Possible involvement of angiotensin-converting enzyme 2 and Mas activation in inhibitory effects of angiotensin II Type 1 receptor blockade on vascular remodeling.

We explored the roles of angiotensin-converting enzyme 2 (ACE2), angiotensin-(1-7), and Mas activation in angiotensin II type 1 receptor blockade-mediated attenuation of vascular remodeling. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. After cuff placement, the mRNA level of both ACE2 and Mas was markedly decreased in wild-type mice, whereas ACE mRNA was not changed. Immunostaining of ACE2 and Mas was observed mainly in the media and was reduced in the injured artery. Administration of angiotensin-(1-7) decreased neointimal formation after cuff placement, whereas administration of [D-Ala(7)] angiotensin-(1-7), a Mas antagonist, increased it. Consistent with these results, we also demonstrated that neointimal formation induced by cuff placement was further increased in ACE2 knockout mice. In angiotensin II type 1a receptor knockout mice, mRNA expression and immunostaining of ACE2 and Mas in the injured artery were greater, with less neointimal formation than in wild-type mice. Increased ACE2 expression in the injured artery was also observed by treatment of wild-type mice with an angiotensin II type 1 receptor blocker, olmesartan. These results suggested that activation of the ACE2-angiotensin-(1-7)-Mas axis is at least partly involved in the beneficial effects of angiotensin II type 1 receptor blockade on vascular remodeling.

Renin inhibitors inhibited the activity of recombinant human renin but not activity in healthy human plasma.

BACKGROUND: Activity of renin substrate cleavage (renin-like activity) was measured in vitro in plasma samples obtained from healthy human volunteers. METHODS: Renin-like activity was determined using FRET (Fluorescence Resonance Energy Transfer) human renin substrate. Recombinant human renin and human plasma showed dose-dependent cleavage activity of FRET human renin substrate. RESULTS: Activity of recombinant human renin was completely inhibited by either a peptidergic or a non-peptidergic renin inhibitor. However, renin-like activity in human plasma was not inhibited by these renin inhibitors. In a mixture of recombinant renin and human plasma, renin inhibitors inhibited only that part of the activity caused by recombinant renin, while the activity in plasma still remained. Human plasma did not show cleavage activity of rat FRET renin substrate. Native human prorenin showed cleavage activity of human renin substrate. This activety was also completely inhibited by renin inhibitors. Immunoprecipitation with anti-renin or anti-prorenin antibodies did not reduce the activity in human plasma. Renin-like activity in human plasma was abolished by degeneration of protein when sample was heated to 95 degrees C. Activity of both recombinant renin and human plasma was significantly inhibited by a protease inhibitor cocktail. CONCLUSIONS: These results suggest that the activity of renin substrate cleavage in human plasma is not mainly caused by the renin or prorenin molecule, but probably by other proteases.

Role of angiotensin-converting enzyme 2 in cardiac hypertrophy induced by nitric oxide synthase inhibition.

OBJECTIVE: Angiotensin-converting enzyme 2 (ACE2) generates angiotensin-(1-7) [Ang-(1-7)], a peptide highlighted as exerting a pivotal role in cardiovascular remodeling. Moreover, the ACE2/Ang-(1-7)/Mas axis directly activates endothelial nitric oxide (NO) synthase and NO generation in the heart. However, the role of ACE2 in cardiovascular remodeling induced by persistent inhibition of NO under chronic activation of the renin-angiotensin system (RAS) remains poorly understood. METHODS AND RESULTS: Chimeric hypertensive mice that exhibit activation of the human RAS were produced by mating human renin (hRN) and human angiotensinogen (hANG) transgenic mice. Persistent NO inhibition with NG-nitro-L-arginine methyl ester (L-NAME) was started at 8 weeks of age for 4 weeks. After administration of L-NAME, blood pressure (BP) markedly increased in the chimeric mice (hRN/hANG-Tg), whereas wild-type mice (C57BL/6J) showed little increase in BP. Cardiovascular remodeling with enhanced oxidative stress in hRN/hANG-Tg was markedly accelerated by NO inhibition compared with that in wild-type mice. Moreover, ACE2 mRNA expression and activity in cardiac tissue were markedly reduced in L-NAME-treated hRN/hANG-Tg. Co-administration of an angiotensin II type 1 (AT1) receptor blocker (ARB), olmesartan, inhibited L-NAME-induced cardiovascular remodeling and improved the reduction in cardiac ACE2. The preventive effect of olmesartan on cardiac hypertrophy was blunted by co-administration of a selective Ang-(1-7) antagonist, [D-Ala7]-Ang-(1-7). CONCLUSION: Our findings demonstrate that cardiovascular remodeling induced by persistent NO inhibition was enhanced in hRN/hANG-Tg. An ARB, olmesartan, blunted cardiac remodeling induced by NO inhibition with RAS activation partially through the ACE2/Ang-(1-7)/Mas axis in addition to directly through its classical ACE/Ang II/AT1 receptor axis-blocking action.

Possible involvement of AT2 receptor dysfunction in age-related gender difference in vascular remodeling.

This study explored the possible involvement of AT(2) receptor stimulation in the age-related gender difference in vascular remodeling of mouse femoral artery induced by cuff placement. In the young adult group of wild-type mice (10 weeks of age), the increase in DNA synthesis, neointimal formation, expression of chemokines, and superoxide anion production in the injured femoral artery were smaller in female than in male mice. These gender differences were smaller in the aged group (50-55 weeks of age) of wild-type mice, because vascular responses of female mice in the aged group were stronger than those in the young group. Treatment with 17ß-estradiol attenuated vascular remodeling in aged female mice. AT(2) receptor expression in the injured artery was higher in female than in male in the young group. AT(2) receptor expression in the injured artery of female mice was lower in the aged group than in the young group. Lack of AT(2) receptor increased neointimal formation in the aged group and reduced the inhibitory action of 17ß-estradiol in aged female mice. Our findings suggest a possibility that the change in AT(2) receptor stimulation by aging might be involved in the response to estrogen and improvement of vascular remodeling in the aged female group.

Irbesartan increased PPARγ activity in vivo in white adipose tissue of atherosclerotic mice and improved adipose tissue dysfunction.

The effect of the PPARγ agonistic action of an AT(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 50mg/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 PPARγ in white adipose tissue and the DNA-binding activity of PPARγ 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 PPARγ and improved adipose tissue dysfunction including insulin resistance.

Temporary treatment with AT1 receptor blocker, valsartan, from early stage of hypertension prevented vascular remodeling.

BACKGROUND: The present study examined the inhibitory action of temporary treatment with an angiotensin type 1 (AT(1)) receptor blocker (ARB) on vascular remodeling using hypertensive mice with overexpression of the human renin (hRN) and angiotensinogen (hANG) genes. METHODS: hRN/hANG transgenic mice (hRN/hANG-Tg) were treated with an ARB, valsartan, from 4 weeks of age. In some mice, valsartan treatment was stopped at 8 weeks of age (temporary treatment). Inflammatory vascular injury was induced by polyethylene-cuff placement around the femoral artery at the age of 10 weeks. RESULTS: Compared with wild-type (WT) mice, hRN/hANG-Tg showed higher blood pressure (BP) and enhancement of oxidative stress and medial thickening even before cuff placement. Inflammatory vascular remodeling and oxidative stress after cuff placement were further enhanced in hRN/hANG-Tg. Temporary treatment with valsartan continuously lowered BP even after cessation of administration, and inhibited these changes. In contrast, administration of hydralazine lowered BP to a similar level to that with valsartan, but did not inhibit medial thickening and inflammatory vascular remodeling. In contrast to the valsartan treatment, BP immediately increased to the untreated level after cessation of hydralazine. CONCLUSIONS: These results indicate that temporary ARB treatment leads to prolonged effect of BP lowering and prevents vascular remodeling in hypertensive mice induced by activation of the human renin-angiotensin system. The inhibitory action of valsartan is not due to the BP lowering but is at least in part due to a decrease in oxidative stress and inflammation.

Nifedipine, a calcium-channel blocker, attenuated glucose intolerance and white adipose tissue dysfunction in type 2 diabetic KK-A(y) mice.

BACKGROUND: To explore the metabolic actions of nifedipine on diabetes, we examined glucose intolerance and white adipose tissue changes in type 2 diabetic KK-A(y) mice. METHODS: Male KK-A(y) mice were treated with nifedipine (1.5 mg/kg/day in lab chow) for 5 weeks, which did not affect blood pressure or feeding of KK-A(y) mice. RESULTS: After treatment with nifedipine, body weight tended to decrease and the weight of white adipose tissue was reduced. Without food restriction, nifedipine decreased plasma insulin level, while plasma glucose level tended to decrease. In oral glucose tolerance test, nifedipine suppressed the increase in glucose level after a glucose load without affecting plasma insulin concentration. Nifedipine also improved the result of insulin tolerance test. In white adipose tissue, nifedipine increased adipocyte number and the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and adipocyte fatty acid-binding protein related to adipocyte differentiation. In addition, expression of adiponectin, insulin receptor, insulin receptor substrate-1, and glucose transporter type-4 was also increased by nifedipine. Nifedipine also increased the expression of NO synthase in white adipose tissue. Nifedipine did not affect expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in white adipose tissue. Such changes in white adipose tissue were apparent in retroperitoneal adipose tissue. Nifedipine did not change the expression of angiotensin receptors, renin receptor, and angiotensinogen in white adipose tissue. Moreover, nifedipine attenuated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and increased superoxide dismutase (SOD) activity in white adipose tissue. CONCLUSION: These results suggest that nifedipine can enhance insulin sensitivity and reduce white adipose tissue, possibly related to stimulation of adipocyte differentiation.

Direct renin inhibition improved insulin resistance and adipose tissue dysfunction in type 2 diabetic KK-A(y) mice.

OBJECTIVE: The renin-angiotensin system affects insulin sensitivity mainly through the angiotensin II type 1 receptor. In this study, the effects of renin inhibition on insulin resistance and adipose tissue dysfunction were explored in type 2 diabetic KK-A(y) mice. METHODS AND RESULTS: Male KK-A mice were treated with a direct renin inhibitor, aliskiren, administered subcutaneously at a dose of 50 mg/kg per day for 14 days using an osmotic minipump. This dose of aliskiren strongly inhibited plasma renin activity and lowered blood pressure about 17% in KK-A(y) mice. Aliskiren decreased body weight and plasma glucose level, and increased plasma insulin level in a fed condition. Aliskiren also lowered the plasma levels of cholesterol, fatty acids and triglycerides. In the oral glucose tolerant test, the plasma glucose elevation after glucose load was reduced by aliskiren, without a significant change in insulin level. Insulin tolerance test showed that aliskiren enhanced insulin's effect on plasma glucose. Aliskiren also reduced the epididymal adipose tissue mass by 25% and retroperitoneal adipose tissue mass by 35%. In adipose tissue, expression of the insulin receptor was not changed by aliskiren; however, expression of insulin receptor substrate-1, glucose transporter type 4, adiponectin, peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding proteindelta was increased by aliskiren. Moreover, NADPH oxidase activity and expression of inflammatory factors were reduced in adipose tissue. Aliskiren increased the pancreatic beta-cell area in KK-A(y) mice. CONCLUSION: These results suggest that renin inhibition by aliskiren improved insulin resistance and adipose tissue dysfunction in type 2 diabetic mice through an increase in insulin sensitivity, insulin secretion and adipocyte differentiation, and a reduction of oxidative stress.

Improvement of glucose intolerance by combination of pravastatin and olmesartan in type II diabetic KK-A(y) mice.

The effects of the coadministration of pravastatin and an angiotensin type 1 (AT(1)) receptor blocker, olmesartan, on glucose intolerance were examined using type II diabetic mice. Male KK-A(y) mice (8 weeks of age) were treated with pravastatin and/or olmesartan for 2 weeks. An oral glucose tolerance test (OGTT) was performed with an administration of 2 g kg(-1) glucose. Tissue glucose uptake was determined using 2-[(3)H]deoxyglucose. The treatment of mice with pravastatin attenuated the increase in the plasma glucose level during OGTT in a dose-dependent manner, without affecting the plasma insulin level. Pravastatin increased glucose uptake in insulin-sensitive tissue such as the skeletal muscle and adipose tissue after treatment at 5-20 mg kg(-1) day(-1) for 2 weeks, but not at 1 mg kg(-1) day(-1). The combination of a noneffective dose of pravastatin (1 mg kg(-1) day(-1)) and a noneffective dose of olmesartan (0.5 mg kg(-1) day(-1)) synergistically improved OGTT without affecting the plasma insulin level. This combination also increased 2-[(3)H]deoxyglucose uptake in the skeletal muscle and adipose tissue. The effects of pravastatin or olmesartan on OGTT and tissue 2-[(3)H]deoxyglucose uptake were significantly enhanced by an antioxidant, tempol, whereas the effects of a pravastatin-olmesartan combination were not further enhanced by tempol. These results indicate that the combination of pravastatin and olmesartan synergistically improves glucose intolerance through an increase in tissue glucose uptake. The effects seem to be mediated by an increase in insulin sensitivity through the inhibition of oxidative stress.

AT2 receptor deficiency attenuates adipocyte differentiation and decreases adipocyte number in atherosclerotic mice.

BACKGROUND: Previous reports indicated that blockade of AT(1) receptor stimulation attenuated adipocyte dysfunction. However, the effects of AT(2) receptor stimulation on adipose tissue were not yet clear. In the present study, we examined the adipose tissue dysfunction in atherosclerotic apolipoprotein E knockout (ApoEKO) mice with AT(2) receptor deficiency. METHODS: Male ApoEKO and AT(2) receptor/ApoE knockout (AT(2)/ApoEKO) mice at 6 weeks of age were treated with a normal diet or a high-cholesterol diet (HCD: 1.25% cholesterol). Markers for adipocyte differentiation and inflammation in adipose tissue were assayed with real-time reverse-transcription-PCR and western blot. RESULTS: Compared with ApoEKO mice, AT(2)/ApoEKO mice with a normal diet showed only a decrease in expression of adiponectin and CCAAT/enhancer binding protein delta (C/EBPdelta) in epididymal adipose tissue without changes in body weight, adipose tissue weight, and adipocyte number even at 6 months of age. After HCD for 4 weeks, the weight of both epididymal and retroperitoneal adipose tissue in AT(2)/ApoEKO mice was greater than that in ApoEKO mice without a change in body weight. Plasma concentrations of cholesterol and fatty acids were higher in AT(2)/ApoEKO mice than in ApoEKO mice. In adipose tissue of AT(2)/ApoEKO mice, the adipocyte number was decreased and the expression of peroxisome proliferator-activated receptor gamma (PPARgamma), C/EBPalpha, and aP2 was lower than that in ApoEKO mice, in association with an increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. CONCLUSIONS: These results suggest that AT(2) receptor stimulation in adipose tissue is involved in the improvement of adipocyte differentiation and adipose tissue dysfunction in atherosclerotic model.

Exaggeration of focal cerebral ischemia in transgenic mice carrying human Renin and human angiotensinogen genes.

BACKGROUND AND PURPOSE: We examined the possibility that activation of the human brain renin-angiotensin system is involved in enhancement of ischemic brain damage using chimeric transgenic mice with human renin (hRN) and human angiotensinogen (hANG) genes. METHODS: Chimeric (hRN/hANG-Tg) mice were generated by mating of hRN and hANG transgenic mice. Permanent occlusion of the middle cerebral artery (MCA) by an intraluminal filament technique induced focal ischemic brain lesions. RESULTS: hRN/hANG-Tg mice showed higher angiotensin II levels in the plasma and brain. The ischemic brain area at 24 hours after MCA occlusion was significantly enlarged in hRN/hANG-Tg mice with an enhanced neurological deficit compared to that in wild-type, hRN-Tg and hANG-Tg mice. The reduction of cerebral blood flow in the periphery region of the MCA territory after MCA occlusion was markedly exaggerated in hRN/hANG-Tg mice. Superoxide anion production in the brain and arteries was also increased significantly in hRN/hANG-Tg mice even before MCA occlusion and was further enhanced after MCA occlusion. Treatment with an AT(1) receptor blocker, valsartan (3.0 mg/kg per day), for 2 weeks significantly reduced the ischemic brain area and improved the neurological deficit after MCA occlusion in hRN/hANG-Tg mice, similar to those in wild-type, hRN-Tg, and hANG-Tg mice, with restoration of cerebral blood flow in the peripheral region and decreases in superoxide anion production and blood pressure. CONCLUSIONS: These results indicate that activation of the human renin-angiotensin system exaggerates ischemic brain damage mainly through stimulation of the AT(1) receptor and marked reduction of cerebral blood flow and enhanced oxidative stress.

Continuous activation of renin-angiotensin system impairs cognitive function in renin/angiotensinogen transgenic mice.

We examined the possibility that continuous activation of the human brain renin-angiotensin system causes cognitive impairment, using human renin (hRN) and human angiotensinogen (hANG) gene chimeric transgenic (Tg) mice. Cognitive function was evaluated by the shuttle avoidance test once a week from 10 to 20 weeks of age. The avoidance rate in wild-type mice gradually increased. In contrast, the avoidance rate in chimeric hRN/hANG-Tg mice also increased; however, no further increase in avoidance rate was observed from 14 weeks of age, and it decreased thereafter. Cerebral surface blood flow was markedly reduced in 20-week-old hRN/hANG-Tg mice. Superoxide anion production in the brain was already higher in 10-week-old hRN/hANG-Tg mice and further increased thereafter with an increase in NADPH oxidase activity. Moreover, expression of p47(phox) and Nox4 in the brain of hRN/hANG-Tg mice also increased. Administration of an angiotensin II type 1 receptor blocker, olmesartan (5.0 mg/kg per day), attenuated the increase in blood pressure and ameliorated cognitive decline with enhancement of cerebral surface blood flow and a reduction of oxidative stress in hRN/hANG-Tg mice. On the other hand, hydralazine (0.5 mg/kg per day) did not improve the decrease in avoidance rate, and did not influence cerebral surface blood flow or oxidative stress in hRN/hANG-Tg mice, in spite of a similar reduction of blood pressure to that by olmesartan. Moreover, we observed that treatment with Tempol improved impaired cognitive function in hRN/hANG-Tg mice. These results suggest that continuous activation of the brain renin-angiotensin system impairs cognitive function via stimulation of the angiotensin II type 1 receptor with a decrease in cerebral surface blood flow and an increase in oxidative stress.

Prevention of vascular injury by combination of an AT1 receptor blocker, olmesartan, with various calcium antagonists.

BACKGROUND: A combination of different types of antihypertensive drugs is widely used for the treatment of hypertension. We examined the inhibitory effects of a combination of an AT(1) receptor blocker (ARB), olmesartan, with various calcium channel blockers (CCBs) on inflammatory vascular remodeling. METHODS: Inflammatory vascular remodeling was induced by polyethylene-cuff placement around the femoral artery of C57BL/6J mice at 10 weeks of age. Olmesartan (0.5 mg/kg/day) was administered intraperitoneally using an osmotic minipump. CCBs (nifedipine 1.0 mg/kg/day, amlodipine 0.1 mg/kg/day, azelnidipine 0.1 mg/kg/day), and hydrochlorothiazide (HCTZ 0.5 mg/kg/day) were administered orally. RESULTS: In the injured artery, superoxide anion production and expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p47(phox) and Rac-1 were markedly increased, together with expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor (TNF)-alpha. Administration of a single drug alone at each concentration did not significantly inhibit these changes in the injured artery. However, a combination of olmesartan with various CCBs inhibited neointimal formation as well as oxidative stress and inflammatory markers in the injured artery. Moreover, among these CCBs, inhibition of these markers by olmesartan with azelnidipine was stronger than that caused by a combination with other CCBs. On the other hand, a combination of subeffective doses of olmesartan and HCTZ did not significantly affect vascular changes after cuff placement. CONCLUSIONS: These results suggest that the combination of ARB with CCB synergistically inhibits vascular remodeling and that the inhibitory actions of ARB on vascular remodeling may vary depending on the combined CCB.