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A high-fat diet (HFD) induces an increase in arterial pressure and a decrease in baroreflex function, which may be associated with increased expression of angiotensin type 1 receptor (AT1R) and pro-inflammatory cytokine genes and reduced expression of the angiotensin type 2 receptor (AT2R) gene within the nucleus of the solitary tract (NTS), a key area of the brainstem involved in cardiovascular control. Thus, in the present study, we evaluated the changes in arterial pressure and gene expression of components of the renin-angiotensin system (RAS) and neuroinflammatory markers in the NTS of rats fed a HFD and treated with either an AT1R blocker or with virus-mediated AT2R overexpression in the NTS. Male Holtzman rats (300-320 g) were fed either a standard rat chow diet (SD) or HFD for 6 weeks before commencing the tests. AT1R blockade in the NTS of HFD-fed rats attenuated the increase in arterial pressure and the impairment of reflex bradycardia, whereas AT2R overexpression in the NTS only improved the baroreflex function. The HFD also increased the hypertensive and decreased the protective axis of the RAS and was associated with neuroinflammation within the NTS. The expression of angiotensin-converting enzyme and neuroinflammatory components, but not AT1R, in the NTS was reduced by AT2R overexpression in this site. Based on these data, AT1R and AT2R in the NTS are differentially involved in the cardiovascular changes induced by a HFD. Chronic inflammation and changes in the RAS in the NTS may also account for the cardiovascular responses observed in HFD-fed rats.

The obesity epidemic is multi-generational and is particularly debilitating in the aging population, necessitating the use of pharmaceutical interventions. Recent evidence suggests that increasing the activity of the angiotensin converting enzyme-2 [ACE2]/angiotensin-(1-7)[Ang-(1-7)]/Mas receptor (MasR) axis in obese animal models leads to significant reductions in body weight. It was hypothesized that activation of ACE2 via diminazene aceturate (DIZE) will significantly reduce body weight of rats fed a high fat diet. Young and old (4 and 23 months, respectively) male Fisher 344 × Brown Norway rats were fed 60% high fat diet for one week, and subsequently given either 15 mg/kg/day DIZE s.c. or vehicle for three weeks. DIZE treatment resulted in a significant reduction of food intake and body weight in both young and old animals. However, that decrease was so dramatic in the older animals that they all nearly stopped eating. Interestingly, the TD-NMR assessments revealed that the weight-loss was primarily a result of decreased body fat percentage, with a relative preservation of lean mass. Tissue weights confirm the significant loss of white adipose tissue (WAT), with no change in muscle weights. Gene expression and serum ACE2 activity analyses implied that increased activation of the ACE2/Ang-(1-7)/MasR axis plays a role in reducing fat mass. Collectively, our results suggest that DIZE may be a useful tool in the study of obesity; however, caution is recommended when using this compound in older animals due to severe anorectic effects, although there is a mechanism by which muscle is preserved.

Sustained increases in the activity of the sympathetic neural pathways that exit the brain and which increase blood pressure (BP) are a major underlying factor in resistant hypertension. Recently available information on the occurrence of angiotensin II type 2 receptors (AT2Rs) within or adjacent to brain cardiovascular control centers is consistent with findings that stimulation of these receptors lowers BP, particularly during hypertension of neurogenic origin. Until recently brain AT2R had not been considered by many to play a role in the central control of BP. Demonstration of these powerful antihypertensive effects of brain AT2R opens the door to reconsideration of their role in BP regulation, and their consideration as a novel therapeutic avenue for resistant hypertension.

Background The majority of studies evaluating neurocognition in humans who had procedures under anesthesia early in life found long-term deficits even though the typical anesthesia duration normalized to the human life span is much shorter than that shown to induce developmental abnormalities in rodents. Therefore, we studied whether subsequent environmental stressors contribute to deficiencies programmed by a brief neonatal etomidate exposure. Methods Postnatal days (P) 4, 5, or 6, Sprague-Dawley rats, pretreated with vehicle or the Na+-K+-2Cl− (NKCC1) inhibitor, bumetanide, received two injections of etomidate resulting in anesthesia for 2 h. To simulate stress after anesthesia, the animals were exposed to a single maternal separation for 3 h at P10. 3–7 days after exposure to etomidate the rats had increased hypothalamic NKCC1 mRNA and corticotropin releasing hormone (CRH) mRNA and decreased K+-2Cl− (KCC2) mRNA levels with greater changes in males. In rats neonatally exposed to both etomidate and maternal separation, these abnormalities persisted into adulthood. These animals also exhibited extended corticosterone responses to restraint stress with increases in total plasma corticosterone more robust in males, as well as behavioral abnormalities. Pretreatment with the NKCC1 inhibitor ameliorated most of these effects. Conclusions Post-anesthesia stressors may exacerbate/unmask neurodevelopmental abnormalities even after a relatively short anesthetic with etomidate, leading to dysregulated stress response systems and neurobehavioral deficiencies in adulthood. Amelioration by bumetanide suggests a mechanistic role for etomidate-enhanced gamma-aminobutyric acid type A receptor-mediated depolarization in initiating long-lasting alterations in gene expression that are further potentiated by subsequent maternal separation.

Made available in DSpace on 2018-11-26T17:33:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) NIH BACKGROUND Macrophage migration inhibitory factor (MIF) is an intracellular inhibitory regulator of the actions of angiotensin II in the central nervous system. Renovascular hypertensive 2-kidney, 1-clip (2K1C) rats have an increased activity of the renin-angiotensin system and a decrease in baroreflex function compared to normotensive (NT) rats. In the present study, we tested the effects of MIF overexpression within the nucleus of the solitary tract (NTS), a key brainstem region for cardiovascular regulation, on the development of hypertension, on baroreflex function, and on water and food intake in 2K1C rats. METHODS Holtzman NT rats received a silver clip around the left renal artery to induce 2K1C hypertension. Three weeks later, rats were microinjected in the NTS with AAV2-CBA-MIF, to increase the expression of MIF, or with the control vector AAV2-CBA-enhanced green fluorescent protein. Mean arterial pressure (MAP) and heart rate were recorded by telemetry. Baroreflex function was tested, and water and food intake were also measured. RESULTS Increasing MIF expression in the NTS of 2K1C rats attenuated the development of hypertension, reversed the impairment of baroreflex function, and reduced the increase in water intake. In contrast to 2K1C rats, similar increases in MIF expression in the NTS of NT rats produced no changes in baseline MAP, baroreflex function, or water intake. CONCLUSIONS These results indicate that an increased expression of MIF within the NTS attenuates the development of hypertension and restores the baroreflex function in 2K1C rats. Sao Paulo State Univ, Sch Dent, Dept Physiol & Pathol, Araraquara, SP, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Physiol, Ribeirao Preto, SP, Brazil Southern Med Univ, Sch Biotechnol, Guangzhou, Guangdong, Peoples R China Univ Florida, Coll Med, Dept Physiol & Funct Genom, Gainesville, FL USA Sao Paulo State Univ, Sch Dent, Dept Physiol & Pathol, Araraquara, SP, Brazil CNPq: 473108/2011-9 CNPq: 304918/2011-3 FAPESP: 2011/50770-1 FAPESP: 2015/23467-7 NIH: HL-076803

Neurons and glia exhibit metabolic imbalances in hypertensive animal models, and loss of metabolic homeostasis can lead to neuroinflammation and oxidative stress. The objective of this study was to determine the effects of the microbial metabolite butyrate on mitochondrial bioenergetics and inflammatory markers in mixed brainstem and hypothalamic primary cultures of astrocytes between normotensive (Sprague‐Dawley, S‐D) and spontaneously hypertensive (SHR) rats. Bioenergetics of mitochondria in astrocytes from normotensive S‐D rats were modified with butyrate, but this was not the case in astrocytes derived from SHR, suggesting aberrant mitochondrial function. Transcripts related to oxidative stress, butyrate transporters, butyrate metabolism, and neuroinflammation were quantified in astrocyte cultures treated with butyrate at 0, 200, 600, and 1000 μmol/L. Butyrate decreased catalase and monocarboxylate transporter 1 mRNA in astrocytes of S‐D rats but not in the SHR. Moreover, while butyrate did not directly regulate the expression of 3‐hydroxybutyrate dehydrogenase 1 and 2 in astrocytes of either strain, the expression levels for these transcripts in untreated cultures were lower in the SHR compared to S‐D. We observed higher levels of specific inflammatory cytokines in astrocytes of SHR, and treatment with butyrate decreased expression of Ccl2 and Tlr4 in SHR astrocytes only. Conversely, butyrate treatment increased expression of tumor necrosis factor in astrocytes from SHR but not from the S‐D rats. This study improves our understanding of the role of microbial metabolites in regulating astrocyte function, and provides support that butyrate differentially regulates both the bioenergetics and transcripts related to neuroinflammation in astrocytes from SHR versus S‐D rats.

Background 1.5 million children under 12 months of age are exposed to general anesthesia annually in the United States alone. Human and especially animal studies provide evidence that exposure to general anesthesia during the early postnatal period may lead to long-term neurocognitive abnormalities via poorly understood mechanisms. We investigated whether an immature stress response system and γ-aminobutyric acid (GABA) type A receptor activities are involved in mediating these abnormalities. Methods Sprague–Dawley rats at postnatal days 4, 5 or 6 were anesthetized with 2.1% sevoflurane for 6 h; maternally separated and house reared rats served as controls. Results Sevoflurane anesthesia markedly increased corticosterone levels in rat pups of both genders. In adulthood, these rats responded to stress with heightened secretion of corticosterone and a greater increase in corticosterone levels in males versus females. Only male rats, previously exposed to neonatal sevoflurane, had a higher frequency of miniature inhibitory postsynaptic currents in CA1 neurons, spent a shorter time in open arms of the elevated plus maze (EPM) and exhibited impaired prepulse inhibition (PPI) of startle. Pretreatment of male rats prior to sevoflurane with the Na + -K + -2Cl − cotransporter inhibitor, bumetanide, or the mineralocorticoid receptor antagonist, RU28318, normalized endocrine responses to stress and the EPM behavior in adulthood, while only those pretreated with bumetanide exhibited normalized PPI of startle responses. Neither bumetanide nor RU28318 altered the effect of sevoflurane on synaptic activity. Conclusions Sevoflurane-enhanced neuronal excitation and elevated corticosteroid levels at the time of anesthesia contribute to the mechanisms initiating neonatal sevoflurane-induced long-term endocrine and neurobehavioral abnormalities.

Macrophage migration inhibitory factor (MIF) counteracts pressor effects of angiotensin II (ANG II) in the paraventricular nucleus of the hypothalamus (PVN) in normotensive rats, but this mechanism is absent in spontaneously hypertensive rats (SHRs) due to a lack of MIF in PVN neurons. Since endogenous ANG II in the PVN modulates stress reactivity, we tested the hypothesis that replacement of MIF in PVN neurons would reduce baseline blood pressure and inhibit stress-induced increases in blood pressure and plasma corticosterone in adult male SHRs. Radiotelemetry transmitters were implanted to measure blood pressure, and then an adeno-associated viral vector expressing either enhanced green fluorescent protein (GFP) or MIF was injected bilaterally into the PVN. Cardiovascular responses to a 15-min water stress (1-cm deep, 25°C) and a 60-min restraint stress were evaluated 3–4 wk later. MIF treatment in the PVN attenuated average restraint-induced increases in blood pressure (37.4 ± 2.0 and 27.6 ± 3.5 mmHg in GFP and MIF groups, respectively, P < 0.05) and corticosterone (42 ± 2 and 36 ± 3 μg/dl in GFP and MIF groups, respectively, P < 0.05). MIF treatment in the PVN also reduced stress-induced elevations in the number of c-Fos-positive cells in the rostral ventrolateral medulla (71 ± 5 in GFP and 47 ± 5 in MIF SHRs, P < 0.01) and corticotropin-releasing factor mRNA expression in the PVN. However, MIF had no significant effects on the cardiovascular responses to water stress in SHRs or to either stress in Sprague-Dawley rats. Therefore, viral vector-mediated restoration of MIF in PVN neurons of SHRs attenuates blood pressure and hypothalamic pituitary adrenal axis responses to stress.

In most situations, the angiotensin AT2-receptor (AT2R) mediates physiological actions opposing those mediated by the AT1-receptor (AT1R), including a vasorelaxant effect. Nevertheless, experimental evidence vastly supports that systemic application of AT2R-agonists is blood pressure neutral. However, stimulation of AT2R locally within the brain or the kidney apparently elicits a systemic blood pressure lowering effect. A systemic effect of AT2R stimulation on blood pressure can also be achieved, when the prevailing effect of continuous background AT1R-stimulation is attenuated by low-dose AT1R blockade. Despite a lack of effect on blood pressure, AT2R stimulation still protects from hypertensive end-organ damage. Current data and evidence therefore suggest that AT2R agonists will not be suitable as future anti-hypertensive drugs, but that they may well be useful for end-organ protection in combination with established anti-hypertensives.

Background and Purpose Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. Experimental Approach A single s.c. injection of MCT (50 mg·kg−1) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg−1 C21, 3 mg·kg−1 PD-123319 or 0.5 mg·kg−1 A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. Key Results Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. Conclusions and Implications Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.

PURPOSE OF REVIEW: The goal of this review is to assess the evidence that activation of angiotensin type 2 receptors (AT2R) in the brain can lower blood pressure and possibly constitute an endogenous anti-hypertensive mechanism.RECENT FINDINGS: Recent studies that detail the location of AT2R in the brain, particularly within or near cardiovascular control centers, mesh well with findings from pharmacological and gene transfer studies which demonstrate that activation of central AT2R can influence cardiovascular regulation. Collectively, these studies indicate that selective activation of brain AT2R causes moderate decreases in blood pressure in normal animals and more profound anti-hypertensive effects, along with restoration of baroreflex function, in rodent models of neurogenic hypertension. These findings have opened the door to studies that can (i) assess the role of specific AT2R neuron populations in depressing blood pressure, (ii) determine the relevance of such mechanisms, and (iii) investigate interactions between AT2R and depressor angiotensin-(1-7)/Mas mechanisms in the brain.

Angiotensin II increases and decreases arterial pressure by acting at angiotensin type 1 and type 2 receptors, respectively. Renovascular hypertensive rats exhibit a high level of activity of the peripheral and central renin–angiotensin system. Therefore, in the present study, we evaluated the effect of increasing the expression of angiotensin type 2 receptors in the solitary–vagal complex (nucleus of the solitary tract/dorsal motor nucleus of the vagus), a key brain stem region for cardiovascular regulation, on the development of renovascular hypertension. Holtzman normotensive rats were implanted with a silver clip around the left renal artery to induce 2-kidney 1-clip renovascular hypertension. Three weeks later, rats were microinjected in the solitary–vagal complex with either an adenoassociated virus to increase the expression of angiotensin type 2 receptors or with a control vector. We observed that increasing angiotensin type 2 receptor expression in the solitary–vagal complex attenuated the development of renovascular hypertension and also reversed the impairment of the baroreflex and the increase in the low-frequency component of systolic blood pressure observed in renovascular hypertensive rats. Furthermore, an observed decrease in mRNA levels of angiotensin-converting enzyme 2 in the solitary–vagal complex of renovascular hypertensive rats was restored to control levels after viral-mediated increases in angiotensin type 2 receptors at this site. Collectively, these data demonstrate specific and beneficial effects of angiotensin type 2 receptors via the brain of hypertensive rats and suggest that central angiotensin type 2 receptors may be a potential target for therapeutics in renovascular hypertension.

Made available in DSpace on 2018-11-26T17:13:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) NIH The aim of this study was to investigate the physiological effects of increased angiotensin II type 2 receptor (AT2R) expression in the solitary-vagal complex (nucleus of the solitary tract/dorsal motor nucleus of the vagus; NTS/DVM) on baroreflex function in non-anaesthetised normotensive (NT) and spontaneously hypertensive rats (SHR). Ten week old NT Holtzman and SHR were microinjected with either an adeno-associated virus expressing AT2R (AAV2-CBA-AT2R) or enhanced green fluorescent protein (control; AAV2-CBA-eGFP) into the NTS/DVM. Baroreflex and telemetry recordings were performed on four experimental groups: 1) NTeGFP, 2) NTAT2R, 3) SHReGFP and 4) SHRAT2R (n = 4-7/group). Following in-vivo experimental procedures, brains were harvested for gene expression analysis. Impaired bradycardia in SHReGFP was restored in SHR rats over-expressing AT2R in the NTS/DMV. mRNA levels of angiotensin converting enzyme decreased and angiotensin converting enzyme 2 increased in the NTS/DMV of SHRAT2R compared to SHReGFP. Increased levels of pro inflammatory cytokine mRNA levels in the SHReGFP group also decreased in the SHRAT2R group. AT2R overexpression did not elicit any significant change in mean arterial pressure (MAP) in all groups from baseline to 4 weeks post viral transfection. Both SHReGFP and SHRAT2R showed a significant elevation in MAP compared to the NTeGFP and NTAT2R groups. Increased AT2R expression within the NTS/DMV of SHR was effective at improving baroreflex function but not MAP. We propose possible mediators involved in improving baroreflex are in the ANG II/ACE2 axis, suggesting a potential beneficial modulatory effect of AT2R overexpression in the NTS/DMV of neurogenic hypertensive rats. (C) 2016 Elsevier Ltd. All rights reserved. Sao Paulo State Univ, Sch Dent, Dept Physiol & Pathol, Araraquara, SP, Brazil Southern Med Univ, Sch Biotechnol, Guangzhou, Guangdong, Peoples R China Univ Florida, Coll Med, Dept Physiol & Funct Genom, Gainesville, FL USA Univ Florida, Coll Med, McKnight Brain Inst, Gainesville, FL USA Sao Paulo State Univ, Sch Dent, Dept Physiol & Pathol, Araraquara, SP, Brazil CNPq: 473108/2011-9 CNPq: 304918/2011-3 FAPESP: 2011/50770-1 FAPESP: 2012/23546-6 FAPESP: 2013/50121-9 CAPES: 2053/2013 NIH: HL-076803

New Findings What is the central question of this study? Activation of angiotensin-converting enzyme 2, resulting in production of angiotensin-(1–7) and stimulation of its receptor, Mas, exerts beneficial actions in a number cardiovascular diseases, including ischaemic stroke. A potential beneficial role for angiotensin-(1–7) in haemorrhagic stroke has not previously been reported. What is the main finding and its importance? Central administration of angiotensin-(1–7) into stroke-prone spontaneously hypertensive rats, a model of haemorrhagic stroke, increases lifespan and improves the neurological status of these rats, as well as decreasing microglial numbers in the striatum (implying attenuation of cerebral inflammation). These actions of angiotensin-(1–7) have not previously been reported and identify this peptide as a potential new therapeutic target in haemorrhagic stroke. Angiotensin-(1–7) [Ang-(1–7)] exerts cerebroprotective effects in ischaemic stroke, and this action is associated with a blunting of intracerebral inflammatory processes and microglial activation. Given that intracerebral inflammation and microglial activation play key roles in the mechanism of injury and brain damage in both ischaemic and haemorrhagic stroke, we have investigated the potential beneficial actions of Ang-(1–7) in stroke-prone spontaneously hypertensive rats (spSHRs), an established animal model of hypertension-induced haemorrhagic stroke. Angiotensin-(1–7) was administered by continuous infusion via the intracerebroventricular route for 6 weeks into spSHRs fed a high-sodium (4%) diet, starting at 49 days of age. This treatment resulted in a significant increase in survival of the spSHRs. Median survival was 108 days in control, artificial cerebrospinal fluid-infused spSHRs and 154 days in Ang-(1–7)-treated spSHRs. This effect was partly reversed by intracerebroventricular infusion of the Mas receptor blocker, A779. This Ang-(1–7) treatment also decreased the number of haemorrhages in the striatum, improved neurological status (reduced lethargy), decreased the number of microglia in the striatum and tended to increase neuron survival at the same site. Importantly, infusions of Ang-(1–7) had no effect on kidney pathology, heart pathology, body weight, serum corticosterone levels or blood pressure. This study is the first to demonstrate the cerebroprotective actions of Ang-(1–7), including increased survival time, in spSHRs. As such, these data reveal a potential therapeutic target for haemorrhagic stroke.

The goal of the present review is to examine the evidence for beneficial actions of manipulation of the RAS (renin–angiotensin system) in stroke, with particular focus on Ang-(1–7) [angiotensin-(1–7)] and its receptor Mas. The RAS appears to be highly involved in the multifactorial pathophysiology of stroke. Blocking the effects of AngII (angiotensin II) at AT1R (AngII type 1 receptor), through the use of commonly prescribed ACE (angiotensin-converting enzyme) inhibitors or AT1R blockers, has been shown to have therapeutic effects in both ischaemic and haemorrhagic stroke. In contrast with the deleterious actions of over activation of AT1R by AngII, stimulation of AT2Rs (AngII type 2 receptors) in the brain has been demonstrated to elicit beneficial effects in stroke. Likewise, the ACE2/Ang-(1–7)/Mas axis of the RAS has been shown to have therapeutic effects in stroke when activated, countering the effects of the ACE/AngII/AT1R axis. Studies have demonstrated that activating this axis in the brain elicits beneficial cerebral effects in rat models of ischaemic stroke, and we have also demonstrated the cerebroprotective potential of this axis in haemorrhagic stroke using stroke-prone spontaneously hypertensive rats and collagenase-induced striatal haemorrhage. The mechanism of cerebroprotection elicited by ACE2/Ang-(1–7)/Mas activation includes anti-inflammatory effects within the brain parenchyma. The major hurdle to overcome in translating these results to humans is devising strategies to activate the ACE2/Ang-(1–7)/Mas cerebroprotective axis using post-stroke treatments that can be administered non-invasively. Abbreviations: ACE, angiotensin-converting enzyme; ACEi, ACE inhibitor(s); aCSF, artificial cerebrospinal fluid; Ang-(1–7), angiotensin-(1–7); AngII, angiotensin II; ARB, AT1R blocker; AT1R, AngII type 1 receptor; AT2R, AngII type 2 receptor; BBB, blood–brain barrier; BP, blood pressure; COX-2, cyclo-oxygenase 2; DIZE, diminazene aceturate; eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cell; ERK1/2, extracellular-signal-regulated kinase 1/2; ET-1, endothelin-1; HPβCD, hydroxypropyl-β-cyclodextran; HSC, haematopoetic stem cell; ICV, intracerebroventricular(ly); IL, interleukin; iNOS, inducible nitric oxide synthase; MCAO, middle cerebral artery occlusion; NF-κB, nuclear factor κB; RAS, renin–angiotensin system; SHR, spontaneously hypertensive rat; spSHR, stroke-prone SHR; TNF-α, tumour necrosis factor-α

1. In recent years it has been firmly established that apart from the classic renin-angiotensin system (RAS) comprising angiotensin (Ang) II, angiotensin converting enzyme (ACE; responsible for AngII generation) and the angiotensin AT(1) receptor (AT(1)R), there also exist protective arms of the RAS that comprise the angiotensin AT(2) receptor (AT(2)R), Ang-(1-7), ACE2 (mainly responsible for Ang-(1-7) synthesis) and Mas, the receptor for Ang-(1-7). 2. Stimulation of AT(2)R promotes neuronal differentiation, neurite outgrowth and axonal regeneration, which results in an acceleration of repair and improved functional outcome after injury of peripheral nerves or the spinal cord. 3. Stimulation of AT(2)R and the receptor Mas has been shown to reduce infarct size and ameliorate neurological deficits in various animal models of stroke. The underlying mechanisms of action are comprised of activation of direct neurotrophic, anti-inflammatory and anti-oxidant pathways, as well as the augmentation of cerebral blood flow. 4. Cognitive function is improved by AT(2)R stimulation due, at least in part, to increased cerebral blood flow. There is indirect evidence that Ang-(1-7) could also play a role in protection against cognitive decline, but studies confirming this have not yet been published. 5. In view of the data reviewed in this article, it can be assumed that the protective arms of the RAS are putative targets in the treatment of neurological diseases, which involve tissue damage or cognitive impairment.

Previously we demonstrated that viral-mediated increased expression of the anti-inflammatory cytokine interleukin-10 within the paraventricular nucleus of the hypothalamus significantly reduces blood pressure in normal rats made hypertensive by infusion of angiotensin II. However, the exact cellular locus of this interleukin-10 action within the paraventricular nucleus is unknown. In the present study we tested whether interleukin-10 exerts direct effects at its receptors located on hypothalamic neurons to offset the neuronal excitatory actions of angiotensin II via its type 1 receptors. The results indicated the presence of immunoreactive interleukin-10 receptors on neurons in normal rat paraventricular nucleus and that receptors for this cytokine were also expressed in neurons cultured from the hypothalamus. Patch-clamp electrophysiological recordings from these cultures revealed that extracellular application of interleukin-10 alone did not exert any alterations in neuronal membrane delayed rectifier or transient potassium currents. However, angiotensin II elicited a significant decrease in delayed rectifier potassium current, an effect that was abolished by interleukin-10 application. Since decreases in delayed rectifier potassium current contribute to increased neuronal excitability, this result is consistent with a direct inhibitory action of interleukin-10 on angiotensin-induced excitation of hypothalamic neurons. As such, these data are the first indication of a neuronal locus of action of interleukin-10 to temper the actions of angiotensin II in the hypothalamus.

Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type 1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1awithin the brain control these processes, we used the Cre/lox system to delete AT1afrom the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1adeletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1ain the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1adeletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1adeletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole-cell patch-clamp recordings confirmed that PVN AT1adeletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1adeletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1aregulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain.

The role of the angiotensin type-2 receptor (AT2R) in the neural control of cardiovascular homeostasis and hypertension is not well-characterized. Using a BAC transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse, dense localization of AT2R to GABA neurons was found within the nucleus of the solitary tract (NTS) of the hindbrain, an area important for regulating baroreflex function and blood pressure. This localization was confirmed by RNAscope fluorescence in situ hybridization. Considering that GABA is pressor in the intermediate NTS (intNTS), and that its effects are enhanced in models of neurogenic hypertension such as SHR and deoxycorticosterone acetate (DOCA)-salt rats, we tested the hypothesis that AT2R on GABA neurons in the NTS constitute a counter-regulatory, blood pressure lowering mechanism. In support of this idea, we have demonstrated that: (i) Optogenetic stimulation of GABA neurons in the intNTS of normal mice elicited a significant increase in blood pressure; (ii) mRNA levels for both the GABA synthetic enzyme Gad1 and for the AT2R are significantly increased in the intNTS of DOCA-salt hypertensive mice; (iii) Intracerebroventricular (ICV) infusion of the AT2R agonist Compound 21 (C21; 7.5 ng/h) into normotensive or DOCA-salt hypertensive mice elicited a significant reduction of systolic blood pressure, an effect that was much larger in hypertensive (126.2 ± 5.0 v. 139.8 ± 3.2 mmHg; n = 14; p = 0.02) than in normotensive (119.8 ± 2.6 v. 126.5 ± 2.5 mmHg; n = 16; p = 0.04) mice and was accompanied by decreased levels of the GABA synthetic enzymes Gad1 and Gad2 in the intNTS; (iv) Finally, the crucial involvement of GABA neurons in the blood pressure lowering effect of AT2R was proven by the lack of any effect of ICV C21 in DOCA-salt hypertensive mice containing a selective knockout of AT2R from GABA neurons. These novel data provide strong evidence for an anti-hypertensive action of AT2R within the intNTS, an effect that is exerted through decreases in GABA transmission.

Levels of angiotensin converting enzyme 2 (ACE2), a cardio and neuro-protective carboxypeptidase, are dynamically altered after stroke in preclinical models. We sought to characterize the previously unexplored changes in serum ACE2 activity of stroke patients and the mechanism of these changes. Serum samples were obtained from patients during acute ischemic stroke ( n=39), conditions mimicking stroke (stroke-alert, n=23), or from control participants ( n=20). Enzyme activity levels were analyzed by fluorometric assay and correlated with clinical variables by regression analyses. Serum ACE2 activity was significantly lower in acute ischemic stroke as compared to both control and stroke-alert patients, followed by an increase to control levels at three days. Serum ACE2 activity significantly correlated with the presence of ischemic stroke after controlling for other factors ( P=0.01). Additional associations with ACE2 activity included a positive correlation with systolic blood pressure at presentation in stroke-alert ( R2=0.24, P=0.03), while stroke levels showed no correlation ( R2=0.01, P=0.50). ACE2 sheddase activity was unchanged between groups. These dynamic changes in serum ACE2 activity in stroke, which concur with preclinical studies, are not likely to be driven primarily by acute changes in blood pressure or sheddase activity. These findings provide new insight for developing therapies targeting this protective system in ischemic stroke.

It is known that angiotensin-II acts at its type-1 receptor to stimulate vasopressin (AVP) secretion, which may contribute to angiotensin-II-induced hypertension. Less well known is the impact of angiotensin type-2 receptor (AT2R) activation on these processes. Studies conducted in a transgenic AT2R enhanced green fluorescent protein reporter mouse revealed that although AT2R are not themselves localized to AVP neurons within the paraventricular nucleus of the hypothalamus (PVN), they are localized to neurons that extend processes into the PVN. In the present set of studies, we set out to characterize the origin, phenotype, and function of nerve terminals within the PVN that arise from AT2R-enhanced green fluorescent protein-positive neurons and synapse onto AVP neurons. Initial experiments combined genetic and neuroanatomical techniques to determine that γ-aminobutyric acid (GABA)ergic neurons derived from the peri-PVN area containing AT2R make appositions onto AVP neurons within the PVN, thereby positioning AT2R to negatively regulate neuroendocrine secretion. Subsequent patch-clamp electrophysiological experiments revealed that selective activation of AT2R in the peri-PVN area using compound 21 facilitates inhibitory (ie, GABAergic) neurotransmission and leads to reduced activity of AVP neurons within the PVN. Final experiments determined the functional impact of AT2R activation by testing the effects of compound 21 on plasma AVP levels. Collectively, these experiments revealed that AT2R expressing neurons make GABAergic synapses onto AVP neurons that inhibit AVP neuronal activity and suppress baseline systemic AVP levels. These findings have direct implications in the targeting of AT2R for disorders of AVP secretion and also for the alleviation of high blood pressure.

Increasing evidence indicates a strong association between inflammatory processes within brain cardiovascular control centers and hypertension that is neurogenic in origin.

Aims The macrophage migration inhibitory factor (MIF) is an intracellular inhibitor of the central nervous system actions of angiotensin II on blood pressure. Considering that angiotensin II actions at the nucleus of the solitary tract are important for the maintenance of hypertension in spontaneously hypertensive rats (SHRs), we tested if increased MIF expression in the nucleus of the solitary tract of SHR alters the baseline high blood pressure in these rats. Methods and results Eight-week-old SHRs or normotensive rats were microinjected with the vector AAV2-CBA-MIF into the nucleus of the solitary tract, resulting in MIF expression predominantly in neurons. Rats also underwent recordings of the mean arterial blood pressure (MAP) and heart rate (via telemetry devices implanted in the abdominal aorta), cardiac- and baroreflex function. Injections of AAV2-CBA-MIF into the nucleus of the solitary tract of SHRs produced significant decreases in the MAP, ranging from 10 to 20 mmHg, compared with age-matched SHRs that had received identical microinjections of the control vector AAV2-CBA-eGFP. This lowered MAP in SHRs was maintained through the end of the experiment at 31 days, and was associated with an improvement in baroreflex function to values observed in normotensive rats. In contrast to SHRs, similar increased MIF expression in the nucleus of the solitary tract of normotensive rats produced no changes in baseline MAP and baroreflex function. Conclusion These results indicate that an increased expression of MIF within the nucleus of the solitary tract neurons of SHRs lowers blood pressure and restores baroreflex function.

We hypothesized that moderate cardiac-selective overexpression of the angiotensin II type 2 receptor (AT2R) would protect the myocardium from ischaemic injury after a myocardial infarction (MI) induced by coronary artery ligation. For in vitro studies, adenoviral vector expressing genomic DNA of AT2R and enhanced green fluorescence protein (EGFP) was used to overexpress AT2R in rat neonatal cardiac myocytes. Expression of AT2R, measured by real-time PCR and immunostaining, demonstrated efficient transduction of AT2R in a dose-dependent pattern. The AT2R constitutively induced apoptosis in rat neonatal cardiac myocytes in dose-dependent patterns. For in vivo studies, 4 × 1010 vector genomes (vg) of recombinant adeno-associated virus serotype 9 (rAAV9)-chicken β actin promoter-AT2R was injected into the left ventricle of 5-day-old Sprague–Dawley rats. At 6 weeks of age, hearts were harvested and expression of AT2R determined by real-time PCR and Western blotting. Expression was increased onefold over control hearts, and no apoptosis was detected. Two subsequent in vivo studies were performed. In a prevention study, 4 × 1010 vg of rAAV9-CBA-AT2R was injected into the left ventricle of 5-day-old Sprague–Dawley rats and MI was induced at 6 weeks of age. For a post-treatment study, 4 × 1010 vg of rAAV9-CBA-AT2R was administrated to the peri-infarcted myocardium area immediately after MI in 6-week-old animals. For both in vivo studies, cardiac functions were assessed using echocardiography and haemodynamic measurements 4 weeks after coronary artery ligation. In the in vivo studies, the rats subjected to MI showed significant decreases in fractional shortening and rate of change of left ventricular pressure, with increased left ventricular end-diastolic pressure and ventricular hypertrophy. For the prevention study, the moderate cardiac-selective overexpression of AT2R attenuated these MI-induced impairments and also caused a decrease in ventricular wall thinning. In the post-treatment study, the overexpression of AT2R partly reversed the MI-induced cardiac dysfunction. Myocardial infarction also induced the upregulation of angiotensin II type 1 receptor, angiotensin-converting enzyme and collagen I mRNA expression, all of which were attenuated by the overexpression of AT2R. It is concluded that moderate cardiac-selective overexpression of AT2R protects heart function from ischaemic injury, which may be mediated, at least in part, through modulation of components of the cardiac renin–angiotensin system and collagen levels in the myocardium.

It is a well-established fact that components of the renin–angiotensin system (RAS) play a critical role in the progression of heart failure. Pharmacotherapeutic interventions with either angiotensin-converting enzyme (ACE) inhibitors (Haywood et al. 1997) or angiotensin type 1 receptor blockers (Steckelings et al. 2005; Ferreira et al. 2009) have demonstrated significant protection against myocardial infarction and heart failure in experimental animal models as well as in patients. Several studies have suggested that the beneficial effects observed with angiotensin-converting enzyme inhibitors are not only due to the reduction in the formation of the detrimental angiotensin II (Ang II), but are also due to significant elevation in the levels of angiotensin-(1–7) (Ang-(1–7); Keidar et al. 2007). Correlative studies have also shown that ACE2 and Ang-(1–7) levels are increased in the heart following myocardial infarction in both humans and rats (Averill et al. 2003; Burrell et al. 2005). A growing body of evidence indicates that Ang-(1–7) plays an active role in maintaining cardiovascular homeostasis, thus providing protection against heart diseases. This conclusion is supported by the following findings. Angiotensin-(1–7) has been shown to attenuate isoprenaline-induced cardiac hypertrophy (Santos et al. 2004). Chronic administration of Ang-(1–7) has been shown to improve coronary artery perfusion and endothelial function in a rat model for heart failure (Loot et al. 2002). Angiotensin-(1–7) was shown to reduce the incidence and the duration of postischaemic reperfusion arrhythmias in the isolated rat heart (Ferreira et al. 2001; De Mello, 2004). Intravenous infusion of Ang-(1–7) resulted in improved contractile function in rat hearts (Sampaio et al. 2003). Angiotensin-(1–7) has been demonstrated to attenuate cardiac remodelling significantly in terms of reducing myocyte hypertrophy and interstitial fibrosis (Santos et al. 2004; Tallant et al. 2005; Grobe et al. 2006, 2007b; Mercure et al. 2008; Ferreira et al. 2010; Giani et al. 2010). Furthermore, AVE0991, a synthetic mimetic of Ang-(1–7), has been shown to exert cardioprotective effects (Ferreira et al. 2007). Although Ang-(1–7) exerts beneficial effects against a range of cardiovascular diseases, the in vivo efficacy of this peptide is severely hampered owing to rapid degradation by peptidases. The half-life for Ang-(1–7) is very short and dependent on species. For example, following intravenous administration the half-life of Ang-(1–7) in humans is approximately 30 min (Kono et al. 1986; Rodgers et al. 2006), whereas in rodents it is approximately 20 s (Iusuf et al. 2008). To overcome this limitation, we have overexpressed Ang-(1–7) in the rodent heart shortly after birth, as we previously demonstrated (Methot et al. 2001; Diez-Freire et al. 2006), to evaluate its cardioprotective effects against myocardial infarction. Also, we hypothesize that Ang-(1–7) may exert its beneficial effects against myocardial infarction by restoring the balance between the deleterious ACE–Ang II–angiotensin II type 1 receptor (AT1R) axis and the beneficial ACE2–Ang-(1–7)–Mas axis of the RAS.

Activation of angiotensin-converting enzyme 2 (ACE2), production of angiotensin-(1–7) [Ang-(1–7)] and stimulation of the Ang-(1–7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang-(1–7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang-(1–7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang-(1–7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET-1-induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang-(1–7) and DIZE were reversed by co-intracerebroventricular administration of the Mas receptor inhibitor, A-779. Neither the Ang-(1–7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET-1. Lastly, intracerebroventricular administration of Ang-(1–7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET-1-induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2–Ang-(1–7)–Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang-(1–7) protective action includes blunting of inducible nitric oxide synthase expression.

Rationale: Despite overwhelming evidence of the importance of brain renin–angiotensin system (RAS), the very existence of intrinsic brain RAS remains controversial. Objective: To investigate the hypothesis that the brain (pro)renin receptor (PRR) is physiologically important in the brain RAS regulation and cardiovascular functions. Methods and Results: PRR is broadly distributed within neurons of cardiovascular-relevant brain regions. The physiological functions of PRR were studied in the supraoptic nucleus (SON) because this brain region showed greater levels of PRR mRNA in the spontaneously hypertensive rats (SHR) compared with normotensive Wistar–Kyoto (WKY) rats. Adeno-associated virus (AAV)-mediated overexpression of human PRR in the SON of normal rats resulted in increases in plasma and urine vasopressin, and decreases in H 2 O intake and urine output without any effects on mean arterial pressure and heart rate. Knockdown of endogenous PRR by AAV-short hairpin RNA in the SON of SHRs attenuated age-dependent increases in mean arterial pressure and caused a decrease in heart rate and plasma vasopressin. Incubation of neuronal cells in culture with human prorenin and angiotensinogen resulted in increased generation of angiotensin I and II. Furthermore, renin treatment increased phosphorylation of extracellular signal-regulated kinase ½ in neurons from both WKY rats and SHRs; however, the stimulation was 50% greater in the SHR. Conclusions: The study demonstrates that brain PRR is functional and plays a role in the neural control of cardiovascular functions. This may help resolve a long-held controversy concerning the existence of intrinsic and functional brain RAS.

Accumulating evidence indicates a key role of inflammation in hypertension and cardiovascular disorders. However, the role of inflammatory processes in neurogenic hypertension remains to be determined. Thus, our objective in the present study was to test the hypothesis that activation of microglial cells and the generation of proinflammatory cytokines in the paraventricular nucleus (PVN) contribute to neurogenic hypertension. Intracerebroventricular infusion of minocycline, an anti-inflammatory antibiotic, caused a significant attenuation of mean arterial pressure, cardiac hypertrophy, and plasma norepinephrine induced by chronic angiotensin II infusion. This was associated with decreases in the numbers of activated microglia and mRNAs for interleukin (IL) 1β, IL-6, and tumor necrosis factor-α, and an increase in the mRNA for IL-10 in the PVN. Overexpression of IL-10 induced by recombinant adenoassociated virus-mediated gene transfer in the PVN mimicked the antihypertensive effects of minocycline. Furthermore, acute application of a proinflammatory cytokine, IL-1β, into the left ventricle or the PVN in normal rats resulted in a significant increase in mean arterial pressure. Collectively, this indicates that angiotensin II induced hypertension involves activation of microglia and increases in proinflammatory cytokines in the PVN. These data have significant implications on the development of innovative therapeutic strategies for the control of neurogenic hypertension.

The centrally mediated cardiovascular regulatory actions of angiotensin II in normal and hypertensive rats include angiotensin II type 1 receptor (AT1R)–mediated actions at the paraventricular nucleus (PVN) of the hypothalamus. Because the PVN consists of multiple neuronal populations, it is important to understand which neuronal types in the PVN are influenced by angiotensin II. Here we have developed a viral vector (Adeno-associated vector 2 [AAV2]-PAG-eGFP [PAG; phosphate-activated glutaminase promoter]) to drive expression of green fluorescent protein (GFP) primarily within glutamate neurons. At 10 to 14 days after bilateral microinjection (200 nL per side; 1.2×10 12 genome copies) of AAV2-PAG-eGFP into adult Sprague-Dawley rat PVN, animals were euthanized and brains removed and used for isolation and culture of PVN neurons. Fluorescence microscopy and immunostaining using neuron and PAG-specific antibodies revealed the presence of GFP-containing glutamatergic neurons in these PVN cultures. Whole-cell patch-clamp recordings demonstrated that angiotensin II (100 nmol/L) produced a 16% decrease in delayed rectifier potassium current in ≈50% of the GFP-containing neurons, an effect that was abolished by the AT1R antagonist losartan (1 μmol/L). Consistently, 9 of 28 GFP/PAG-expressing neurons contained AT1R mRNA, as indicated by single-cell RT-PCR. Furthermore, specific GFP/PAG-positive neurons in the PVN that project to the rostral ventrolateral medulla of the brain stem express immunoreactive AT1R. In conclusion, we have demonstrated the presence of functional AT1R on PAG-positive (largely glutamate) neurons within rat PVN, certain of which project to the rostral ventrolateral medulla.

Macrophage migration inhibitory factor (MIF) expression is increased by angiotensin II (Ang II) within paraventricular nucleus (PVN) neurons of normotensive rats and acts via its intrinsic thiol protein oxidoreductase (TPOR) to counterregulate the central nervous system-mediated pressor action of Ang II. Considering that the PVN-mediated actions of Ang II are enhanced in spontaneously hypertensive rats (SHRs) and contribute to the development of hypertension in these animals, we investigated this MIF regulatory mechanism in SHRs. Here, we have demonstrated that Ang II failed to increase MIF protein expression in the PVN of SHRs. Furthermore, although basal levels of MIF protein and mRNA were similar in the PVN of SHRs and normotensive rats, immunostaining revealed that MIF was either absent from or diminished in PVN neurons of SHRs. AAV2-mediated increases in MIF expression within PVN neurons of young (8 wk old) SHRs produced a chronic attenuation of hypertension and cardiac hypertrophy. However, similar AAV2-mediated transduction of [C60S]-MIF, which lacks TPOR activity, did not alter the development of hypertension or cardiac hypertrophy in SHRs. Collectively, these findings suggest that a lack of MIF expression within PVN neurons contributes to the development of hypertension and cardiac hypertrophy in SHRs.—Li, H., Gao, Y., Qi, Y., Katovich, M. J., Jiang, N., Braseth, L. N., Scheuer, D. A., Shi, P., Sumners, C. Macrophage migration inhibitory factor in hypothalamic paraventricular nucleus neurons decreases blood pressure in spontaneously hypertensive rats.

Recent discovery of (pro)renin receptor (PRR) has revolutionized the field of renin–angiotensin system (RAS) physiology and has underscored the critical role of the local and tissue RAS in the development and establishment of cardiac, vascular and renal pathophysiology linked to hypertension and diabetes (Ichihara et al. 2007; Nguyen, 2007). (Pro)renin receptor is a 350 amino acid protein with a single transmembrane domain that binds and actives prorenin. Prorenin contains a ‘handle’ region that conceals the active site of renin (Suzuki et al. 2003). Prorenin binding to PRR results in non-proteolytic activation of the enzyme by inducing a conformational change at the ‘handle’ region, exposing the active site (Nguyen et al. 2002; Danser et al. 2007). In addition, renin binding to the PRR increases its intrinsic activity. Thus, prorenin bound to PRR is many-fold more efficient in the conversion of angiotensinogen to angiotensin I, subsequently leading to increased angiotensin II (Ang II) at the tissue level (Nguyen et al. 2002; Nguyen, 2007). Studies have established that PRR plays a dual role in the regulation of RAS activity: it binds to prorenin/renin to facilitate the formation of Ang I and II, and the binding initiates an intracellular signal transduction pathway involving mitogen-activated protein kinases (MAPK). The latter action is presumably associated with increased synthesis of profibrotic molecules such as plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen and transforming growth factor-β (TGF-β; Huang et al. 2006; Danser et al. 2007; Nguyen, 2007). The importance of PRR in the cardiovascular system is further evident from pathophysiological and transgenic studies. For example: (i) inhibition of PRR by a ‘handle’ region peptide has been shown to protect the heart and kidney against tissue damage induced by hypertension (Ichihara et al. 2004, 2006); (ii) activation of PRR selectively promotes pathological retinal neovascularization (Satofuka et al. 2007); and (iii) overexpression of PRR in rats results in elevated blood pressure and increased plasma aldosterone (Burckle et al. 2006). Collectively, these observations indicate that PRR may be a critical member of the RAS in the maintenance of normal cardiovascular physiology. In spite of its emerging involvement and importance in cardiac, renal and vascular pathophysiology, nothing is known about the role of PRR in the brain. This is particularly relevant in view of the fact that the brain RAS is integral in neural control of cardiovascular functions and its hyperactivity is linked to hypertension (Veerasingham & Raizada, 2003; Peterson et al. 2006; Osborn et al. 2007). Although the presence of all the components of the RAS is unquestionable in the brain, their cellular distribution and the interplay of different cell types (neurons, glia, etc.) in the generation of Ang II and the access of this hormone to the Ang II type 1 (AT1) receptor in cardiovascular-relevant neurons remains elusive. In addition, the presence of low levels of renin in the brain has been a consistent enigma in establishing an independent role of the brain RAS (Dzau et al. 1986; Baltatu et al. 1998). Discovery of the PRR is of great significance in this regard and may be critical in solving the puzzle of an intrinsic RAS in the brain and delineating the mechanism of neuronal Ang II actions. Thus, our objective in the present study was to determine whether neurons within the brain express a functional PRR, in at attempt to elucidate the role of the brain RAS in cardiovascular functions.

In previous studies, we determined that macrophage migration inhibitory factor (MIF), acting intracellularly via its intrinsic thiol-protein oxidoreductase (TPOR) activity, stimulates basal neuronal delayed-rectifier K+ current ( IKv) and inhibits basal and angiotensin (ANG) II-induced increases in neuronal activity. These findings are the basis for our hypothesis that MIF is a negative regulator of ANG II actions in neurons. MIF has recently been recategorized as a member of the thioredoxin (Trx) superfamily of small proteins. In the present study we have examined whether Trx influences basal and ANG II-modulated IKv in an effort to determine whether the Trx superfamily can exert a general regulatory influence over neuronal activity and the actions of ANG II. Intracellular application of Trx (0.8–80 nM) into rat hypothalamic/brain stem neurons in culture increased neuronal IKv, as measured by voltage-clamp recordings. This effect of Trx was abolished in the presence of the TPOR inhibitor PMX 464 (800 nM). Furthermore, the mutant protein recombinant human C32S/C35S-Trx, which lacks TPOR activity, failed to alter neuronal IKv. Trx applied at a concentration (0.08 nM) that does not alter basal IKv abolished the inhibition of neuronal IKv produced by ANG II (100 nM). Given our observation that ANG II increases Trx levels in neuronal cultures, it is possible that Trx (like MIF) has a negative regulatory role over basal and ANG II-stimulated neuronal activity via modulation of IKv. Moreover, these data suggest that TPOR may be a general mechanism for negatively regulating neuronal activity.

Our previous studies demonstrated that peripheral overexpression of angiotensin II (ANG II) type 2 receptors (AT2R) prevents hypertension-induced cardiac hypertrophy and remodeling without altering high blood pressure. This, coupled with the observations that AT2R play a role in the antihypertensive actions of ANG II type 1 receptor (AT1R) blockers (ARBs), led us to propose that peripheral overexpression of AT2R would improve the antihypertensive action of losartan (Los) in Sprague-Dawley (SD) rats made hypertensive via chronic infusion of ANG II. Here we utilized adenoviral vector-mediated AT2R gene transfer to test this hypothesis. A single intracardiac injection of adenoviral vector containing genomic AT2R (G-AT2R) DNA and enhanced green fluorescent protein (EGFP) gene controlled by cytomegalovirus (CMV) promoters (Ad-G-AT2R-EGFP; 5 × 109 infectious units) into adult SD rats produced robust AT2R overexpression in cardiovascular tissues (kidney, lung, heart, aorta, mesenteric artery, and renal artery) that persisted for 3–5 days postinjection. By 7 days post viral injection, the overexpressed AT2R are reduced toward basal values in certain tissues (lung, kidney, and heart) and are undetectable in others (kidney and blood vessels). In two separate protocols, we demonstrated that the hypotensive effect of Los (0.125, 0.5, and 1.0 mg/kg iv) was significantly greater in the AT2R-overexpressing animals (−40.7 ± 4.3, −41.8 ± 4.8, and −48.1 ± 2.6 mmHg, respectively) compared with control vector (Ad-CMV-EGFP)-treated rats (−12.4 ± 2.2, −20.2 ± 3.4, and −27.3 ± 3.4 mmHg, respectively). These results provide support for a depressor role of AT2R and the proposal that combined AT2R agonist and ARB treatment may be an improved therapeutic strategy for controlling hypertension.

Much evidence indicates that pro-inflammatory effects of the renin-angiotensin system within the hypothalamus, including microglial activation and production of pro-inflammatory cytokines, play a role in chronic neurogenic hypertension. Our objective here was to examine whether angiotensin-(1-7) [Ang-(1-7)], a protective component of the renin-angiotensin system, exerts direct actions at microglia to counteract these pro-inflammatory effects. Mas, the Ang-(1-7) receptor, was shown to be present on cultured hypothalamic microglia. Treatment of these cells with Ang-(1-7) (100-1000 nM, 3-12 h) elicited significant decreases in basal levels of mRNAs for the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor-necrosis factor α (TNFα) and of the microglia-macrophage marker CD11b, and increases in basal levels of the anti-inflammatory cytokine interleukin-10. Incubation of microglial cultures with (pro)renin (PRO) (10-50 nM; 6 h) elicited significant increases in mRNAs for IL-1β, TNFα and CD11b. The effects of PRO (10 nM) on IL-1β and TNFα mRNAs, and TNFα protein, were significantly attenuated by co-treatment with Ang-(1-7) (100 nM). Lastly, these actions of Ang-(1-7) were abolished by the Mas antagonist A-779, and were associated with reductions in NF-κB subunit expression. Collectively, these data provide the first evidence that Ang-(1-7) can exert direct effects at microglia to lower baseline and counteract PRO-induced increases in pro-inflammatory cytokines. Renin-Angiotensin system mediated microglial activation and pro-inflammatory cytokine production within the hypothalamus are components of the chronic neuroinflammation associated with 'neurogenic' hypertension. We demonstrated that angiotension-(1-7) acting via its receptor Mas on hypothalamic microglia lessens baseline and (pro)renin-induced increases in pro-inflammatory cytokine production by these cells. This is the first evidence that angiotensin-(1-7) has direct anti-inflammatory effects via microglia.

Over activation of renin angiotensin system (RAS) signaling in cardiovascular control centers of the brain causes inflammatory responses that are associated with the onset of neurogenic hypertensio...

The discovery of beneficial neuroprotective effects of the angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis [ACE2-Ang-(1-7)-Mas] in ischemic and hemorrhagic stroke has spurred interest in a more complete characterization of its mechanisms of action. Here, we summarize findings that describe the protective role of the ACE2-Ang-(1-7)-Mas axis in stroke, along with a focused discussion on the potential mechanisms of neuroprotective effects of Ang-(1-7) in stroke. The latter incorporates evidence describing the actions of Ang-(1-7) to counter the deleterious effects of angiotensin II (AngII) via its type 1 receptor, including anti-inflammatory, anti-oxidant, vasodilatory, and angiogenic effects, and the role of altered kinase-phosphatase signaling. Interactions of Mas with other receptors, including bradykinin receptors and AngII type 2 receptors are also considered. A more complete understanding of the mechanisms of action of Ang-(1-7) to elicit neuroprotection will serve as an essential step toward research into potential targeted therapeutics in the clinical setting.

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.

The cellular characteristics of the beta-adrenoreceptor in glial and neuronal cells from the newborn rat brain were determined by (-)-[125I]iodocyanopindolol binding. In membranes from both cell types, the binding was saturable and from competition assays the potency series of (-)-isoproterenol greater than (-)-epinephrine = (-)-norepinephrine greater than (+)-isoproterenol was observed. 5'-Guanylyl-imidodiphosphate reduced the affinity of (-)-isoproterenol for the beta-adrenoreceptor from glial cells but had no effect on agonist affinity in neuronal cells. Chronic treatment of both cell types with (-)-isoproterenol reduced the receptor content and the capacity of the agonist to increase the cellular cyclic AMP content. However, the receptor recovery after chronic agonist treatment was faster in glial cells (72 h) than neuronal cells (120 h) and was blocked by cycloheximide. Treatment of both types with the irreversible beta-blocker bromoacetylalprenololmentane (2 microM) reduced the receptor content by 78% but no receptor recovery was observed for 120 h after the initial receptor loss. The data indicated that the majority of beta-adrenoreceptors in both cell types are the beta-1 subtype, but show some differences in receptor-agonist interactions. Furthermore, these CNS cells may be useful models for regulatory studies on the beta-adrenoreceptor.

This paper reviews recent results of our investigation of the mechanisms whereby hyperphenylalaninemia may cause brain dysfunction in classical phenylketonuria (PKU). Acute applications of L-Phe in rat and mouse hippocampal and cerebrocortical cultured neurons, at a range of concentrations found in PKU brain, significantly and reversibly depressed glutamatergic synaptic transmission by a combination of pre- and postsynaptic actions: (1) competition for the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptors; (2) attenuation of neurotransmitter release; (3) competition for the glutamate-binding site of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid and kainate (AMPA/kainate) receptors. Unlike L-Phe, its non-tyrosine metabolites, phenylacetic acid, phenylpyruvic acid, and phenyllactic acid, did not produce antiglutamatergic effects. L-Phe did not affect inhibitory gamma-aminobutyric (GABA)-ergic transmission. Consistent with this specific pattern of effects caused by L-Phe in neuronal cultures, the expression of NMDA receptor NR2A and AMPA receptor Glu1 and Glu2/3 subunits in brain of hyperphenylalaninemic PKU mice (Pah(enu2) strain) was significantly increased, whereas expression of the NMDA receptor NR2B subunit was decreased. There was no change in GABA alpha1 subunit expression. Considering the important role of glutamatergic synaptic transmission in normal brain development and function, these L-Phe-induced changes in glutamatergic synaptic transmission in PKU brain may be a critical element of the neurological symptoms of PKU.

It is well established that the central cardiovascular effects of angiotensin II (Ang II) involve superoxide production. However, the intracellular mechanism by which reactive oxygen species (ROS) signaling regulates neuronal Ang II actions remains to be elucidated. In the present study, we have used neuronal cells in primary cultures from the hypothalamus and brain stem areas to study the role of ROS on the cellular actions of Ang II. Ang II increases neuronal firing rate, an effect mediated by the AT 1 receptor subtype and involving inhibition of the delayed rectifier potassium current ( I Kv ). This increase in neuronal activity was associated with increases in NADPH oxidase activity and ROS levels within neurons, the latter evidenced by an increase in ethidium fluorescence. The increases in NADPH oxidase activity and ethidium fluorescence were blocked by either the AT 1 receptor antagonist losartan or by the selective NAD(P)H oxidase inhibitor gp91ds-tat. Extracellular application of the ROS scavenger, Tempol, attenuated the Ang II–induced increase in neuronal firing rate by 70%. In addition, gp91ds-tat treatment resulted in a 50% inhibition of Ang II–induced increase in firing rate. In contrast, the ROS generator Xanthine-Xanthine oxidase significantly increased neuronal firing rate. Finally, Ang II inhibited neuronal I Kv, and this inhibition was abolished by gp91ds-tat treatment. These observations demonstrate, for the first time, that Ang II regulates neuronal activity via a series of events that includes ROS generation and inhibition of I Kv . This signaling seems to be a critical cellular event in central Ang II regulation of cardiovascular function.

Angiotensin II (Ang II) elicits Ang II type 1 receptor (AT1-R)-mediated increases in neuronal firing within the hypothalamus and brainstem that are ultimately responsible for physiological actions such as increased blood pressure and fluid intake. Although there is a growing literature on the intracellular mechanisms that mediate the actions of Ang II via AT1-R in neurons, little is known about the mechanisms that diminish or “switch-off” the neuronal chronotropic action of Ang II. In the present study, we identified macrophage migration inhibitory factor (MIF) as an intracellular inhibitor of the actions of Ang II in neurons. The evidence is as follows. First, Ang II, acting via AT1-R, increases the intracellular levels of MIF in neurons cultured from rat hypothalamus and brainstem. Second, elevation of intracellular MIF by Ang II prevents further chronotropic actions of this peptide. Third, intracellular application of exogenous recombinant MIF abolishes the Ang II-induced chronotropic action in neurons. Finally, intracellular application of the MIF peptide fragment MIF-(50-65), which harbors the thiol oxidoreductase property of the MIF molecule, mimics the inhibitory actions of MIF on Ang II-stimulated neuronal firing. Thus, this study is the first to demonstrate the existence of an intracellular negative regulator of Ang II-induced actions in neurons and indicates that MIF may act as a physiological brake for the chronotropic effects of Ang II in rat neurons.

Most of our knowledge of the function of the angiotensin type 2 receptor (AT(2)R) has been obtained from transgenic mouse models. The aim of the present study was to investigate the role of the AT(2)R in normotensive Sprague-Dawley (SD) rats by using antisense gene transfer technology to 'knockdown' this specific receptor subtype. A retroviral vector containing full-length AT(2)R antisense cDNA (AT(2)R-AS) was constructed and the effectiveness of the transduction of AT(2)R-AS was studied in vitro. In subsequent in vivo studies, 5-day-old normotensive SD rats received a single intracardiac bolus (25 microl) of AT(2)R-AS viral particles. When animals reached adulthood, direct blood pressure (BP), and both pressor and dipsogenic responses to angiotensin II were investigated. Long-lasting expression of the AT(2)R-AS transcript and a reduction in mRNA and binding of the AT(2)R was observed in vitro. Expression of AT(2)R-AS transcript was maintained for 90 days in heart, kidney, lung and brain, indicating a high degree of transgene transduction in vivo. As adults, systolic BP and the pressor responses to angiotensin were significantly elevated in AT(2)R-AS-treated rats. However, AT(2)R-AS-treated rats displayed significantly reduced dipsogenic responses to both angiotensin and water deprivation. Collectively, these data demonstrate that a single neonatal injection of the retroviral vector containing antisense to the AT(2) receptors in rats results in similar cardiovascular and dipsogenic responses as reported in AT(2)R knockout mice. The actions of the AT(2) receptors appear to be antagonistic to the cardiovascular actions of the AT(1) receptors, whereas AT(1) and AT(2) receptors appear to act synergistically in the regulation of water intake.

Angiotensin II acting centrally contributes to the regulation of blood pressure and water intake and stimulates the release of catecholamines from the adrenal medulla. We hypothesized that the central angiotensin II is one mediator of biosynthesis of catecholamines in the adrenal medulla. Rats were administered i.c.v. angiotensin II or saline, and TH mRNA and protein levels in adrenal medulla were measured 1 or 3 h later. Angiotensin II did not change TH mRNA or protein 1 h later. However, by 3 h, angiotensin II increased TH mRNA and protein levels. Centrally administered angiotensin II elevates TH mRNA expression and protein levels in the adrenal medulla. In conclusion, one component of central angiotensin II elevation of blood pressure may be the result of increased catecholamine synthesis in the adrenal gland and elevated TH synthesis represents one underlying mechanism.

Angiotensin II (Ang II) modulates, via Ang II type 1 (AT(1)) receptors, the activity of brain catecholaminergic neurons. Here we utilized catecholaminergic CATH.a cells to define the effects of Ang II on delayed rectifier K(+) current (I(Kv)), one of the factors that determines changes in neuronal activation. Receptor binding analyses demonstrated the presence of AT(1) receptors in CATH.a cells. Whole cell voltage clamp experiments in these cells revealed that Ang II (100nM) produced a significant inhibition of I(Kv), that was abolished by the AT(1) receptor blocker, losartan (1 microM), or by inhibition of phospholipase C (PLC) with U73122 (10 microM). Furthermore, this action of Ang II was completely abolished by co-inhibition of protein kinase C (PKC) and calcium/calmodulin protein kinase II (CaMKII). These results demonstrate that Ang II produces an inhibition of I(Kv) in CATH.a cells, via an intracellular pathway that includes PLC, PKC, and CaMKII.

Prior studies utilizing neurons cultured from the hypothalamus and brain stem of newborn rats have demonstrated that ANG II-induced modulation of neuronal firing involves activation of both protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaMKII). The present studies were performed to determine whether these signaling molecules are also involved in physiological responses elicited by ANG II in the brain in vivo. Central injection of ANG II (10 ng/2 microl) into the lateral cerebroventricle (icv) of Sprague-Dawley rats increased water intake in a time-dependent manner. This ANG II-mediated dipsogenic response was attenuated by central injection of the PKC inhibitors chelerythrine chloride (0.5-50 microM, 2 microl) and Go-6976 (2.3 nM, 2 microl) and by the CaMKII inhibitor KN-93 (10 microM, 2 microl). Conversely, icv injection of chelerythrine chloride (50 microM, 2 microl) and KN-93 (10 microM, 2 microl) had no effect on the dipsogenic response elicited by central injection of carbachol (200 ng/2 microl). Furthermore, injection of ANG II (10 ng/2 microl) icv increases the activity of both PKC-alpha and CaMKII in rat septum and hypothalamus. These data suggest that signaling molecules involved in ANG II-induced responses in vitro are also relevant in physiological responses elicited by ANG II in the whole animal model.

This study examines the angiotensin II (Ang II) regulation of intracellular free calcium concentration ([Ca 2+ ] i ) in astroglia cultured from the hypothalamus and brainstem of the adult rat. Bath perfusion or rapid puffer application of angiotensin II (Ang II) (1-100 nM) increased [Ca 2+ ] i in both polygonal and stellate astroglia when measured using fura-2 imaging fluorescence microscopy. Ang II increased [Ca 2+ ] i in 96.1 and 95.6% of the polygonal and stellate glial cells, respectively. In normal Tyrode's solution (containing 2 mM CaCl 2 ), the Ang II-stimulated increase in [Ca 2+ ] i characteristically showed a biphasic response, i.e., an initial rapid transient peak followed by a sustained, steady-state plateau of free Ca 2+ . In both cell types, the selective Ang II type 1 receptor subtype (AT 1 ) antagonist losartan (1 μM) inhibited the Ang II-stimulated increase in [Ca 2+ ] i . The selective AT 2 antagonist PD 123319 (1 μM) did not inhibit the Ang II-stimulated increase in [Ca 2+ ] i in either cell type. To define the sources of Ca 2+ that participate in the Ang 11-stimulated increase in [Ca 2+ ] i in astroglia, experiments were performed in a nominally Ca 2+ -free Tyrode's solution. In either cell type, this resulted in only an initial transient increase of Ca 2+ and no sustained plateau of Ca 2+ when challenged with Ang II. Thapsigargin (5 μM), cyclopiazonic acid (10 μM), and ryanodine (10 μM), but not caffeine (1-10 mM), inhibited the initial rise in [Ca 2+ ] i . The plateau increase of [Ca 2+ ] i caused by Ang II (100 nM) was reversibly inhibited by both cadmium (100 μM) and nifedipine (10 μM) ; in contrast, gadolinium (100 μM) had no effect on the plateau increase of [Ca 2+ ] i . These results indicate that Ang II, in physiological concentrations, can activate AT 1 receptors to stimulate both Ca 2+ release from intracellular stores and Ca 2+ influx from the extracellular space to increase [Ca 2+ ] i of polygonal and stellate astroglia.