Our laboratory is focused on the study of the central functions of the hormone and brain modulator Angiotensin II, and in particular on its role in the regulation of the cerebral circulation and the reaction to stress. We have earlier discovered that pre-treating spontaneously (genetic) hypertensive rats (SHR) with an inhibitor of the peripheral, cerebrovascular and brain Angiotensin II AT1 receptors, candesartan, protected against subsequent brain ischemia resulting from occlusion of a major cerebral artery, the middle cerebral artery. Such protection paralleled the reversal of the hypertension-induced shifts in cerebral blood flow autoregulation. We later found that protection from ischemia and normalization of cerebral blood flow autoregulation is associated with preservation of blood flow above a critical threshold at the periphery of the ischemic zone after middle cerebral artery occlusion. We interpreted these results as a normalization of cerebrovascular compliance, and increased capacity to dilate when confronted with ischemia. Our finding of reversal of the hypertension-induced cerebrovascular remodeling by candesartan supports this hypothesis. Thus, blockade of AT1 receptors not only decreases blood pressure, but also inhibits the vasoconstrictor and growth-promoting actions of Angiotensin II in the cerebral vasculature. We also demonstrated, by comparison with other anti-hypertensive medications, that inhibition of the Angiotensin II system appeared superior, in its therapeutic potential for brain ischemia and stroke. We initiated a series of studies to clarify the mechanisms of action of the AT1 receptor inhibitors, and we hypothesized that this treatment might influence production of NO in the cerebral vasculature. Indeed, we discovered that in brain microvessels and in major cerebral arteries of the Willis polygon, the expression of eNOS mRNA and protein was lower in SHR when compared to normotensive controls, WKY rats. Conversely, the expression of iNOS mRNA and protein was higher in microvessels and Willis polygon of SHR when compared to WKY rats. Further, we demonstrated that pretreatment with candesartan normalized the expression of eNOS and iNOS in microvessels and large brain arteries of SHR. We hypothesize that, by reversing the altered eNOS/iNOS ratio, blockade of AT1 receptors enhances the vasodilating, anti-remodeling actions of NO, while decreasing its pro-inflammatory, pro-remodeling, deleterious effects. To further explore the effects of hypertension, and the mechanisms of the protective effects of AT1 blockade, we initiated experiments with the use of Gene Arrays. We analyzed gene expression in microvessels from SHR and WKY rats, treated with vehicle or with the AT1 antagonist. A preliminary analysis reveals that several hundred genes, from a total of over eight thousand genes analyzed, are apparently significantly increased or decreased when SHR are compared to WKY, when normotensive rats treated with vehicle are compared to rats treated with the AT1 antagonist, and when SHR treated with vehicle are compared with SHR treated with the AT1 antagonist. This database is the basis for several studies. After confirmation of the initial results by data analysis, we will study specific groups of genes selected on the basis of carefully researched specific questions, by means of RT-PCR, Western blot and immunohistochemistry. In addition to the AT1 receptors, there is a second type of Angiotensin II receptors, the AT2 receptors. We have found that in mice gene-deficient for AT2 receptors, the expression of AT1 receptors is increased. This is the explanation for the enhanced sensitivity to Angiotensin II and enhanced stress response in this model, supporting the hypothesis of a cross talk between the two Angiotensin II receptor types. While the AT1 receptors are involved in vasoconstriction, the AT2 receptors have been proposed as vasodilators. We found increased expression of AT2 receptors in the kidney of female mice when compared to male controls, and that the expression of renal AT2 receptors is dramatically increased by estrogen treatment of ovariectomized mice. These findings support a physiological role for AT2 receptors, and may explain why females are more resistant to renal ischemia and fibrosis than males. We are presently studying whether or not these findings extend to the brain circulation. In addition to its vasoconstrictive, pro-hypertensive, pro-ischemic properties, Angiotensin II is an important stress hormone. We have earlier demonstrated that pretreatment with a peripheral and central AT1 receptor antagonist completely prevents the sympathoadrenal response to isolation stress, including a prevention of the increase in adrenomedullary tyrosine hydroxylase mRNA. We have further analyzed the mechanism of action of the anti-stress property of the AT1 antagonists. The molecular mechanism of the modulation of tyrosine hydroxylase mRNA expression 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. Our results also indicate that AT1 and AT2 receptors regulate basal norepinephrine production, because blockade of either one of these receptor types decreases adrenomedullary norepinephrine and tyrosine hydroxylase mRNA expression. The AT1 and AT2 mechanisms partially overlap and are partially different. AT1 receptor blockade decreases adrenomedullary tyrosine hydroxylase mRNA, Fra-2, pCREB and pERK2, whereas AT2 receptor blockade only decreases adrenomedullary Fra-2. We are continuing our studies to further elucidate the mechanism of control of catecholamine production during basal conditions and during stress, by AT1 and AT2 Angiotensin II receptors. Treatment with AT1 receptor blockers prior to stress also 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 Angiotensin II AT1 receptors modulate multiple central CRH systems. In addition, AT1 receptor blockade has an anxiolytic effect in rodents, as determined by behavioral studies using the Plus-Maze. These findings, and the modulation of brain CRH receptors, may indicate that AT1 receptor antagonists may be considered as a possible novel class of anti-anxiety medications. Because these compounds are devoid of addictive properties, development of new compounds of this class may result in anti-anxiety medications of great therapeutic potential. Finally, we studied whether AT1 receptor inhibition could prevent the development of stress-related disorders. We found that pretreatment with AT1 receptor antagonists totally prevented the development of stress-induced gastric ulcers in the rat. The mechanism of this protective effect appears to be the combination of local increase in blood flow to the stomach mucosa and of selective inhibition of the sympathetic response to stress. In conclusion, our studies indicate that non-peptidic antagonists of the AT1 receptor with central effects are among the drugs of choice in the treatment of cardiovascular disease and brain ischemia, may protect against stress-related disorders, and may be useful compounds to develop effective and non-addictive anti-anxiety drugs.
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