Brain inflammation. ARBs reverse cerebrovascular inflammation and limit the peripheral and brain innate immune response resulting from systemic administration of the bacterial endotoxin lipopolysaccharide (LPS). In FY 2009 we focused on the mechanisms of the anti-inflammatory effects of ARBs, selecting rodent brain target areas and human circulating target cells for study. Circulating pro-inflammatory cytokines and LPS induce a brain innate immune response by stimulating specific target sites, cerebrovascular endothelial cells, the paraventricular nucleus (PVN) and the subfornical organ (SFO). Peripheral administration of a centrally acting ARB decreased the LPS-induced gene transcription of multiple inflammatory markers in the PVN and SFO by processes including nitric oxide, prostaglandins, the nuclear factor kappa-B (NFkappaB) activation, and LPS molecular recognition. Moreover, ARBs decreased microglial activation in many brain areas including cortical structures. This indicates that the anti-inflammatory effect of ARBs in the brain is widespread. We established a link between AT1 receptors and serotonin in the regulation of the innate immune response. ARBs reduce free radical generation by preventing the LPS-induced up-regulation of the kynurenine pathway, a pro-inflammatory pathway contributing to serotonin metabolism. We have used gene-deletion (gene knock-out, k.o.) mouse models. In AT2 k.o. female mice, LPS enhances pro-inflammatory cytokines in the circulation and up-regulates the immune response and apoptotic programs. In normal rats, an AT2 agonist has anti-inflammatory properties in vivo, including reduction of the kynurenine pathway activity. Our observations established AT2 receptors as major regulators of the innate immune response and AT2 receptor agonists as lead compounds for new series of anti-inflammatory compounds of possible clinical use. Using AT1 receptor k.o. mice, we are attempting to determine whether life-long absence of active AT1 receptors changes the response to immune challenge. We are studying the processes of ARB reduction of microglia activation during brain inflammation using primary microglial cultures. We study the processes of neuronal response to inflammation in neuronal cultures of cerebellar granule cells. We are investigating the factors involved in the ARB protection from inflammation in cultures of human cerebrovascular endothelial cells with transfection and silencing of the AT1 receptor gene and phenotype rescue studies. We want to determine the extent and mechanisms of anti-inflammatory effects of ARBs on human circulating monocytes, target cells for LPS and a major factor in the response of the brain to peripheral inflammation. We previously found that ARBs rapidly reduced the LPS-induced gene expression and secretion of pro-inflammatory cytokines in unstimulated human monocytes. In FY 2009 we focused on the molecular processes leading to this effect. We determined that multiple mechanisms were responsible for the anti-inflammatory effect of ARBs in human monocytes, including inhibition of LPS-induced reactive oxygen species, nitric oxide and prostaglandin E2 formation. The ARB effects are decreased by a specific antagonist of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor down regulating pro-inflammatory gene expression. The conclusion is that ARBs are multifunctional anti-inflammatory compounds with dual AT1 receptor blockade and PPARgamma agonist effects in human cells. Our findings are of translational importance, because ARBs are clinically safe and may be tested for the prevention and treatment of inflammatory conditions of the brain. In our search for powerful, safe, centrally-acting anti-inflammatory compounds, we recently studied minocycline, a tetracycline antibiotic reported to be anti-inflammatory and neuroprotective. We found that minocycline prevents LPS-induced inflammation in human monocytes, increasing phosphorylation of Akt, a negative regulator of LPS inflammatory pathways. A specific inhibitor of phosphoinositide-3-kinase (PI3K) reduces minocycline effects, indicating that the PI3K-Akt pathway is involved in the anti-inflammatory effect of minocycline. Stress. ARBs limited the HPA axis stimulation, sympathetic activation and the alterations in expression of cortical benzodiazepine-1 receptors during isolation in rodent models. The benzodiazepine-1 receptor is part of the gamma amino butyric acid A (GABAA) receptor complex, the major inhibitory system in the brain. During FY 2009, we found that ARBs prevent the cortical benzodiazepine receptor response to restraint stress in rodents. These results explain the anti-anxiety effects of ARBs. Increased AT1 receptor gene transcription in the PVN is a common feature of all types of stress studied, supporting our hypothesis that AT1 receptors participate in the HPA axis stimulation during stress. The inflammatory and stress responses to immune challenge are closely related. We had previously found that ARBs decrease the response to LPS-induced stress and inflammation in the adrenal gland. We now report that ARBs decrease the LPS-induced upregulation of selective pro-inflammatory factor gene transcription in the pituitary gland, through processes involving nitric oxide production and NFkappaB activation. We have found evidence of a role for AT2 receptors during stress, and of cross-talk between brain AT1 and AT2 receptors. Peripheral administration of an AT2 receptor blocker with central AT2 blocking effects decreases the HPA axis basal activity and brain tyrosine hydroxylase transcription. Moreover, AT1 receptors are selectively expressed in dorsal root ganglia, sensory pathways in the spinal cord;they are transported in the sciatic nerve, and are expressed in sensory organs. These results suggest that AT1 receptors participate in the regulation of sensory information during stress. We continue our research on the mechanisms of the HPA axis response to stress and on the regulation of the cortical GABAA system by ARBs. In addition, we focus on two fundamental supra-hypothalamic structures, the hippocampus and the locus coeruleus. The hippocampus regulates the HPA axis response to stress and an important site for glucocorticoid regulatory feedback. This structure expresses large numbers of AT1 receptors involved in hippocampal function. The locus coeruleus is a principal player in the central sympathetic activation during stress. During isolation and cold restraint ARBs prevent the stress-dependent up-regulation of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine production. Further understanding of the processes involved in the regulation of the HPA axis, tyrosine hydroxylase and GABAA systems by ARBs will clarify the mechanisms of the anti-stress and anti-anxiety effects of these compounds. We are using laser microdissection techniques to identify selective neuronal populations within the PVN, hippocampus and locus coeruleus, and gene microarrays to identify regulatory pathways under the influence of Ang II AT1 and AT2 receptors. In conclusion, we clarified some of the modes of action of ARB anti-inflammatory, anti-stress and anti-anxiety effects, and further defined the role of AT2 receptors. Our translational research suggests that ARBs may be considered as a novel class of safe, multitasking medications for the treatment of psychiatric disorders including anxiety, depression and PTSD. Elucidation of their mechanisms of action may lead to the development of compounds of therapeutic potential.
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