Pretreatment with eplerenone reduces stroke volume in mouse middle cerebral artery occlusion model.

Eplerenone, a mineralocorticoid receptor antagonist, is reported to be effective to prevent end-stage cardiovascular damage induced by aldosterone. However, the effect of eplerenone on brain damage is not fully understood. Here, we investigated whether pretreatment with eplerenone attenuates stroke size in mice subjected to middle cerebral artery occlusion. Middle cerebral artery occlusion with a microfilament technique induced focal ischemia, to approximately 25% of the total area in a coronal section of the brain. Treatment with eplerenone at a dose of 1.67 mg/g chow significantly reduced the ischemic area, ischemic volume, and neurological deficit, without a blood pressure-lowering effect. Laser-Doppler flowmetry analysis showed a decrease in surface cerebral blood flow in the peripheral region after 1 h of middle cerebral artery occlusion. This decrease was smaller in mice treated with eplerenone. Superoxide production evaluated by staining with dihydroethidium was attenuated in the ischemic area of the brain in eplerenone-treated mice. Taken together, our findings suggest that eplerenone has a protective effect on ischemic brain damage, at least partly due to improvement of cerebral blood flow in the penumbra and reduction of oxidative stress.

Amlodipine treatment reduces stroke size in apolipoprotein E-deficient mice.

BACKGROUND: This study investigated the effects of amlodipine, an L-type calcium channel blocker, on stroke size after focal brain ischemia in apolipoprotein E-deficient (ApoE KO) mice. METHODS: Mice were subjected to middle cerebral artery (MCA) occlusion after being given a high-cholesterol (HCD) or normal diet for 10 weeks with or without amlodipine at a nonhypotensive dose of 3 mg/kg/day. Ischemic brain area was measured by 2,3,5-triphenyltetrazolium chloride staining. Cerebral blood flow was analyzed by laser-Doppler flowmetry. Superoxide anion production in the brain was detected by dihydroethidium staining. RESULTS: The ApoE KO mice given HCD for 10 weeks showed a larger ischemic lesion size than mice with a normal diet. Amlodipine treatment in parallel with HCD feeding reduced the ischemic lesion size in ApoE KO mice. Interestingly, amlodipine treatment for only the last 2 weeks was also effective in reducing the ischemic lesion size in HCD-fed ApoE KO mice. The neurologic deficit after MCA occlusion was also improved by amlodipine treatment for either 10 weeks or 2 weeks. The decrease in surface cerebral blood flow after MCA occlusion was significantly attenuated in the peripheral region of the MCA territory in amlodipine-treated mice. Amlodipine treatment in HCD-fed ApoE KO mice also reduced superoxide production in the ischemic area of the brain. CONCLUSIONS: These results suggest that amlodipine treatment reduces stroke size and neurologic deficit after focal brain ischemia, possibly through an increase in cerebral blood flow and inhibition of superoxide production.

The calcium-channel blocker, azelnidipine, enhances the inhibitory action of AT1 receptor blockade on ischemic brain damage.

OBJECTIVE: The combined effects of a calcium-channel blocker (CCB) with an angiotensin (Ang) II type 1 (AT1) receptor blocker were investigated in focal brain ischemia induced by middle cerebral artery (MCA) occlusion. METHODS AND RESULTS: In male C57BL/6J mice, permanent occlusion of the MCA-induced focal cerebral ischemia and neurological deficit after 24 h, accompanied by a reduction of cerebral blood flow and an increase in superoxide production in the ischemic area. Administration of azelnidipine, a CCB, at 1.0 mg/kg per day for 10 days significantly suppressed these changes after MCA without affecting systolic blood pressure. Such inhibitory effects of azelnidipine on brain ischemia could be observed in AT1a receptor-deficient mice. In addition, olmesartan, an AT1 receptor blocker, at 3.0 mg/kg per day also diminished the ischemic brain area and neurological score, as well as superoxide production and the reduction of cerebral surface blood flow in C57BL/6 mice. The combination of lower doses of azelnidipine (0.1 mg/kg per day) and olmesartan (0.5 mg/kg per day) significantly attenuated the ischemic brain area, neurological score, superoxide production and the reduction of cerebral surface blood flow after MCA occlusion in C57BL/6 mice, whereas either of these agents alone at these doses did not affect brain ischemia. CONCLUSION: These results indicate that azelnidipine inhibited ischemic brain damage induced by MCA occlusion, at least in part, through suppression of blood flow change and oxidative stress via a signaling mechanism independent of AT1 receptor stimulation. Moreover, azelnidipine synergistically enhanced the inhibitory action of olmesartan on brain ischemia, suggesting beneficial combined effects of a CCB with an AT1 receptor blocker on ischemic brain damage.

Regression of atherosclerosis by amlodipine via anti-inflammatory and anti-oxidative stress actions.

We examined whether amlodipine, an L-type calcium channel blocker (CCB), has an inhibitory effect on oxidative stress and inflammatory response, and thereby atherosclerosis, in apolipoprotein E-deficient (ApoEKO) mice. Adult male ApoEKO mice (6 weeks of age) were fed a high-cholesterol diet (HCD) for 8 or 10 weeks with or without oral administration of amlodipine (3 mg/kg/day) for 10 weeks or for only the last 2 weeks of the HCD. After HCD feeding, atherosclerotic lesion formation, in situ superoxide production and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity were evaluated in the proximal aorta. The expressions of NADPH oxidase subunits (p47(phox) and rac-1), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were determined with immunohistochemistry and quantitative real-time reverse-transcription polymerase chain reaction. After 8 to 10 weeks of HCD administration to ApoEKO mice, marked atherosclerotic lesion formation was observed in the proximal aorta. In the atherosclerotic lesion, superoxide production, the expression of NADPH oxidase subunits, and NADPH oxidase activity were enhanced, and the expressions of MCP-1, ICAM-1, and VCAM-1 were increased. These changes were suppressed in mice that were treated with amlodipine for 10 weeks concomitant with HCD administration, with no significant change in blood pressure and plasma cholesterol level. We also observed that treatment with amlodipine for only the last 2 weeks regressed the atherosclerotic lesions with a decrease in oxidative stress and vascular inflammation. Inhibition of the atherosclerotic lesion area and lipid area in the proximal aorta by amlodipine was correlated with its inhibitory actions on oxidative stress, inflammation and the production of adhesive molecules. These results suggest that amlodipine not only inhibits atherosclerotic lesion formation, but also regresses atherosclerosis, and that these effects are at least partly due to inhibition of oxidative stress and inflammatory response.

Attenuation of inflammatory vascular remodeling by angiotensin II type 1 receptor-associated protein.

To explore the role of angiotensin II Type 1 receptor-associated protein (ATRAP) in vascular remodeling, we developed transgenic mice for mouse ATRAP cDNA and examined remodeling after inflammatory vascular injury induced by polyethylene cuff placement. In ATRAP transgenic (ATRAP-Tg) mice, ATRAP mRNA was increased 3- to 4-fold in the heart, aorta, and femoral artery. ATRAP-Tg mice showed no significant change in body weight, systolic blood pressure, heart rate, and heart/body weight ratio. However, cell proliferation and neointimal formation in the injured artery were attenuated in ATRAP-Tg mice. The increase in NADPH oxidase activity and the expression of p22(phox), a reduced nicotinamide-adenine dinucleotide/reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit, after cuff placement was also attenuated in ATRAP-Tg mice. Moreover, activation of extracellular signal-regulated kinase, signal transducer and activator of transcription 1, and signal transducer and activator of transcription 3 after cuff placement was significantly reduced in ATRAP-Tg mice. Pressor response and cardiac hypertrophy induced by angiotensin II infusion and pressure overload were also attenuated in ATRAP-Tg mice. These results suggest that ATRAP plays an important role in vascular remodeling as a negative regulator.

Comparison of inhibitory action of candesartan and enalapril on brain ischemia through inhibition of oxidative stress.

The effects of an angiotensin II type 1 (AT1) receptor blocker (ARB) on ischemic brain damage induced by middle cerebral artery (MCA) occlusion were compared with those of an angiotensin converting enzyme (ACE) inhibitor. Treatment of male C57BL/6J mice with an ARB, candesartan, reduced the brain ischemic area and neurological deficit after MCA occlusion at a non-hypotensive dose. In contrast, an ACE inhibitor, enalapril, did not reduce the brain ischemic area, and neurological deficit even at a hypotensive dose. Candesartan improved the reduction of brain surface blood flow after MCA occlusion, and inhibited the increase in superoxide production both in the cortex and brain arterial wall at non-hypotensive and hypotensive doses. However, enalapril did not affect the changes in blood flow and superoxide production in the brain after MCA occlusion. AT2 receptor expression in the ischemic area was increased at 3 h after MCA occlusion by pretreatment with candesartan, but not that with enalapril. AT1 receptor expression was neither affected by candesartan nor by enalapril. These results suggest that candesartan attenuated ischemic brain damage, at least partly, through inhibition of oxidative stress.

Role of angiotensin II receptor subtypes in conjunctival wound healing.

PURPOSE: To investigate the role of angiotensin II (Ang II) receptor subtypes in subconjunctival injury. METHODS: A wound-healing model was developed by subconjunctival blunt dissection in male wild-type, AT1a receptor-deficient (AT1aKO) and AT2 receptor-deficient (AT2KO) mice. Collagen deposition and cell infiltration were evaluated histologically. Expression of collagen, matrix metalloproteinase (MMP), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by real-time PCR. RESULTS: Subconjunctival injury increased the infiltration of inflammatory cells, collagen deposition in the subconjunctival space, and the expression of collagen type I and type III, TIMP-1 and MMP2. In AT1aKO mice, collagen deposition, cell infiltration, and expression of collagen and TIMP-1 were inhibited, but MMP2 expression was enhanced. In contrast, in AT2KO mice, the increase in collagen deposition, cell infiltration, and expression of collagen and TIMP-1 were further enhanced. CONCLUSIONS: These results indicate that AT1a and AT2 receptor stimulation may in addition to other mechanisms be antagonistically involved in the wound-healing process after subconjunctival injury.

Deletion of angiotensin II type 2 receptor exaggerated atherosclerosis in apolipoprotein E-null mice.

BACKGROUND: The role of angiotensin II (Ang II) type 2 (AT2) receptor in atherosclerosis was explored with the use of AT2 receptor/apolipoprotein E (ApoE)-double-knockout (AT2/ApoE-DKO) mice, with a focus on oxidative stress. METHODS AND RESULTS: After treatment with a high-cholesterol diet (1.25% cholesterol) for 10 weeks, ApoE-knockout (KO) mice developed atherosclerotic lesions in the aorta. In AT2/ApoE-DKO mice receiving a high-cholesterol diet, the atherosclerotic changes were further exaggerated, without significant changes in plasma cholesterol level and blood pressure. In the atherosclerotic lesion, an increase in superoxide production, NADPH oxidase activity, and expression of p47phox was observed. These changes were also greater in AT2/ApoE-DKO mice. An Ang II type 1 (AT1) receptor blocker, valsartan, inhibited atherosclerotic lesion formation, superoxide production, NADPH oxidase activity, and p47phox expression; these inhibitory effects were significantly weaker in AT2/ApoE-KO mice. We further examined the signaling mechanism of the AT2 receptor-mediated antioxidative effect in cultured fetal vascular smooth muscle cells. NADPH oxidase activity and phosphorylation and translocation of p47phox induced by Ang II were inhibited by valsartan but enhanced by an AT2 receptor blocker, PD123319. CONCLUSIONS: These results suggest that AT2 receptor stimulation attenuates atherosclerosis through inhibition of oxidative stress and that the antiatherosclerotic effect of valsartan could be at least partly due to AT2 receptor stimulation by unbound Ang II.

Sex difference in vascular injury and the vasoprotective effect of valsartan are related to differential AT2 receptor expression.

The angiotensin II type 2 (AT2) receptor is upregulated in pathological conditions such as vascular injury and exerts antagonistic effects against AT1 receptor-mediated actions. We examined the possibility that the sex difference in vascular remodeling is associated with altered AT2 receptor expression, which is located on the X chromosome. In this study, we examined this possibility by using AT2 receptor-null (Agtr2-) mice. Vascular injury was induced by polyethylene cuff placement around the femoral artery of wild-type (Agtr2+) and Agtr2- mice. In Agtr2+ mice, AT2 receptor expression in the injured artery was enhanced, and this increase was greater in female than in male mice, with no significant difference in AT1 receptor expression between male and female mice. Increases in neointimal formation, DNA synthesis, expression of monocyte chemoattractant protein-1, production of superoxide anion, and NADPH oxidase activity in the injured artery were attenuated in female compared with male mice. These parameters were augmented in Agtr2- mice, whereas the sex differences in these parameters were smaller in Agtr2- than in Agtr2+ mice. Treatment with a nonhypotensive dose of the AT1 receptor blocker valsartan decreased these parameters significantly in Agtr2+ mice, and these inhibitory effects of valsartan were greater in female mice. This sex difference in valsartan's inhibitory effect was less marked in Agtr2- mice. Our results suggest that the sex difference in response to vascular injury could be at least partially attributed to the exaggerated AT2 receptor expression in the injured vessel in female mice.

Effect of combination of calcium antagonist, azelnidipine, and AT1 receptor blocker, olmesartan, on atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: Angiotensin II type 1 receptor blockers (ARB) are widely recognized to have a vasculoprotective effect. Accumulating data have revealed that calcium antagonists also retard atherosclerosis. We examined the possibility that combination therapy of ARB and calcium antagonists could more effectively prevent atherosclerosis than monotherapy. METHODS AND RESULTS: We observed a marked increase in the atherosclerotic area, associated with the exaggerated expression of nicotinamide adenine dinucleotide (phosphate), reduced form [NAD(P)H] oxidase subunits (p22 and p47) and superoxide anion production, in the aorta of apolipoprotein E-deficient mice maintained on a 1.25% high-cholesterol diet for 10 weeks. A calcium antagonist, azelnidipine, at a dose of 1 mg/kg a day or an ARB, olmesartan, at a dose of 3 mg/kg a day, significantly inhibited these parameters, with no change in systolic blood pressure and the blood cholesterol level. Moreover, the co-administration of lower doses of azelnidipine (0.1 mg/kg a day) and olmesartan (1 mg/kg a day) significantly inhibited the atherosclerotic area and oxidative stress, whereas azelnidipine or olmesartan alone at these doses did not affect these parameters. Furthermore, we observed similar inhibitory effects of azelnidipine or olmesartan on angiotensin II-induced NADPH oxidase activity and Akt activity in cultured vascular smooth muscle cells. CONCLUSION: These results suggest that the co-administration of calcium antagonists and ARB synergistically blunts oxidative stress at least partly through the inhibition of Akt activity and enhances the beneficial effects of these drugs on atherosclerosis compared with monotherapy.

Inhibitory effects of AT1 receptor blocker, olmesartan, and estrogen on atherosclerosis via anti-oxidative stress.

The present study explored the possibility that estrogen enhances the inhibitory effect of an angiotensin II type-1 (AT1) receptor blocker (ARB), olmesartan, on atherosclerosis, focusing on oxidative stress using apolipoprotein E knockout mice (ApoEKO). After 6 weeks on a high-cholesterol diet, marked atherosclerotic lesion formation with an increase in oxidative stress, such as superoxide production, NAD(P)H oxidase activity and expression of p47phox mRNA and rac-1 mRNA, were observed in the proximal aorta in both male and female ApoEKO mice, whereas these changes were less marked in female mice. Ovariectomy enhanced these parameters, the changes of which were reversed by 17beta-estradiol (80 microg/kg per day) replacement. Treatment with olmesartan (3 mg/kg per day) significantly inhibited oxidative stress and atherosclerosis, whereas its inhibitory effects were more marked in female than in male or ovariectomized mice. Smaller doses of olmesartan (0.5 mg/kg per day) or 17beta-estradiol (20 microg/kg per day) did not influence atherosclerosis and oxidative stress in ovariectomized mice, whereas co-administration of olmesartan and 17beta-estradiol at these doses attenuated these parameters. An angiotensin-converting enzyme (ACE) inhibitor, temocapril, also inhibited atherosclerotic changes similarly to olmesartan. Moreover, angiotensin II-mediated activation of NAD(P)H oxidase in cultured vascular smooth muscle cells was attenuated by 17beta-estradiol. These results indicate that estrogen and an ARB synergistically attenuate atherosclerosis at least partly via inhibition of oxidative stress.

Possible inhibition of focal cerebral ischemia by angiotensin II type 2 receptor stimulation.

BACKGROUND: The role of angiotensin II receptor subtypes was investigated in focal brain ischemia induced by middle cerebral artery (MCA) occlusion. METHODS AND RESULTS: In Agtr2+ (wild-type) mice, MCA occlusion induced focal ischemia of approximately 20% to 30% of the total area in coronal section of the brain. The ischemic area was significantly larger in angiotensin II type 2 receptor-deficient (Agtr2-) mice than in Agtr2+ mice. The neurological deficit after MCA occlusion was also greater in Agtr2- mice than in Agtr2+ mice. The decrease in surface cerebral blood flow after MCA occlusion was significantly exaggerated in the peripheral region of the MCA territory in Agtr2- mice. Superoxide production and NADPH oxidase activity were enhanced in the ischemic area of the brain in Agtr2- mice. An AT1 receptor blocker, valsartan, at a nonhypotensive dose significantly inhibited the ischemic area, neurological deficit, and reduction of cerebral blood flow as well as superoxide production and NADPH oxidase activity in Agtr2+ mice. These inhibitory actions of valsartan were weaker in Agtr2- mice. CONCLUSIONS: These results suggest that AT2 receptor stimulation has a protective effect on ischemic brain lesions, at least partly through the modulation of cerebral blood flow and superoxide production.