Our laboratory is focused on the study of the central functions of the hormone and brain modulator Angiotensin II (Ang II), and in particular on its role in the regulation of the cerebral circulation and the reaction to stress. In hypertension, excess AT1 receptor stimulation promotes cerebrovascular growth and vasoconstriction, with decreased arterial compliance and susceptibility to ischemia. We found that in spontaneously (genetic) hypertensive rats (SHR), AT1 antagonism with a non-peptidic, selective, potent and insurmountable antagonist of the physiologically active Ang II AT1 receptors, candesartan, remodels brain arteries, increasing their compliance and their resistance to ischemia. When male untreated SHR, submitted to experimental stroke by occlusion of the middle cerebral artery, are pretreated with AT1 receptor antagonists for two to four weeks before ischemia, the blood flow is preserved above a critical threshold in the periphery of the ischemic zone, with a very significant reduction of the cortical volume of ischemia and in a decrease in brain edema. The effects of AT1 antagonists are more pronounced than those of inhibitors of Ang II formation (ACE inhibitors) and far superior than those of calcium channel or adrenergic receptor blockers. We also demonstrated an important role of brain AT1 receptors by intracerebral administration of an AT1 receptor antisense oligonucleotide, which protects against cerebral ischemia. We conclude that AT1 receptor inhibitors may be the treatment of choice for patients with increased risk of cerebral ischemia, and that blockade of brain AT1 receptors decreases neuronal death during ischemia. Ang II is an important stress hormone. We found that pretreatment with a peripheral and central AT1 receptor antagonist completely prevents the sympathoadrenal response to isolation stress, decreasing the formation of the catecholamine rate-limiting enzyme, tyrosine hydroxylase (TH), the adrenal catecholamine content, and the adrenal catecholamine release during stress. The molecular mechanism involves a reduction of the stress-induced increase in Fra-2, a protein that interacts with the AP-1 binding site in the TH promoter region, probably through AT1/AT2 receptor cross talk, because inhibition of AT2 receptors reduces basal Fra-2 levels. In addition, AT1 and AT2 receptors are colocalized in adrenomedullary neurons, and they mutually regulate their expression. In AT2 receptor gene-deficient model, absence of AT2 receptor expression results in increased AT1 receptors in the paraventricular nucleus, and this correlates with activation of their pituitary-adrenal axis. This explains the increased sensitivity to stress in this mice model. Pretreatment with AT1 receptor blockers also prevents the hypothalamic-pituitary hormonal response to isolation stress, and reverses the decrease in CRH mRNA in rat paraventricular nucleus produced by isolation stress. This indicates that inhibition of CRH formation is an important component of the central protective action of AT1 antagonists. Inhibition of AT1 receptors completely prevents the stress-induced decrease in CRH1 receptors in rat frontal, parietal and cingulate cortex and the stress-induced increase in CRH2beta receptors in the choroids plexus, suggesting that Ang II AT1 receptors modulate multiple CRH systems. Finally, AT1 receptor inhibition prevents the development of stress-induced gastric ulcers and exerts anxiolytic effects in the rat. Our observations strongly indicate that inhibition of AT1 receptors has therapeutic potential in stress-related disorders. In AT2 receptor gene-deficient mice, selective populations of AT1 receptors are upregulated, not only in the adrenal gland and brain but also in other organs such the kidney, spleen and lung, but not in the liver. Many of the cells expressing higher AT1 receptor number do not express AT2 receptors, raising the possibility of extracellular and/or intercellular cross-talk mechanisms for AT1/AT2 receptors. Increased kidney AT1 receptors in AT2 receptor gene-deficient mice partially explain the phenotype in this model, higher blood pressure and increased response to exogenously administered Ang II. These findings also raise the possibility of a protective function of AT2 receptors in the kidney. In the inner medulla of the kidney of female mice, AT2 receptors are highly expressed, and their expression is strongly estrogen-dependent. This may explain gender differences in renal vasodilation and function. If AT2 receptors are regulated by estrogen in other organs, and in particular in the brain, this could explain some of the protective effect of estrogens in cerebral ischemia. Non-peptidic antagonists of the AT1 receptor are among the drugs of choice in the treatment of cardiovascular disease. We have shown that, in addition, these compounds may exert therapeutic benefits in the protection against brain ischemia and stress-related disorders. For this reason, an in-depth analysis of the molecular requirements of the binding site for non-peptidic antagonists of the AT1 receptor was important. We took advantage of two naturally occurring mutations, in the two gerbil AT1 receptor subtypes, gAT1A and gAT1B, cloned in our laboratory. The gAT1A receptor has an affinity for non-peptidic antagonists about 400-fold lower than that of the hAT1 receptor, while the affinity for these compounds in the gAT1B receptor is about 40-fold lower when compared to the hAT1 receptor. Gerbil receptors have very few non-conserved amino acids when compared to AT1 receptors from other mammalian species, including human. With the use of gain-of-function and loss-of-function mutations in the gAT1A, gAT1B and hAT1 receptors, we found that positions 107, 108, and 195 are crucial for determination of affinity of the AT1 receptor to non-peptidic antagonists of therapeutic efficacy. These observations may be of importance for the design of new, more potent and selective non-peptidic AT1 antagonists of therapeutic efficacy.

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National Institute of Mental Health (NIMH)
Intramural Research (Z01)
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Elkahloun, Abdel G; Hafko, Roman; Saavedra, Juan M (2016) An integrative genome-wide transcriptome reveals that candesartan is neuroprotective and a candidate therapeutic for Alzheimer's disease. Alzheimers Res Ther 8:5
Villapol, Sonia; Kryndushkin, Dmitry; Balarezo, Maria G et al. (2015) Hepatic expression of serum amyloid A1 is induced by traumatic brain injury and modulated by telmisartan. Am J Pathol 185:2641-52
Wang, Juan; Pang, Tao; Hafko, Roman et al. (2014) Telmisartan ameliorates glutamate-induced neurotoxicity: roles of AT(1) receptor blockade and PPAR? activation. Neuropharmacology 79:249-61
Sanchez-Lemus, Enrique; Benicky, Julius; Pavel, Jaroslav et al. (2009) Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen. Am J Physiol Regul Integr Comp Physiol 296:R1376-84
Pavel, Jaroslav; Benicky, Julius; Murakami, Yuki et al. (2008) Peripherally administered angiotensin II AT1 receptor antagonists are anti-stress compounds in vivo. Ann N Y Acad Sci 1148:360-6
Sommer, Wolfgang H; Saavedra, Juan M (2008) Targeting brain angiotensin and corticotrophin-releasing hormone systems interaction for the treatment of mood and alcohol use disorders. J Mol Med 86:723-8
Armando, Ines; Volpi, Simona; Aguilera, Greti et al. (2007) Angiotensin II AT1 receptor blockade prevents the hypothalamic corticotropin-releasing factor response to isolation stress. Brain Res 1142:92-9
Saavedra, Juan M; Benicky, Julius (2007) Brain and peripheral angiotensin II play a major role in stress. Stress 10:185-93
Saavedra, Juan M (2007) The challenge of genetic studies in hypertension. Circ Res 100:1389-93
Zorad, Stefan; Dou, Jing-tao; Benicky, Julius et al. (2006) Long-term angiotensin II AT1 receptor inhibition produces adipose tissue hypotrophy accompanied by increased expression of adiponectin and PPARgamma. Eur J Pharmacol 552:112-22

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