Jun Iwanami

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.

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.

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.

Regulation of angiotensin II receptors beyond the classical pathway

The RAS (renin-angiotensin system) plays a role not only in the cardiovascular system, including blood pressure regulation, but also in the central nervous system. AngII (angiotensin II) binds two major receptors: the AT(1) receptor (AngII type 1 receptor) and AT(2) receptor (AngII type 2 receptor). It has been recognized that AT(2) receptor activation not only opposes AT(1) receptor actions, but also has unique effects beyond inhibitory cross-talk with AT(1) receptor signalling. Novel pathways beyond the classical actions of RAS, the ACE (angiotensin-converting enzyme)/AngII/AT(1) receptor axis, have been highlighted: the ACE2/Ang-(1-7) [angiotensin-(1-7)]/Mas receptor axis as a new opposing axis against the ACE/AngII/AT(1) receptor axis, novel AngII-receptor-interacting proteins and various AngII-receptor-activation mechanisms including dimer formation. ATRAP (AT(1)-receptor-associated protein) and ATIP (AT(2)-receptor-interacting protein) are well-characterized AngII-receptor-associated proteins. These proteins could regulate the functions of AngII receptors and thereby influence various pathophysiological states. Moreover, the possible cross-talk between PPAR (peroxisome-proliferator-activated receptor)-γ and AngII receptor subtypes is an intriguing issue to be addressed in order to understand the roles of RAS in the metabolic syndrome, and interestingly some ARBs (AT(1)-receptor blockers) have been reported to have an AT(1)-receptor-blocking action with a partial PPAR-γ agonistic effect. These emerging concepts concerning the regulation of AngII receptors are discussed in the present review.

Peroxisome Proliferator-Activated Receptor-γ Activation With Angiotensin II Type 1 Receptor Blockade Is Pivotal for the Prevention of Blood-Brain Barrier Impairment and Cognitive Decline in Type 2 Diabetic Mice

We reported previously that an angiotensin II type 1 receptor blocker, telmisartan, improved cognitive decline with peroxisome proliferator-activated receptor-&ggr; activation; however, the detailed mechanisms are unclear. Enhanced blood-brain barrier (BBB) permeability with alteration of tight junctions is suggested to be related to diabetes mellitus. Therefore, we examined the possibility that telmisartan could attenuate BBB impairment with peroxisome proliferator-activated receptor-&ggr; activation to improve diabetes mellitus–induced cognitive decline. Type 2 diabetic mice KKAy exhibited impairment of cognitive function, and telmisartan treatment attenuated this. Cotreatment with GW9662, a peroxisome proliferator-activated receptor-&ggr; antagonist, interfered with these protective effects of telmisartan against cognitive function. BBB permeability was increased in both the cortex and hippocampus in KKAy mice. Administration of telmisartan attenuated this increased BBB permeability. Coadministration of GW9662 reduced this effect of telmisartan. Significant decreases in expression of tight junction proteins and increases in matrix metalloproteinase expression, oxidative stress, and proinflammatory cytokine production were observed in the brain, and treatment with telmisartan restored these changes. Swollen astroglial end-feet in BBB were observed in KKAy mice, and this change in BBB ultrastructure was decreased in telmisartan. These effects of telmisartan were weakened by cotreatment with GW9662. In contrast, administration of another angiotensin II type 1 receptor blocker, losartan, was less effective compared with telmisartan in terms of preventing BBB permeability and astroglial end-foot swelling, and coadministration of GW9662 did not affect the effects of losartan. These findings are consistent with the possibility that, in type 2 diabetic mice, angiotensin II type 1 receptor blockade with peroxisome proliferator-activated receptor-&ggr; activation by telmisartan may help with protection against cognitive decline by preserving the integrity of the BBB.

Direct Angiotensin II Type 2 Receptor Stimulation Ameliorates Insulin Resistance in Type 2 Diabetes Mice with PPARγ Activation

Objectives The role of angiotensin II type 2 (AT2) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue. Methods T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[3H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined. Results Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored β cell damage in diabetic pancreatic tissue. Conclusion The present study demonstrated that direct AT2 receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic β cells.

Low dose of telmisartan prevents ischemic brain damage with peroxisome proliferator-activated receptor-γ activation in diabetic mice

Background Telmisartan is a unique AT1 receptor blocker with a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonistic action. Activation of PPAR-γ could prevent inflammation and brain damage. Method We investigated the beneficial effect of telmisartan on ischemic brain damage via PPAR-γ activation as well as AT1 receptor blockade. Eight-week-old male KK-Ay mice were subjected to middle cerebral artery occlusion. Before middle cerebral artery occlusion, they were administered telmisartan or losartan, with or without GW9662, a PPAR-γ antagonist, for 2 weeks. Ischemic area, neurological score, oxidative stress, inflammation and cerebral blood flow were assessed 24 h after middle cerebral artery occlusion. Results Administration of telmisartan, losartan, GW9662 and these AT1 receptor blockers with GW9662 had no significant effect on blood pressure. KK-Ay mice exhibited a significant increase in the ischemic area compared with C57BL6 mice. Treatment with telmisartan decreased the ischemic area and improved the neurological score compared with the no-treatment group, with an increase in cerebral blood flow and a reduction in superoxide production and expression of inflammatory cytokines. These protective effects of telmisartan were partially attenuated by coadministration of GW9662, although GW9662 treatment alone had no significant effect on ischemic area. Losartan treatment showed a reduction in ischemic area compared with nontreated KK-Ay mice. However, coadministration of GW9662 had no effect on the losartan-mediated reduction in ischemic area. Conclusion These results suggest that telmisartan has a beneficial effect on stroke partly due to activation of PPAR-γ as well as AT1 receptor blockade.

Direct stimulation of angiotensin II type 2 receptor initiated after stroke ameliorates ischemic brain damage.

BACKGROUND Stroke is a leading cause of death and disability; however, meta-analysis of randomized controlled trials of blood pressure-lowering drugs in acute stroke has shown no definite evidence of a beneficial effect on functional outcome. Accumulating evidence suggests that angiotensin II type 1 receptor blockade with angiotensin II type 2 (AT2) receptor stimulation could contribute to protection against ischemic brain damage. We examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) initiated even after stroke can prevent ischemic brain damage. METHODS Stroke was induced by middle cerebral artery (MCA) occlusion, and the area of cerebral infarction was measured by magnetic resonant imaging. C21 (10 µg/kg/day) treatment was initiated immediately after MCA occlusion by intraperitoneal injection followed by treatment with C21 once daily. RESULTS We observed that ischemic area was enlarged in a time dependent fashion and decreased on day 5 after MCA occlusion. Treatment with C21 initiated after MCA occlusion significantly reduced the ischemic area, with improvement of neurological deficit in a time-dependent manner without affecting blood pressure. The decrease of cerebral blood flow after MCA occlusion was also ameliorated by C21 treatment. Moreover, treatment with C21 significantly attenuated superoxide anion production and expression of proinflammatory cytokines, monocyte chemoattractant protein 1, and tumor necrosis factor α. Interestingly, C21 administration significantly decreased blood-brain barrier permeability and cerebral edema on the ischemic side. CONCLUSIONS These results provide new evidence that direct AT2 receptor stimulation with C21 is a novel therapeutic approach to prevent ischemic brain damage after acute stroke.