Brain inflammation. In previous work, we had found that sustained in vivo AT1 receptor blockade with the ARB candesartan reversed, in a rat model of genetic hypertension, the cerebrovascular inflammation which is characteristic of hypertension in both rodents and humans. We asked the question whether ARBs could reduce other types of brain inflammation. During FY 2008, we focused on the effect of ARBs on the rat innate immune response, produced by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS). We found that ARBs limited the peripheral and brain inflammatory responses to systemic immune challenge in the rat. In vivo, ARBs reduced the LPS-induced inflammatory reaction in rodent spleen, adrenal gland, pituitary gland, and in multiple brain areas including the hypothalamus, olfactory system, hippocampus and cortex. Anti-inflammatory effects of ARBs were more pronounced in areas expressing large numbers of neuronal AT1 receptors. In addition, ARBs reduced the LPS-induced increase of the pro-inflammatory mineralocorticoid hormone aldosterone and enhanced the production of the anti-inflammatory hormone adiponectin. Moreover, ARBs rapidly reduced, in vitro, the LPS-induced pro-inflammatory gene expression and secretion of pro-inflammatory cytokines in cultured human monocytes. We next focused on the elucidation of the mechanisms of the anti-inflammatory effects of ARBs, and we asked the question which of the major mechanisms of inflammation stimulated by LPS were reduced or eliminated by ARB administration. We found that ARBs reduced, in vivo, the LPS-induced production of multiple pro-inflammatory factors, including reactive oxygen species, nitric oxide and prostaglandin E2. This implicates mechanisms involving Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase, cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS) and transcription factors such as NF-kappaB. We found similar ARB effects, in vitro, in cultured human circulating monocytes. A repeated finding after ARB administration was the decrease in the constitutive and in the LPS-induced expression of the LPS receptor CD14. We hypothesize that interference with the LPS molecular recognition complex may explain part of the anti-inflammatory effects of ARBs.? We are now focusing on the further clarification of the mechanisms of action of ARBs. We use a mouse model of AT1 receptor gene-deletion, to determine the effect of LPS when AT1 receptors are not expressed from birth. Cerebral endothelium plays a crucial role in the propagation of inflammatory signals throughout the brain and is critical for activation of specific groups of neurons involved in central response to systemic inflammation. We use cultures of cerebral endothelial cells and isolated microglia to study the relationship between LPS and AT1 receptors, to determine cellular colocalization and direct protein-protein interactions. Studies include gene transfections and gene silencing techniques in phenotype rescue studies and to identify critical components mediating the effect of ARBs in particular cell types. These experiments will further clarify the mechanisms of the anti-inflammatory effects of ARBs. In previous work, we have reported that CGP42112, an agonist of a second type of Ang II receptors, the AT2 receptors, actively inhibits the LPS-induced inflammation in human monocytes. During FY 2008 we discovered that, in rodents, CGP42112 effectively decreases LPS-induced inflammation in vivo. The anti-inflammatory effects of CGP42112 could be the result of AT2 receptor stimulation and/or binding to a novel macrophage receptor that we have previously discovered and reported, localized to brain and circulating macrophages. This novel non-Angiotensin receptor has not yet been characterized. In collaboration with Drs. Markey, NIMH, we are attempting to identify proteins which bind to CGP42112 using biotin-labeled CGP42112 and isolation by streptavidin coupling, and by laser capture microdissection, immunohistochemistry and proteomics. In other studies we will characterize the mechanisms of CGP42112 novel anti-inflammatory effects using AT2 gene-deleted mice and human circulating monocytes. ? ? Stress. We previously demonstrated that orally administered ARBs limited the HPA axis, sympathetic and cortical responses to isolation stress, and that these compounds reduced anxiety in rodents. On this basis, we previously proposed the first clinical protocol to evaluate the effects of AT1 receptor antagonists in the fear-startle response in human volunteers, with the goal to determine if AT1 receptor antagonists are effective in reducing anxiety and stress in humans. During FY 2008, we further explored the anti-stress effects of ARBs in response to immune challenge. We now find that ARBs limit the HPA axis response to the endotoxin lipopolysaccharide (LPS) in the adrenal gland, pituitary and hypothalamus. ARBs decreased the LPS-induced upregulation of the early transcription factor c-fos and microglial activation in the paraventricular nucleus. While eliminating the LPS-induced up-regulation of the hormone aldosterone, ARBs do not decrease LPS-induced ACTH and corticosterone responses, demonstrating that ARBs do not limit the anti-inflammatory effects of LPS-induced corticosteroid release. We found that the effect of ARBs on the HPA axis depend on the type of stress. During isolation stress, ARBs prevent HPA axis activation, but they do not prevent the HPA axis response to inflammation. We have also found that ARBs prevent the stress-induced increase in brain sympathetic activity by suppressing the stress-induced tyrosine hydroxylase gene transcription in the rat locus coeruleus. Our experiments are now focused on the elucidation of the mechanisms of the ARB control of tyrosine hydroxylase transcription in the locus coeruleus, the regulation of corticotrophin-releasing factor in the paraventricular nucleus, and the determination of additional brain sites involved on the anti-stress effects of ARBs. To this end we combine selective dissection techniques, gene microarray and proteomic studies. ? In conclusion, we demonstrated during the FY 2008 that ARBs may be considered as a novel class of multitasking anti-stress, anti-anxiety, anti-inflammatory medications in the treatment of brain disorders. They are already widely used to treat high blood pressure in humans and are safe and devoid of addictive properties, Elucidation of their mechanisms of action may lead to the development of other compounds of great therapeutic potential.
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