Masaki Mogi

Aldosterone and Angiotensin II Synergistically Induce Mitogenic Response in Vascular Smooth Muscle Cells

Interaction between aldosterone (Aldo) and angiotensin II (Ang II) in the cardiovascular system has been highlighted; however, its detailed signaling mechanism is poorly understood. Here, we examined the cross-talk of growth-promoting signaling between Aldo and Ang II in vascular smooth muscle cells (VSMC). Treatment with a lower dose of Aldo (10−12 mol/L) and with a lower dose of Ang II (10−10 mol/L) significantly enhanced DNA synthesis, whereas Aldo or Ang II alone at these doses did not affect VSMC proliferation. This effect of a combination of Aldo and Ang II was markedly inhibited by a selective AT1 receptor blocker, olmesartan, a mineralocorticoid receptor antagonist, spironolactone, an MEK inhibitor, PD98059, or an EGF receptor tyrosine kinase inhibitor, AG1478. Treatment with Aldo together with Ang II, even at noneffective doses, respectively, synergistically increased extracellular signal-regulated kinase (ERK) activation, reaching 2 peaks at 10 to 15 minutes and 2 to 4 hours. The early ERK peak was effectively blocked by olmesartan or an EGF receptor kinase inhibitor, AG1478, but not by spironolactone, whereas the late ERK peak was completely inhibited by not only olmesartan, but also spironolactone. Combined treatment with Aldo and Ang II attenuated mitogen-activated protein kinase phosphatase-1 (MKP-1) expression and increased Ki-ras2A expression. The late ERK peak was not observed in VSMC treated with Ki-ras2A-siRNA. Interestingly, the decrease in MKP-1 expression and the increase in Ki-ras2A expression were restored by PD98059 or AG1478. These results suggest that Aldo exerts a synergistic mitogenic effect with Ang II and support the notion that blockade of both Aldo and Ang II could be more effective to prevent vascular remodeling.

Cognitive Deficit in Amyloid-β–Injected Mice Was Improved by Pretreatment With a Low Dose of Telmisartan Partly Because of Peroxisome Proliferator-Activated Receptor-γ Activation

The pathological hallmark of Alzheimer disease is deposition of amyloid-&bgr; protein (A&bgr;) in the brain. Telmisartan is a unique angiotensin II receptor blocker with peroxisome proliferator-activated receptor-&ggr; (PPAR-&ggr;)–stimulating activity. Activation of PPAR-&ggr; is expected to prevent inflammation and A&bgr; accumulation in the brain. We investigated the possible preventive effect of telmisartan on cognitive decline in an Alzheimer disease mouse model via PPAR-&ggr; activation. Here, male ddY mice underwent ICV injection of A&bgr; 1-40. Cognitive function was evaluated by the Morris water maze test. A low dose of telmisartan (0.35 mg/kg per day) was administered in drinking water with or without GW9662, a PPAR-&ggr; antagonist. Cerebral blood flow was evaluated by laser speckle flowmetry. Inflammatory cytokine levels were measured by quantitative RT-PCR. A&bgr; 1-40 ICV injection significantly impaired cognitive function. Pretreatment with telmisartan improved this cognitive decline to a similar level to that in control mice. Cotreatment with GW9662, a PPAR-&ggr; antagonist, attenuated this telmisartan-mediated improvement of cognition. Treatment with telmisartan enhanced cerebral blood flow and attenuated the A&bgr;-induced increase in expression of cytokines, such as tumor necrosis factor-&agr; and inducible NO synthase in the brain. Interestingly, coadministration of GW9662 cancelled these beneficial effects of telmisartan. A&bgr; 1-40 concentration in the brain was significantly decreased by treatment with telmisartan, whereas administration of GW9662 attenuated the decrease in telmisartan-mediated A&bgr; 1-40 concentration. Taken together, our findings suggest that even a low dose of telmisartan had a preventive effect on cognitive decline in an Alzheimer disease mouse model, partly because of PPAR-&ggr; activation.

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.

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 17&bgr;-estradiol (80 &mgr;g/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 17&bgr;-estradiol (20 &mgr;g/kg per day) did not influence atherosclerosis and oxidative stress in ovariectomized mice, whereas co-administration of olmesartan and 17&bgr;-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 17&bgr;-estradiol. These results indicate that estrogen and an ARB synergistically attenuate atherosclerosis at least partly via inhibition of oxidative stress.

Angiotensin II Type-2 Receptor Stimulation Prevents Neural Damage by Transcriptional Activation of Methyl Methanesulfonate Sensitive 2

The molecular mechanisms of the contribution of angiotensin II type-1 receptor blockers to neuronal protection are still unclear. Here, we investigated the effect of angiotensin II type-2 (AT2) receptor stimulation on neurons and cognitive function involving a new neuroprotective factor, methyl methanesulfonate sensitive 2 (MMS2). Angiotensin II treatment of neurospheres enhanced their differentiation and increased MMS2 expression. Knockdown of the MMS2 gene by small interference RNA (siRNA) significantly reduced the number of neurospheres, with loss of sphere formation. An angiotensin II type-1 receptor blocker, valsartan, enhanced such neurosphere differentiation and MMS2 induction, whereas an AT2 receptor antagonist, PD123319, inhibited them. After mice underwent permanent middle cerebral artery occlusion, AT2 receptor mRNA expression was significantly increased in the ischemic side of the brain. Passive avoidance rate to evaluate cognitive function was significantly impaired in AT2 receptor null (Agtr2−) mice compared with wild-type mice. Treatment with valsartan prevented the cognitive decline in wild-type mice, but this effect was weaker in Agtr2− mice. In ischemic brain regions, MMS2 was increased in wild-type mice, but not in Agtr2− mice. Valsartan also enhanced MMS2 expression to a greater degree in wild-type mice. Finally, intracerebroventricular administration of MMS2 siRNA showed more impaired avoidance rate after middle cerebral artery occlusion compared with that in control siRNA–transfected mice. These findings experimentally support the clinical evidence and indicate a unique mechanism of the AT2 receptor in brain protection.

Telmisartan prevented cognitive decline partly due to PPAR-γ activation

Telmisartan is a unique angiotensin receptor blocker (ARB) and partial agonist of peroxisome proliferator-activated receptor (PPAR)-gamma. Here, we investigated the preventive effect of telmisartan on cognitive decline in Alzheimer disease. In ddY mice, intracerebroventricular injection of Abeta 1-40 significantly attenuated their cognitive function evaluated by shuttle avoidance test. Pretreatment with a non-hypotensive dose of telmisartan significantly inhibited such cognitive decline. Interestingly, co-treatment with GW9662, a PPAR-gamma antagonist, partially inhibited this improvement of cognitive decline. Another ARB, losartan, which has less PPAR-gamma agonistic effect, also inhibited Abeta-injection-induced cognitive decline; however the effect was smaller than that of telmisartan and was not affected by GW9662. Immunohistochemical staining for Abeta showed the reduced Abeta deposition in telmisartan-treated mice. However, this reduction was not observed in mice co-administered GW9662. These findings suggest that ARB has a preventive effect on cognitive impairment in Alzheimer disease, and telmisartan, with PPAR-gamma activation, could exert a stronger effect.

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 p47phox 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.

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 (p47phox 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.

Clinical Interaction between Brain and Kidney in Small Vessel Disease.

Patients with chronic kidney disease (CKD) are well known to have a higher prevalence of cardiovascular disease from epidemiological studies. Recently, CKD has also been shown to be related to neurological disorders, not only ischemic brain injury but also cognitive impairment. This cerebrorenal connection is considered to involve small vessel disease in both the kidney and brain, based on their hemodynamic similarities. Clinical studies suggest that markers for CKD such as estimated glomerular filtration rate (eGFR), proteinuria, and albuminuria may be helpful to predict brain small vessel disease, white matter lesions (WMLs), silent brain ischemia (SBI), and microhemorrhages. Recently, changes in the vascular system of the brain have been shown to contribute to the onset and progression of cognitive impairment, not only vascular dementia but also Alzheimers disease. Patients with CKD are also reported to have higher risk of impaired cognitive function in the future compared with non-CKD subjects. These results indicate that CKD markers may be helpful to predict the future risk of neuronal disease.

Direct stimulation of angiotensin II type 2 receptor enhances spatial memory

We examined the possibility that direct stimulation of the angiotensin II type 2 (AT2) receptor by a newly generated direct AT2 receptor agonist, Compound 21 (C21), enhances cognitive function. Treatment with C21 intraperitoneal injection for 2 weeks significantly enhanced cognitive function evaluated by the Morris water maze test in C57BL6 mice, but this effect was not observed in AT2 receptor-deficient mice. However, C21-induced cognitive enhancement in C57BL6 mice was attenuated by coadministration of icatibant, a bradykinin B2 receptor antagonist. Administration of C21 dose dependently increased cerebral blood flow assessed by laser speckle flowmetry and hippocampal field-excitatory postsynaptic potential (f-EPSP) determined by electrophysiological techniques in C57BL6 mice. Furthermore, activation of the AT2 receptor by C21 promoted neurite outgrowth of cultured hippocampal neurons prepared from fetal transgenic mice expressing green fluorescent protein. Finally, we investigated the pathologic relevance of C21 for spatial learning using an Alzheimers disease mouse model with intracerebroventricular injection of amyloid-β (1 to 40). We observed that treatment with C21 prevented cognitive decline in this model. These results suggest that a direct AT2 receptor agonist, C21, enhances cognitive function at least owing to an increase in CBF, enhancement of f-EPSP, and neurite outgrowth in hippocampal neurons.