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Beyond blood pressure control, angiotensin receptor blockers reduce common injury mechanisms, decreasing excessive inflammation and protecting endothelial and mitochondrial function, insulin sensitivity, the coagulation cascade, immune responses, cerebrovascular flow, and cognition, properties useful to treat inflammatory, age-related, neurodegenerative, and metabolic disorders of many organs including brain and lung.

This special Issue presents comprehensive and state-of-the-art advances in supporting the crucial role of the bidirectional interactions between the Brain-Gut Axis in health and diseases with an emphasis on the microbiome-gut-brain axis and its implications in variety of neurological disorders. There are intimate connections between the brain and the digestive system. Gut microbiota dysbiosis activates the intestinal immune system, enhances intestinal permeability and bacterial translocation, leading to neuroinflammation, epigenetic changes, cerebrovascular alterations, amyloid β formation and α-synuclein protein aggregates. These alterations may participate in the development of hypertension, Alzheimer, Parkinson, stroke, epilepsy and autism. Brainstem nuclei such as the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMV) regulate gastric motor function by way of bidirectional inputs through the vagus nerve.

Preclinical experiments and clinical trials demonstrated that angiotensin II AT1 receptor overactivity associates with aging and cellular senescence and that AT1 receptor blockers (ARBs) protect from age-related brain disorders. In a primary neuronal culture submitted to glutamate excitotoxicity, gene set enrichment analysis (GSEA) revealed expression of several hundred genes altered by glutamate and normalized by candesartan correlated with changes in expression in Alzheimer's patient's hippocampus. To further establish whether our data correlated with gene expression alterations associated with aging and senescence, we compared our global transcriptional data with additional published datasets, including alterations in gene expression in the neocortex and cerebellum of old mice, human frontal cortex after age of 40, gene alterations in the Werner syndrome, rodent caloric restriction, Ras and oncogene-induced senescence in fibroblasts, and to tissues besides the brain such as the muscle and kidney. The most significant and enriched pathways associated with aging and senescence were positively correlated with alterations in gene expression in glutamate-injured neurons and, conversely, negatively correlated when the injured neurons were treated with candesartan. Our results involve multiple genes and pathways, including CAV1, CCND1, CDKN1A, CHEK1, ICAM1, IL-1B, IL-6, MAPK14, PTGS2, SERPINE1, and TP53, encoding proteins associated with aging and senescence hallmarks, such as inflammation, oxidative stress, cell cycle and mitochondrial function alterations, insulin resistance, genomic instability including telomere shortening and DNA damage, and the senescent-associated secretory phenotype. Our results demonstrate that AT1 receptor blockade ameliorates central mechanisms of aging and senescence. Using ARBs for prevention and treatment of age-related disorders has important translational value.

Graphical abstract, Highlights • Candesartan downregulates expression of many glutamate upregulated genes in cultured neurons. • Glutamate upregulated genes in neurons positively correlate with COVID-19 upregulated genes. • Candesartan gene downregulation negatively correlates with COVID-19 upregulated genes. • Candesartan downregulates genes involved in the COVID-19 cytokine storm., Background Angiotensin receptor blockers (ARBs) reducing inflammation and protecting lung and brain function, could be of therapeutic efficacy in COVID-19 patients. Methods Using GSEA, we compared our previous transcriptome analysis of neurons injured by glutamate and treated with the ARB Candesartan (GSE67036) with transcriptional signatures from SARS-CoV-2 infected primary human bronchial epithelial cells (NHBE) and lung postmortem (GSE147507), PBMC and BALF samples (CRA002390) from COVID-19 patients. Results Hundreds of genes upregulated in SARS-CoV-2/COVID-19 transcriptomes were similarly upregulated by glutamate and normalized by Candesartan. Gene Ontology analysis revealed expression profiles with greatest significance and enrichment, including proinflammatory cytokine and chemokine activity, the NF-kappa B complex, alterations in innate and adaptive immunity, with many genes participating in the COVID-19 cytokine storm. Conclusions There are similar injury mechanisms in SARS-CoV-2 infection and neuronal injury, equally reduced by ARB treatment. This supports the hypothesis of a therapeutic role for ARBs, ameliorating the COVID-19 cytokine storm.

ImportanceAssessment of the use of angiotensin receptor blockers (ARBs) in the United States provides insight into prescribing practices, and may inform guidelines, policy measures and research during the COVID-19 pandemic.ObjectiveTo evaluate trends in ARB use among adults in the United States who have preexisting conditions and sociodemographic risk factors that put them at a higher risk of SARS-CoV-2 infection and COVID-19-related complications and mortality.Design, setting and participantsThis study uses the nationally representative cross-sectional data from the 2005-2018 National Health and Nutrition Examination Survey (NHANES). Participants included 39,749 non-institutionalized U.S. civilian adults who were 20 years and older and those with the most common preexisting conditions and risk factors reported among patients with COVID-19.Main outcomes and measuresUse of ARBs in the prior 30 days from survey interview.ResultsARB use ranged from 7.4% [95% CI, 6.5%-8.4%] to 26.2% [95% CI, 19.4%-34.4%] among those with one or two metabolic, renal, respiratory, and/or cardiovascular diseases. Among individuals with the three most common preexisting conditions in patients with COVID-19 including hypertension, diabetes and obesity, ARB use was higher among the elderly, females, non-Hispanic whites, and those with health insurance coverage.Conclusions and relevanceIn this nationally representative survey, ARB use was found to be widespread, but unevenly distributed among individuals with conditions and sociodemographic risk factors that place them at a higher risk of COVID-19 morbidity and mortality.Key PointsQuestionWhat is the prevalence of angiotensin receptor blocker (ARB) use among individuals at higher risk of COVID-19-related complications?FindingsIn a cross-sectional study with data from 39,749 adult participants of the National Health and Nutrition Examination Survey, ARB use ranged between 7.4% and 26.2% among those with one or two respiratory, metabolic, renal and/or cardiovascular diseases. Significant disparities in ARB use were found in participants with preexisting conditions and sociodemographic factors that place them at a higher risk of COVID-19 morbidity and mortality.MeaningARB use is widespread and disproportionate in the United States among people at higher risk of COVID-19 complications.

Trace amines, including β-phenylethylamine, p-octopamine, p-tyramine, and tryptamine, are produced in high levels in invertebrates where they play major roles in homeostasis regulation in a manner similar to that of adrenergic systems in mammals (Rutigliano et al. in Front Pharmacol 8:987, 2017; Gainetdinov et al. in Pharmacol Rev 70(3):549-620, 2018; Nagaya et al. in Neurosci Lett 329(3):324-328, 2002). In mammals, however, their levels are very low, initially prompting these molecules to be termed "trace" or "minor" amines in mammals with only a secondary role in the regulation of more abundant biogenic amines including catecholamines and serotonin (Gainetdinov et al. in Pharmacol Rev 70(3):549-620, 2018). The more recent discovery of trace amine-associated receptors (TAARs) revealed major, previously unsuspected roles of the trace amines and has led to increasing interest within the scientific community. For example, TAARs have been proposed to modulate signaling through dopamine (Schwartz et al. in Expert Opin Ther Targets 22(6):513-526, 2018). Furthermore, these receptors are implicated in both numerous physiological functions including regulation of sleep, olfaction, metabolism, and immunity as well in disease (e.g., substance abuse, neuropsychiatric disorders) (Gainetdinov et al. in Pharmacol Rev 70(3):549-620, 2018; Rutigliano et al. in Front Pharmacol 8:987, 2017). Consequently, trace amine and TAAR research is rapidly growing and is of great translational relevance. In this Special Issue, leaders in trace amine and TAAR research offer both reviews and original research papers that cover a wide range of topics from involvement of TAAR signaling in metabolic regulation and neurophysiology to implications of this signaling in neuropsychiatric diseases including substance abuse and schizophrenia. While a diverse range of topics is covered by these works, the common theme running through all of them is the increasing awareness that trace amine and TAAR signaling represent novel signaling mechanisms in the brain and periphery. These topics are both highly timely and of considerable importance not only for those working in the field but also for the neuroscience community at large.

Aims: Microglia-mediated neuroinflammation plays an important role in focal ischemic stroke, a disorder with no effective therapeutic agents. Since microglial polarization to the M2 phenot...

Neuroinflammation plays a vital role in the pathological process of cerebral ischemic stroke, but currently there is no effective treatment. After ischemia, microglia-produced proinflammatory mediator expression contributes to the aggravation of neuroinflammation, while anti-inflammatory activation of microglia develops an anti-neuroinflammatory effect via secretion of anti-inflammatory factor. Promoting the anti-inflammatory activation of microglia might be an effective treatment of stroke. Previously, we discovered one derivative of the natural product (+)-balasubramide, compound 3C, that exhibits a remarkably anti-neuroinflammatory effect in vitro with unknown mechanisms. Thus in this study, we aimed to clarify its molecular mechanisms and determine whether compound 3C has a neuroprotective effect after ischemia via regulation on microglial inflammation. We found that compound 3C promoted the anti-inflammatory mediator expression and reduced the proinflammatory mediator expression in LPS-stimulated BV2 cells and mouse primary microglia cells, which were reversed by AMP-activated protein kinase (AMPK) inhibition or AMPK upstream calmodulin-dependent protein kinase kinase beta (CaMKKβ) inhibition. Compound 3C also prevented LPS-stimulated JNK activation and enhanced PGC-1α activation in microglia, which was attenuated by AMPK inhibition. Additionally, compound 3C ameliorated depressive behaviors in LPS-induced neuroinflammatory mice by promoting the anti-inflammatory activation of microglia. Furthermore, we found that compound 3C markedly reduced brain infarct volume, improved the neurological deficit in rats with ischemia and reduced the activated microglia/macrophage cells in the ischemic area, which concomitantly enhanced the anti-inflammatory mediator expression. A mechanistic study showed that the compound 3C-mediated activation of CaMKKβ, AMPK and PGC-1α is involved in the anti-neuroinflammatory and neuroprotective effects of 3C in the brain of LPS-treated mice and ischemic rats. Taken together, our results show that compound 3C could suppress neuroinflammation in vitro and in vivo by modulating microglial activation state through the CaMKKβ-dependent AMPK/PGC-1α signaling pathway, and maybe further be developed as a promising new drug candidate for the treatment of brain disorders such as stroke associated with brain inflammation.

Angiotensin II, through AT1 receptor stimulation, mediates multiple cardiovascular, metabolic, and behavioral functions including the response to stressors. Conversely, the function of Angiotensin II AT2 receptors has not been totally clarified. In adult rodents, AT2 receptor distribution is very limited but it is particularly high in the adrenal medulla. Recent results strongly indicate that AT2 receptors contribute to the regulation of the response to stress stimuli. This occurs in association with AT1 receptors, both receptor types reciprocally influencing their expression and therefore their function. AT2 receptors appear to influence the response to many types of stressors and in all components of the hypothalamic–pituitary–adrenal axis. The molecular mechanisms involved in AT2 receptor activation, the complex interactions with AT1 receptors, and additional factors participating in the control of AT2 receptor regulation and activity in response to stressors are only partially understood. Further research is necessary to close this knowledge gap and to clarify whether AT2 receptor activation may carry the potential of a major translational advance.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA contributing to protect the blood-brain barrier (BBB) after stroke. We searched for small molecules that may up-regulate MALAT1 and focused on polydatin (PD), a natural product, as a possible candidate. PD enhanced MALAT1 gene expression in rat brain microvascular endothelial cells, reducing cell toxicity and apoptosis after oxygen and glucose deprivation (OGD). These effects correlated with reduction of inflammatory factors and enhancement of expression of BBB markers. We found opposite changes after MALAT1 silencing. We determined that C/EBPβ is a key transcription factor for PD-mediated MALAT1 expression. PPARγ activity is involved in MALAT1 protective effects through its coactivator PGC-1α and the transcription factor CREB. This suggests that PD activates the MALAT1/CREB/PGC-1α/PPARγ signaling pathway to protect endothelial cells against ischemia. PD administration to rats subjected to brain ischemia by transient middle cerebral artery occlusion (tMCAO) reduced cerebral infarct volume and brain inflammation, protected cerebrovascular endothelial cells and BBB integrity. These effects correlated with increased expression of MALAT1, C/EBPβ, and PGC-1α. Our results strongly suggest that the beneficial effects of PD involve the C/EBPβ/MALAT1/CREB/PGC-1α/PPARγ pathway, which may provide a novel therapeutic strategy for brain ischemic stroke.

The Angiotensin II Receptor Blocker (ARB) Telmisartan reduces inflammation through Angiotensin II AT1 receptor blockade and peroxisome proliferator-activated receptor gamma (PPARγ) activation. However, in a mouse microglia-like BV2 cell line, imitating primary microglia responses with high fidelity and devoid of AT1 receptor gene expression or PPARγ activation, Telmisartan reduced gene expression of pro-injury factors, enhanced that of anti-inflammatory genes, and prevented LPS-induced increase in inflammatory markers. Using global gene expression profiling and pathways analysis, we revealed that Telmisartan normalized the expression of hundreds of genes upregulated by LPS and linked with inflammation, apoptosis and neurodegenerative disorders, while downregulating the expression of genes associated with oncological, neurodegenerative and viral diseases. The PPARγ full agonist Pioglitazone had no neuroprotective effects. Surprisingly, the PPARγ antagonists GW9662 and T0070907 were neuroprotective and enhanced Telmisartan effects. GW9226 alone significantly reduced LPS toxic effects and enhanced Telmisartan neuroprotection, including downregulation of pro-inflammatory TLR2 gene expression. Telmisartan and GW9662 effects on LPS injury negatively correlated with pro-inflammatory factors and upstream regulators, including TLR2, and positively with known neuroprotective factors and upstream regulators. Gene Set Enrichment Analysis (GSEA) of the Telmisartan and GW9662 data revealed negative correlations with sets of genes associated with neurodegenerative and metabolic disorders and toxic treatments in cultured systems, while demonstrating positive correlations with gene sets associated with neuroprotection and kinase inhibition. Our results strongly suggest that novel neuroprotective effects of Telmisartan and GW9662, beyond AT1 receptor blockade or PPARγ activation, include downregulation of the TLR2 signaling pathway, findings that may have translational relevance.

The natural product totarol, a phenolic diterpenoid and a major constituent isolated from the sap of Podocarpus totara, has been reported to have a potent antimicrobial activity. In this study, we determined whether totarol possessed an additional neuroprotective activity in vitro and in vivo. We found that totarol prevented glutamate- and oxygen and glucose deprivation-induced neuronal death in primary rat cerebellar granule neuronal cells and cerebral cortical neurons. Totarol increased Akt and GSK-3β phosphorylation, Nrf2 and heme oxygenase-1 (HO-1) protein expressions and suppressed oxidative stress by increasing GSH and SOD activities. The PI3K/Akt inhibitor LY294002 prevented totarol neuroprotective effect by suppressing the totarol-induced changes in HO-1 expression and the activities of GSH and SOD. The HO-1 inhibitor ZnPPIX also prevented totarol-increased GSH and SOD activities. In a model of acute cerebral ischemic injury in Sprague-Dawley rats, produced by occlusion of the middle cerebral artery for 2h followed by 22 h or 46 h of reperfusion, totarol significantly reduced infarct volume and improved the neurological deficit. In this model, totarol increased HO-1 expression and the activities of GSH and SOD. These observations suggest that totarol may be a novel activator of the Akt/HO-1 pathway protecting against ischemic stroke through reduction of oxidative stress.

Traumatic brain injury affects the whole body in addition to the direct impact on the brain. The systemic response to trauma is associated with the hepatic acute-phase response. To further characterize this response, we performed controlled cortical impact injury on male mice and determined the expression of serum amyloid A1 (SAA1), an apolipoprotein, induced at the early stages of the acute-phase response in liver and plasma. After cortical impact injury, induction of SAA1 was detectable in plasma at 6 hours post-injury and in liver at 1 day post-injury, followed by gradual diminution over time. In the liver, cortical impact injury increased neutrophil and macrophage infiltration, apoptosis, and expression of mRNA encoding the chemokines CXCL1 and CXCL10. An increase in angiotensin II AT1 receptor mRNA at 3 days post-injury was also observed. Administration of the AT1 receptor antagonist telmisartan 1 hour post-injury significantly decreased liver SAA1 levels and CXCL10 mRNA expression, but did not affect CXCL1 expression or the number of apoptotic cells or infiltrating leukocytes. To our knowledge, this is the first study to demonstrate that SAA1 is induced in the liver after traumatic brain injury and that telmisartan prevents this response. Elucidating the molecular pathogenesis of the liver after brain injury will assist in understanding the efficacy of therapeutic approaches to brain injury.

See Moon (doi:10.1093/awv239) for a scientific commentary on this article.Traumatic brain injury frequently leads to long-term cognitive problems and physical disability yet remains without effective therapeutics. Traumatic brain injury results in neuronal injury and death, acute and prolonged inflammation and decreased blood flow. Drugs that block angiotensin II type 1 receptors (AT1R, encoded by AGTR1) (ARBs or sartans) are strongly neuroprotective, neurorestorative and anti-inflammatory. To test whether these drugs may be effective in treating traumatic brain injury, we selected two sartans, candesartan and telmisartan, of proven therapeutic efficacy in animal models of brain inflammation, neurodegenerative disorders and stroke. Using a validated mouse model of controlled cortical impact injury, we determined effective doses for candesartan and telmisartan, their therapeutic window, mechanisms of action and effect on cognition and motor performance. Both candesartan and telmisartan ameliorated controlled cortical impact-induced injury with a therapeutic window up to 6 h at doses that did not affect blood pressure. Both drugs decreased lesion volume, neuronal injury and apoptosis, astrogliosis, microglial activation, pro-inflammatory signalling, and protected cerebral blood flow, when determined 1 to 3 days post-injury. Controlled cortical impact-induced cognitive impairment was ameliorated 30 days after injury only by candesartan. The neurorestorative effects of candesartan and telmisartan were reduced by concomitant administration of the peroxisome proliferator-activated receptor gamma (PPARγ, encoded by PPARG) antagonist T0070907, showing the importance of PPARγ activation for the neurorestorative effect of these sartans. AT1R knockout mice were less vulnerable to controlled cortical impact-induced injury suggesting that the sartan's blockade of the AT1R also contributes to their efficacy. This study strongly suggests that sartans with dual AT1R blocking and PPARγ activating properties have therapeutic potential for traumatic brain injury.

Detailed molecular mechanisms underpinning enzymatic reactions are still a central problem in biochemistry. The need for active site flexibility to sustain catalytic activity constitutes a notion of wide acceptance, although its direct influence remains to be fully understood. With the aim of studying the relationship between structural dynamics and enzyme catalysis, the cellulase Cel5A from Bacillus agaradherans is used as a model for in silico comparative analysis with mesophilic and psychrophilic counterparts. Structural features that determine flexibility are related to kinetic and thermodynamic parameters of catalysis. As a result, three specific positions in the vicinity of the active site of Cel5A are selected for protein engineering via site-directed mutagenesis. Three Cel5A variants are generated, N141L, A137Y and I102A/A137Y, showing a concomitant increase in the catalytic activity at low temperatures and a decrease in activation energy and activation enthalpy, similar to cold-active enzymes. These results are interpreted in structural terms by molecular dynamics simulations, showing that disrupting a hydrogen bond network in the vicinity of the active site increases local flexibility. These results provide a structural framework for explaining the changes in thermodynamic parameters observed between homologous enzymes with varying temperature adaptations.

A canonical brain Renin-Angiotensin System (RAS), like the canonical peripheral RAS, has long been proposed as a major regulator of brain function. Because of methodological limitations, however, this system is in urgent need of critical revision. The Angiotensin II AT1 receptors have been confirmed as key factors in the regulation of brain function, and AT1 receptor over activity has been established as a major and early injury factor in the development of many brain diseases. Consequently, Angiotensin Receptor Blockers (ARBs), compounds widely used to treat cardiovascular and metabolic disorders, are excellent candidates for repurposing for the treatment of brain disorders. This review will discuss some of the reasons why revisiting brain RAS is a pressing necessity, will present evidence for a participation of AT1 receptor over activity in the development of major brain disorders, and will present definite evidence of ARBs neuroprotective effects. The review will focus on the beneficial effects of ARB therapy in stroke, neurodegenerative disorders including Alzheimer's and Parkinson's diseases, traumatic brain injury, radiation- induced brain damage, stress and mood disorders.

Microglia, of myeloid origin, play fundamental roles in the control of immune responses and the maintenance of central nervous system homeostasis. These cells, just like peripheral macrophages, may be activated into M1 pro-inflammatory or M2 anti-inflammatory phenotypes by appropriate stimuli. Microglia do not respond in isolation, but form part of complex networks of cells influencing each other. This review addresses the complex interaction of microglia with each cell type in the brain: neurons, astrocytes, cerebrovascular endothelial cells, and oligodendrocytes. We also highlight the participation of microglia in the maintenance of homeostasis in the brain, and their roles in the development and progression of age-related neurodegenerative disorders.

Sartans (Angiotensin II AT1 Receptor Blockers, ARBs) are powerful neuroprotective agents in vivo and protect against IL-1β neurotoxicity in vitro. The purpose of this research was to determine the extent of sartan neuroprotection against glutamate excitotoxicity, a common cause of neuronal injury and apoptosis. The results show that sartans are neuroprotective, significantly reducing glutamate-induced neuronal injury and apoptosis in cultured rat primary cerebellar granule cells (CGCs). Telmisartan was the most potent sartan studied, with an order of potency telmisartan > candesartan > losartan > valsartan. Mechanisms involved reduction of pro-apoptotic caspase-3 activation, protection of the survival PI3K/Akt/GSK-3β pathway and prevention of glutamate-induced ERK1/2 activation. NMDA receptor stimulation was essential for glutamate-induced cell injury and apoptosis. Participation of AT1A receptor was supported by glutamate-induced upregulation of AT1A gene expression and AT1 receptor binding. Conversely, AT1B or AT2 receptors played no role. Glutamate-induced neuronal injury and the neuroprotective effect of telmisartan were decreased, but not abolished, in CGCs obtained from AT1A knock-out mice. This indicates that although AT1 receptors are necessary for glutamate to exert its full neurotoxic potential, part of the neuroprotective effect of telmisartan is independent of AT1 receptor blockade. PPARγ activation was also involved in the neuroprotective effects of telmisartan, as telmisartan enhanced PPARγ nuclear translocation and the PPARγ antagonist GW9662 partially reversed the neuroprotective effects of telmisartan. The present results substantiate the therapeutic use of sartans, in particular telmisartan, in neurodegenerative diseases and traumatic brain disorders where glutamate neurotoxicity plays a significant role.

Traumatic brain injury (TBI) results in complex pathological reactions, the initial lesion worsened by secondary inflammation and edema. Angiotensin II (Ang II) is produced in the brain and Ang II receptor type 1 (AT₁R) overstimulation produces vasoconstriction and inflammation. Ang II receptor blockers (ARBs) are neuroprotective in models of stroke but little is known of their effect when administered in TBI models. We therefore performed controlled cortical impact (CCI) injury on mice to investigate whether the ARB candesartan would mitigate any effects of TBI. We administered candesartan or vehicle to mice 5 h before CCI injury. Candesartan treatment reduced the lesion volume after CCI injury by approximately 50%, decreased the number of dying neurons, lessened the number of activated microglial cells, protected cerebral blood flow (CBF), and reduced the expression of the cytokine TGFβ1 while increasing expression of TGFβ3. Candesartan-treated mice also showed better motor skills on the rotarod 3 days after injury, and improved performance in the Morris water maze 4 weeks after injury. These results indicate that candesartan is neuroprotective, reducing neuronal injury, decreasing lesion volume and microglial activation, protecting CBF and improving functional behavior in a mouse model of TBI. Co-treatment with a peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist significantly reduced some of the beneficial effects of candesartan after CCI, suggesting that PPARγ activation may contribute to part or to all of the neuroprotective effect of candesartan. Overall, our data suggest that ARBs with dual AT₁R-blocking and PPARγ activation properties may have therapeutic value in treating TBI.

The effects of brain AngII (angiotensin II) depend on AT1 receptor (AngII type 1 receptor) stimulation and include regulation of cerebrovascular flow, autonomic and hormonal systems, stress, innate immune response and behaviour. Excessive brain AT1 receptor activity associates with hypertension and heart failure, brain ischaemia, abnormal stress responses, blood–brain barrier breakdown and inflammation. These are risk factors leading to neuronal injury, the incidence and progression of neurodegerative, mood and traumatic brain disorders, and cognitive decline. In rodents, ARBs (AT1 receptor blockers) ameliorate stress-induced disorders, anxiety and depression, protect cerebral blood flow during stroke, decrease brain inflammation and amyloid-β neurotoxicity and reduce traumatic brain injury. Direct anti-inflammatory protective effects, demonstrated in cultured microglia, cerebrovascular endothelial cells, neurons and human circulating monocytes, may result not only in AT1 receptor blockade, but also from PPARγ (peroxisome-proliferator-activated receptor γ) stimulation. Controlled clinical studies indicate that ARBs protect cognition after stroke and during aging, and cohort analyses reveal that these compounds significantly reduce the incidence and progression of Alzheimer's disease. ARBs are commonly used for the therapy of hypertension, diabetes and stroke, but have not been studied in the context of neurodegenerative, mood or traumatic brain disorders, conditions lacking effective therapy. These compounds are well-tolerated pleiotropic neuroprotective agents with additional beneficial cardiovascular and metabolic profiles, and their use in central nervous system disorders offers a novel therapeutic approach of immediate translational value. ARBs should be tested for the prevention and therapy of neurodegenerative disorders, in particular Alzheimer's disease, affective disorders, such as co-morbid cardiovascular disease and depression, and traumatic brain injury.

Exposure to stress during early development causes long-lasting alterations in behaviour and hypothalamic pituitary adrenal (HPA) axis activity, including increased expression of corticotrophin-releasing hormone (CRH). To determine whether early-life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription, 8-week old female and male rats, subjected to maternal deprivation (MD) between days 2 and 13 post-birth, were studied for HPA axis responses to stress and CRH promoter methylation in the hypothalamic paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA). Plasma corticosterone and PVN CRH heteronuclear (hn)RNA responses to acute restraint stress were higher in MD rats of both sexes. DNA methylation analysis of the CRH promoter revealed a significantly lower percentage of methylation in two CpGs preceding (CpG1) and inside (CpG2) the cyclic AMP-response element (CRE) at -230 bp in the CRH promoter in the PVN but not the CeA of MD rats. Gel-shift assays, using nuclear proteins from forskolin-treated hypothalamic 4B cells and CRH promoter CRE oligonucleotides, unmethylated or methylated at CpG1, revealed a strong band that was supershifted by phospho-cAMP response element-binding antibody. This band was 50% weaker using oligonucleotides methylated at CpG2 (intra-CRE), or methylated at both CpG1 and CpG2. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the CRH promoter CRE, a region critical for CRH transcriptional activation, could serve as a mechanism for the increased transcriptional responses to stress observed in MD rats.

Centrally acting Angiotensin II AT(1) receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 μg/kg) with or without 3 days of pretreatment with the ARB candesartan (1mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ(1) receptors and reduced mRNA expression of the GABA(A) receptor γ(2) subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ(1) receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ(1) binding, and decreased γ(2) subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF(1) receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF(2) receptor binding was undetectable, but CRF(2) mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ(1) receptor expression; and that the stress-induced BZ(1) receptor expression is under the control of AT(1) receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ(1) receptors.

Angiotensin II type 1 receptor (AT1) blockers (ARBs) reduce the bacterial endotoxin lipopolysaccharide (LPS)-induced innate immune response in human circulating monocytes expressing few AT1. To clarify the mechanisms of anti-inflammatory effects of ARBs with different peroxisome proliferator-activated receptor-γ (PPARγ)-activating potencies, we focused our study on telmisartan, an ARB with the highest PPARγ-stimulating activity.Human circulating monocytes and monocytic THP-1 (human acute monocytic leukemia cell line) cells were exposed to 50 ng/ml LPS with or without pre-incubation with telmisartan. AT1 mRNA and protein expressions were determined by real-time PCR and membrane receptor binding assay, respectively. The expression of pro-inflammatory factors was determined by real-time PCR, western blot analysis and ELISA. PPARγ activation was measured by electrophoretic mobility shift assay and its role was determined by pharmacological inhibition and PPARγ gene silencing.In human monocytes, telmisartan significantly attenuated the LPS-induced expression of pro-inflammatory factors, the release of pro-inflammatory cytokines and prostaglandin E2, nuclear factor-κB activation and reactive oxygen species formation. In THP-1 cells, telmisartan significantly reduced LPS-induced tumor necrosis factor-α, inhibitor of κB-α, monocyte chemotactic protein-1 (MCP-1) and lectin-like oxidized low-density lipoprotein receptor-1 gene expression and MCP-1-directed migration. Telmisartan also stimulated the expression of the PPARγ target genes cluster of differentiation 36 and ATP-binding cassette subfamily G member 1 in monocytes. The anti-inflammatory effects of telmisartan were prevented by both PPARγ antagonism and PPARγ gene silencing. Anti-inflammatory effects of ARBs correlated with their PPARγ agonist potency.Our observations demonstrate that in human monocytes, ARBs inhibit the LPS-induced pro-inflammatory response to a major extent through the PPARγ activation pathway and may be beneficial for the treatment of cardiovascular and metabolic disorders in which inflammation plays a major role.

Inhibition of angiotensin II receptor type 1 (AT1) reduces chronic inflammation associated with hypertension. We asked whether AT1 receptor inhibition would reduce the innate inflammatory response induced by bacterial lipopolysaccharide (LPS).We used unstimulated human circulating monocytes obtained from healthy donors by counterflow centrifugal elutriation. Monocytes were studied in vitro after incubation with LPS (50 ng/ml) with and without 1 mumol/l candesartan, an AT1 receptor blocker. Angiotensin II receptor mRNA expression was determined by reverse transcriptase-PCR and receptor binding by autoradiography; inflammatory factor mRNA expression was studied by reverse transcriptase-PCR and cytokine release by ELISA.Human monocytes did not express detectable AT1 receptors, and angiotensin II did not induce inflammatory factor mRNA expression or cytokine release. However, candesartan substantially reduced the LPS-induced expression of the mRNAs for the LPS recognition protein cluster of differentiation 14, the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 and the lectin-like oxidized low-density lipoprotein receptor. In addition, candesartan reduced the activation of the nuclear factor kappa B pathway, the tumor necrosis factor alpha and interleukin-6 secretion, and the ROS formation induced by LPS, without affecting the secretion of interleukin-10.We hypothesize that the anti-inflammatory effects of candesartan in these cells are likely mediated by mechanisms unrelated to AT1 receptor blockade. Our results demonstrate that candesartan significantly reduces the innate immune response to LPS in human circulating monocytes. The anti-inflammatory effects of candesartan may be of importance not only in hypertension but also in other inflammatory disorders.

Genetic influences contribute upto 30–50% to blood pressure variability in human essential hypertension [1]. Intense efforts have been dedicated to unraveling the role of genes in this disease, following two parallel and interacting tracks. Studies on human populations have mostly focused on the candidate gene linkage approach, associating single nucleotide polymorphic (SNP) markers with the hypertensive phenotype. In spite of some promising observations [2], most attempts to replicate association studies have not been encouraging [3]. There are calls for application of stricter quality criteria for genetic studies, including better definition of phenotype characteristics, increased attention to environmental factors, sharing and combining data resources and more emphasis on whole genome-linkage scans in the context of collaborative multidisciplinary approaches [4–17]. Recently, however, there has been impressive progress in designing studies, understanding the mechanisms of disease and genotyping technology [17]. The second parallel track is the development and study of rodent experimental models of disease. Genetically hypertensive rat strains have been developed over the past 60 years. The models were obtained from initial random rat populations by brother—sister mating selecting for a high blood pressure phenotype. The better characterized genetically hypertensive strains are the spontaneously hypertensive rat (SHR) [18], stroke-prone SHR (SPSHR) [19], Dahl saltsensitive [20], Milan hypertensive (MHS) [21], New Zealand genetically hypertensive [22], Sabra hypertensive (SBH) [23] and the Lyon hypertensive rats [24]. Comparisons are generally made with inbred normotensive strains derived from the original rat population and sharing most of the genetic background with the corresponding hypertensive strain. The most common normotensive strains are the Dahl salt-resistant, Wistar—Kyoto (WKY), Milan normotensive (MNS), genetically normotensive, Sabra normotensive (SBN) and Lyon normotensive and Lyon low blood pressure rats [18–24]. Physiological and pharmacological approaches to the study of these models have enhanced our understanding of many systems for blood pressure control. During the 1990s, technological advances allowed the use of quantitative trait locus (QTL) mapping to identify chromosomal regions with possible genetic variants affecting blood pressure. Many candidate genes for a role in human hypertension were identified as a consequence of findings in rodent animal models [25–30].

ANG II AT1 receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT1 receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 μg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-α, IL-1β, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-α, IL-1β, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT1 receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT1 receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-α and IL-6 mRNA and protein; expression of IL-1β and IκB-α mRNA; COX-2 mRNA and protein expression and PGE2 concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT1 receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

The physiological actions of brain Angiotensin II AT(2) receptors and their relationship to Angiotensin II AT(1) receptors remain controversial. To further clarify their role, we determined to what extent systemic administration of an AT(2) receptor antagonist affected AT(2) receptor binding within the brain and the expression of AT(1) receptors. For this purpose, we subcutaneously administered the AT(2) receptor antagonist PD123319 (1 mg/kg/day) to adult male rats for two weeks via osmotic minipumps. We also studied the content of pituitary adrenocorticotropic hormone and vasopressin, representative of hypothalamic-pituitary-adrenal axis activation, and the tyrosine hydroxylase gene expression in the locus coeruleus as a measure of central norepinephrine function. We found significant decreases in AT(2) receptor binding in brain areas inside the blood brain barrier, the inferior olive and the locus coeruleus. AT(2) receptor blockade increased AT(1) receptor binding and mRNA expression not only in the subfornical organ and the median eminence, situated outside the blood brain barrier, but also in the hypothalamic paraventricular nucleus, located inside the blood brain barrier. These changes paralleled decreased expression of tyrosine hydroxylase mRNA in the locus coeruleus and decreased pituitary adrenocorticotropic and vasopressin content. Our results demonstrate that sustained peripheral administration of an AT(2) antagonist decreases binding to brain AT(2) receptors, indicating that this drug is a useful tool for the study of their central role. AT(2) receptor activity inhibition up-regulates AT(1) receptor expression in specific brain areas. Blockade of brain AT(2) receptors is compatible with enhanced hypothalamic-pituitary-adrenal axis and decreased central sympathetic system activity.

To clarify the role of Angiotensin II in the regulation of peripheral sensory and motor systems, we initiated a study of the expression, localization and transport of Angiotensin II receptor types in the rat sciatic nerve pathway, including L(4)-L(5) spinal cord segments, the corresponding dorsal root ganglia (DRGs) and the sciatic nerve. We used quantitative autoradiography for AT(1) and AT(2) receptors, and in situ hybridization to detect AT(1A), AT(1B) and AT(2) mRNAs. We found substantial expression and discrete localization of Angiotensin II AT(1) receptors, with much higher numbers in the grey than in the white matter. A very high AT(1) receptor expression was detected in the superficial dorsal horns and in neuronal clusters of the DRGs. Expression of AT(1A) mRNA was significantly higher than that of AT(1B). AT(1) receptor binding and AT(1A) and AT(1B) mRNAs were especially prominent in ventral horn motor neurons, and in the DRG neuronal cells. Unilateral dorsal rhizotomy significantly reduced AT(1) receptor binding in the ipsilateral side of the superficial dorsal horn, indicating that a substantial number of dorsal horn AT(1) receptors have their origin in the DRGs. After ligation of the sciatic nerve, there was a high accumulation of AT(1) receptors proximal to the ligature, a demonstration of anterograde receptor transport. We found inconsistent levels of AT(2) receptor binding and mRNA. Our results suggest multiple roles of Angiotensin II AT(1) receptors in the regulation of sensory and motor functions.

Peripheral administration of bacterial endotoxin [lipopolysaccharide (LPS)] to rodents produces an innate immune response and hypothalamic-pituitary-adrenal axis stimulation. Renin-angiotensin-aldosterone system inhibition by angiotensin II AT1 receptor blockade has antiinflammatory effects in the vasculature. We studied whether angiotensin II receptor blockers (ARBs) prevent the LPS response. We focused on the adrenal gland, one organ responsive to LPS and expressing a local renin-angiotensin-aldosterone system. LPS (50 μg/kg, ip) produced a generalized inflammatory response with increased release of TNF-α and IL-6 to the circulation, enhanced adrenal aldosterone synthesis and release, and enhanced adrenal cyclooxygenase-2, IL-6, and TNF-α gene expression. ACTH and corticosterone release were also increased by LPS. Pretreatment with the ARB candesartan (1 mg/kg·d, sc for 3 d before the LPS administration) decreased LPS-induced cytokine release to the circulation, adrenal aldosterone synthesis and release, and cyclooxygenase-2 and IL-6 gene expression. Candesartan did not prevent the LPS-induced ACTH and corticosterone release. Our results suggest that AT1 receptors are essential for the development of the full innate immune and stress responses to bacterial endotoxin. The ARB decreased the general peripheral inflammatory response to LPS, partially decreased the inflammatory response in the adrenal gland, prevented the release of the pro-inflammatory hormone aldosterone, and protected the antiinflammatory effects of glucocorticoid release. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that the ARBs may have therapeutic effects on inflammatory conditions.

The brain renin-angiotensin system (RAS) participates importantly in the regulation of endocrine, autonomic, and behavioral response to stress. Recent data indicate that central action of AT(1) receptor antagonists can reduce anxiety symptoms in experimental animals. Furthermore, central inhibition of RAS activity decreases ethanol intake in an animal model of alcoholism. Pathological anxiety responses and the development of substance dependence are both critically mediated through corticotrophin-releasing hormone (CRH) systems, and the RAS is positioned to interact both with hypothalamic as well as extrahypothalamic CRH systems. The thesis of this paper is that the RAS is part of the neurochemical dysregulation underlying negative affective states, anxiety disorders, and ethanol dependence and that medications targeting the RAS should be considered to augment the treatment of these disorders.

We studied the effect of ovariectomy and estrogen replacement on expression of adrenal angiotensin II AT1 and AT2 receptors, aldosterone content, catecholamine synthesis, and the transcription factor Fos-related antigen 2 (Fra-2). Ovariectomy increased AT1 receptor expression in the adrenal zona glomerulosa and medulla, and decreased adrenomedullary catecholamine content and Fra-2 expression when compared to intact female rats. In the zona glomerulosa, estrogen replacement normalized AT1 receptor expression, decreased AT1B receptor mRNA, and increased AT2 receptor expression and mRNA. Estrogen treatment decreased adrenal aldosterone content. In the adrenal medulla, the effects of estrogen replacement were: normalized AT1 receptor expression, increased AT2 receptor expression, AT2 receptor mRNA, and tyrosine hydroxylase mRNA, and normalized Fra-2 expression and catecholamine content. We demonstrate that the constitutive adrenal expression of AT1 receptors, catecholamine synthesis and Fra-2 expression are partially under the control of reproductive hormones. Our results suggest that estrogen treatment decreases aldosterone production through AT1 receptor downregulation and AT2 receptor upregulation. AT2 receptor upregulation and modulation of Fra-2 expression may participate in the estrogen-dependent normalization of adrenomedullary catecholamine synthesis in ovariectomized rats. The AT2 receptor upregulation and the decrease in AT1 receptor function and in the production of the fluid-retentive, pro-inflammatory hormone aldosterone partially explain the protective effects of estrogen therapy.

Angiotensin II (Ang II), the active principle of the renin-angiotensin system (RAS), was discovered as a vasoconstrictive, fluid retentive circulating hormone. It was revealed later that there are local RAS in many organs, including the brain. The physiological receptor for Ang II, the AT(1) receptor type, was found to be highly expressed in many tissues and brain areas involved in the hypothalamic-pituitary-adrenal axis response to stress and in the sympathoadrenal system. The production of circulating and local Ang II, and the expression of AT(1) receptors increase during stress. Blockade of peripheral and brain AT(1) receptors with receptor antagonists administered peripherally prevented the hormonal and sympathoadrenal response to isolation stress, the stress-related alterations in cortical CRF(1) and benzodiazepine receptors, part of the GABA(A) complex, and reduced anxiety in rodents. AT(1) receptor blockade prevented the ulcerations of the gastric mucosa produced by cold-restraint stress, by preservation of the gastric blood flow, prevention of the stress-induced inflammatory response of the gastric mucosa, and partial blockade of the sympathoadrenal response to the stress. Our observations demonstrate that Ang II is an important stress hormone, and that blockade of AT(1) receptors could be proposed as a potentially useful therapy for stress-induced disorders.

Brain inflammation plays an important role in the pathophysiology of many psychiatric and neurological diseases. During brain inflammation, microglia cells are activated, producing neurotoxic molecules and neurotrophic factors depending on their pro-inflammatory M1 and anti-inflammatory M2 phenotypes. It has been demonstrated that Angiotensin II type 1 receptor blockers (ARBs) ameliorate brain inflammation and reduce M1 microglia activation. The ARB telmisartan suppresses glutamate-induced upregulation of inflammatory genes in cultured primary neurons. We wished to clarify whether telmisartan, in addition, prevents microglia activation through polarization to an anti-inflammatory M2 phenotype. We found that telmisartan promoted M2 polarization and reduced M1 polarization in LPS-stimulated BV2 and primary microglia cells, effects partially dependent on PPARγ activation. The promoting effects of telmisartan on M2 polarization, were attenuated by an AMP-activated protein kinase (AMPK) inhibitor or AMPK knockdown, indicating that AMPK activation participates on telmisartan effects. Moreover, in LPS-stimulated BV2 cells, telmisartan enhancement of M2 gene expression was prevented by the inhibitor STO-609 and siRNA of calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. Furthermore, telmisartan enhanced brain AMPK activation and M2 gene expression in a mouse model of LPS-induced neuroinflammation. In addition, telmisartan reduced the LPS-induced sickness behavior in this in vivo model, and this effect was prevented by prior administration of an AMPK inhibitor. Our results indicate that telmisartan can be considered as a novel AMPK activator, suppressing microglia activation by promoting M2 polarization. Telmisartan may provide a novel, safe therapeutic approach to treat brain disorders associated with enhanced inflammation.