od fed an ascorbic acid-supplemented diet is over 190-200 mmHg, while it decreased to around 120 mmHg at 4-5 weeks after the cessation of ascorbic acid supplementation. With regard to possible mechanisms of blood pressure lowering, we focused on catecholamine synthesis in adrenal glands, since catecholamine is a major factor for blood pressure regulation and ascorbic acid is a co-factor of dopamine beta-hydroxylase (DBH) in catecholamine biosynthesis. Male SHR-od (25-week-old) and normotensive ODS rats with a defect in ascorbic acid biosynthesis (25-week-old) were fed a Funabashi-SP diet with or without ascorbic acid (300 mg/kg diet) for 28 days or 35 days. In SHR-od, systolic blood pressure (191 +/- 6 mmHg) began to decrease from day 21 in the ascorbic acid-deficient group, whereas no significant difference was found in ODS rats. In spite of significant lowering of blood pressure, no significant differences were found in catecholamine levels in serum, adrenal glands and brain on day 28. On day 35, however, urinary excretion of norepinephrine and epinephrine in the ascorbic acid-deficient SHR-od were higher at 490% (P < 0.05) and 460% (P < 0.05) of the respective control. Serum catecholamine concentrations and the adrenal catecholamine content tended to be higher in the ascorbic acid-deficient SHR-od than the control of SHR-od and reached to similar level in ODS rats. The administration of ascorbic acid (intraperitoneal injection, 60 mg ascorbic acid/kg body weight, once a day) to the ascorbic acid-deficient SHR-od restored blood pressure to the range 180-190 mmHg within two days. These findings indicate that ascorbic acid deficiency affects catecholamine metabolism in the adrenal glands of SHR-od in response to blood pressure lowering, suggesting catecholamines are not involved in the mechanism for the remarkable reduction in blood pressure in response to ascorbic acid deficiency.

Effects of Angiotensin II Type 1 receptor blocker and adiponectin on adipocyte dysfunction in stroke-prone spontaneously hypertensive rats

BackgroundHypoadiponectinemia in lipoatrophy may be related to worsening of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). One of the beneficial effects of candesartan (Angiotensin II Type 1 receptor blocker) for preventing hypertension may be increasing of adiponectin due to improvement of adipocyte dysfunction. In this study, we determined the effects of candesartan or adiponectin on pathophysiologic features and adipocyte dysfunction in SHRSP.MethodsCandesartan was administered to male SHRSP from 16 to 20 weeks of age (2 mg/kg/day). Adiponectin was cloned and intravenously administered to male SHRSP from 16 to 20 weeks of age. We examined biological parameters, as well as the expression and release of adipokines.ResultsThe SHRSP exhibited severe atrophy of visceral fat and progression of severe hypertension. The expression and release of leptin and adiponectin were impaired at 6 and 20 weeks of age. Candesartan suppressed the development of lipoatrophy and reduced the incidence of stroke at 20 weeks of age. Candesartan also enhanced the expression of adiponectin and leptin by inducing the overexpression of peroxisome proliferator activated receptor γ. Circulating level of leptin was significantly higher in candesartan group than in the control group, whereas adiponectin was similar in both groups. Intravenous administration of adiponectin resulted in enhancement of adiponectin expression in adipose tissue, but no remarkable effects were found in pathophysiology in SHRSP.ConclusionsOur results indicate that candesartan protects against hypertension and adipocyte dysfunction in SHRSP. The induction of leptin expression appeared to be important factor in the inhibition of stroke lesions, whereas adiponectin was not a major regulator of blood pressure in SHRSP with genetic hypertension. Further studies are needed to elucidate the role of the renin–angiotensin system in adipose tissue dysfunction in relation to hypertensive end-organ damage.