Our first specific aim is to clarify the mechanisms involved in the beneficial effects of compounds which have not, until very recently, been considered of interest to the therapy of brain disorders. The second specific aim is to further establish the extent of therapeutic benefits of such compounds in diseases of the brain. We study a group of compounds collectively named sartans, or Angiotensin II AT1 receptor blockers (ARBs). Sartans are biphenyl derivatives with an excellent margin of safety, extensively used to treat high blood pressure because they antagonize Angiotensin II-induced vasoconstriction. We have previously discovered that sartan treatment reduces brain ischemia, stress, and anxiety, and increases lifespan in rodent models. During the current fiscal year, we studied the mechanisms of anti-inflammatory effects of sartans in the brain. There was a dual impact of sartan treatment in rats subjected to systemic administration of bacterial endotoxin, lipopolysaccharide (LPS). First, we observed that sartans decreased LPS-induced peripheral overproduction of pro-inflammatory cytokines (interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, fractalkine). Enhanced cytokine production and release to the circulation is partially responsible for the central initial inflammatory response to LPS. Second, we found direct anti-inflammatory effects of sartans in brain parenchyma, evidenced by decreased LPS-induced activation of inflammatory cascades. Following sartan treatment in vivo, we found a decline in the LPS-induced activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NFkappaBalpha) and activator protein-1 (AP-1). Sartans also reduced expression of inducible nitric oxide synthase, cyclooxygenase-2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase induced by LPS, enzymes involved in the production of excess nitric oxide, prostaglandin E2, and reactive oxygen species leading to brain inflammation and neuronal injury. In addition, treatment with sartans produced a widespread decrease in LPS-induced microglia activation throughout the brain. Using cell cultures of human circulating monocytes and primary neuronal cultures, we discovered direct anti-inflammatory effects of ARBs against LPS and interleukin-1beta. These effects involve decreased activation of the protein kinases and the transcription factor NFkappaBalpha. In addition, sartans neuroprotective and anti-inflammatory effects were clearly established by cell viability assays and determination of inflammatory factors in cultured neurons and cerebral microvascular endothelial cells. We hypothesized that the major anti-inflammatory and neuroprotective effects of sartans may not be the exclusive result of AT1 receptor inhibition. In human circulating monocytes, cells expressing very few AT1 receptors, the anti-inflammatory effects of sartans were partially dependent on peroxisome proliferator-activated receptor gamma (PPARgamma) activation. We have found that some sartans may have dual mechanisms of action: anti-hypertensive, anti-growth and anti-inflammatory effects related to their inhibition of AT1 receptors, and metabolic and anti-inflammatory effects, partially the consequence of direct PPARgamma activation. Studies on rodent models of systemic inflammation established that anti-inflammatory effects of sartans in the brain are widespread, and not only related to classical brain targets for circulating inflammatory factors such as the hypothalamic paraventricular nucleus and areas outside the blood brain barrier. Reduced inflammation was also noted in multiple cortical areas including the prefrontal cortex, a key regulatory structure controlling the emotional response to stress. Our demonstration of protection from endotoxin in cerebrovascular endothelial cells indicated that the widespread anti-inflammatory effect of sartans involves AT1 receptor blockade in brain endothelial cells. These receptors, first characterized in our laboratory, are fundamental elements regulating the brain response to inflammation and the reaction to stress. In addition, sartans limit the exaggerated hormonal and sympathetic response to stress, effects dependent on the type and intensity of the challenge. For instance, while sartans completely prevent the hormonal and sympathetic response to isolation, these compounds are able to preserve the anti-inflammatory corticosterone response in inflammatory stress models. Moreover, sartans can prevent formation and release of aldosterone, a pro-inflammatory hormone stimulating brain mineralocorticoid receptors in the hippocampus. We also found, in rats submitted to acute restraint, that the anti-stress and anti-anxiety effects of sartan administration included prevention of the cortical gamma-aminobutyric acid (GABAA) receptor alterations characteristic of stress. Prevention of decreased activation of a major inhibitory system such as GABAA, explains the consistent anti-anxiety and anti-stress effects of sartan administration. These discoveries highlight the multiple central roles of AT1 receptors in the control of stress, inflammation, and mood. The anti-stress effects of ARBs have behavioral correlates. Sartan administration prevented LPS-induced sickness behavior, characterized by anorexia and weight loss, which occurs with systemic inflammation. In addition, sartans eliminated the anxiety responses produced by systemic LPS administration. In collaborative studies, we are analyzing the anti-depressant effects of sartan treatment, and the degree of involvement of the brain Angiotensin II system in the mechanisms of susceptibility to chronic variable stress. We have continued to clarify the mechanisms of neuroprotection and prolongation of lifespan produced by life-long sartan administration. We have found profound anti-inflammatory effects in the brain, including prevention of NADPH activation, which is responsible for age-dependent generation of neurotoxic reactive oxygen species, and a reversal of age-related cerebrovascular remodeling. We also found that the anti-anxiety effects of sartans persist throughout life. Our findings may, at least in part, explain the major beneficial effects of sartan therapy in Alzheimers disease. Our work continues with more advanced mechanistic studies including the use of gene knockout mouse models, microarray assays, and selective gene-silencing and phenotype-rescue experiments in cultured cells, and studies on animal models to determine the precise therapeutic range of sartans and related molecules. We are also interested in determining the extent of AT1 receptor blockade-dependent and independent effects of sartans. This information is very valuable for further discovery of sartan molecules of even better therapeutic value. Our mechanistic and translational studies will hopefully lead to novel treatment for disorders of the brain.
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