Search

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.

The angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis represents a promising target for inducing stroke neuroprotection. Here, we explored stroke-induced changes in expression and activity of endogenous angiotensin-converting enzyme 2 and other system components in Sprague–Dawley rats. To evaluate the clinical feasibility of treatments that target this axis and that may act in synergy with stroke-induced changes, we also tested the neuroprotective effects of diminazene aceturate, an angiotensin-converting enzyme 2 activator, administered systemically post stroke. Among rats that underwent experimental endothelin-1–induced ischemic stroke, angiotensin-converting enzyme 2 activity in the cerebral cortex and striatum increased in the 24 hours after stroke. Serum angiotensin-converting enzyme 2 activity was decreased within 4 hours post stroke, but rebounded to reach higher than baseline levels 3 days post stroke. Treatment after stroke with systemically applied diminazene resulted in decreased infarct volume and improved neurological function without apparent increases in cerebral blood flow. Central infusion of A-779, a Mas receptor antagonist, resulted in larger infarct volumes in diminazene-treated rats, and central infusion of the angiotensin-converting enzyme 2 inhibitor MLN-4760 alone worsened neurological function. The dynamic alterations of the protective angiotensin-converting enzyme 2 pathway after stroke suggest that it may be a favorable therapeutic target. Indeed, significant neuroprotection resulted from poststroke angiotensin-converting enzyme 2 activation, likely via Mas signaling in a blood flow–independent manner. Our findings suggest that stroke therapeutics that target the angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis may interact cooperatively with endogenous stroke-induced changes, lending promise to their further study as neuroprotective agents.

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.

Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells. Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.

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.

The baroreflex is an essential regulator of blood pressure (BP); whereby, baroreceptors sense acute changes in BP and convey this information, via the nodose ganglia (NG), to the nucleus of the solitary tract (NTS). Manipulation of the baroreflex may provide a novel strategy in the treatment of hypertension. To that end, our neuroanatomical studies revealed a dense localization of angiotensin type 1a receptor (AT1a)-containing neuronal cell bodies in the NG and terminals in the NTS, thereby positioning them to play a role in the baroreflex. We, therefore, hypothesized that AT1a neurons residing in the NG potently influence cardiovascular function. Male and female mice expressing channelrhodopsin-2 (ChR2) and yellow fluorescent protein (eYFP) specifically in AT1a-expressing neurons (AT1aR-ChR2-eYFP) were used to determine the function of these neurons. In vitro patch clamp electrophysiological recordings from neurons in the NTS receiving axons expressing ChR2-eYFP revealed that optogenetic stimulation (473nm) reliably evoked excitatory postsynaptic currents (EPSCs). In concurrence with studies demonstrating that baroreceptor afferents utilize glutamate in the NTS, these EPSCs were eliminated by the presence of glutamate receptor antagonists. Next, the NG of anesthetized mice were subjected to optogenetic stimulation of varying frequencies (1, 15, 30Hz) for 1 min, and BP and HR responses were assessed. Optogenetic stimulation of these AT1a-expressing neurons led to significant decreases in mean arterial pressure (ΔMAP= -16±3*, -36±4*, -44±7* mmHg) and HR (ΔHR= -13±7, -104±44*, -163±59* bpm) in AT1a-ChR2-eYFP mice (n=6), but there was no effect on control mice harboring only the stop-FLOX-ChR2-eYFP gene (n=5; ΔMAP= 0±2, -2±1, 5±5 mmHg; ΔHR= 4±4, 3±4, -13±21 bpm). Additionally, AT1a-ChR2-eYFP mice rendered hypertensive via DOCA-salt (n=7) exhibited a dampened response to optogenetic stimulation (ΔMAP= -7±1#, -14±3*#, -30±6*# mmHg; ΔHR= -29±34, -71±37*, -178±50* bpm). Collectively, these data suggest that AT1a neurons in the NG are key regulators of the baroreflex, and may serve as a target for antihypertensive therapeutics. (p

It is accepted that activation of angiotensin type 1a receptors (AT1a) within the CNS elevates blood pressure by influencing sympathetic outflow and vasopressin (VP) secretion; however, the neuronal circuits mediating these effects are not completely understood. The present studies characterize the structure and function of AT1a neurons residing in the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT), thereby evaluating their potential role in blood pressure control. Using male mice that express Cre-recombinase prior to the STOP codon of the AT1a gene, initial studies combined genetic reporting with in situ hybridization to reveal that AT1a neurons in the MnPO and OVLT are largely excitatory (87±4% express vesicular glutamate transporter 2). Subsequently, AT1a-Cre mice were delivered a Cre-inducible adeno-associated virus to induce expression of channelrhodopsin-2 (ChR2) and enhanced yellow fluorescent protein (eYFP) specifically within AT1a neurons of the MnPO/OVLT (AAV-ChR2-eYFP; n = 4). Control mice were delivered AAV-eYFP (n = 4). Analysis of eYFP immunofluorescence revealed that neurons within the MnPO/OVLT that express AT1a send projections to the paraventricular nucleus of the hypothalamus (PVN; an area involved in sympathetic outflow and VP secretion) that appear to synapse onto VP synthesizing neurons. To evaluate the functionality of this connection, we optogenetically stimulated AT1a neurons in the region while recording cardiovascular parameters in anesthetized mice. Ten-minutes of optogenetic stimulation (473 nM;15 Hz; 20 ms pulse width; 60 s ON/OFF) robustly elevated systolic blood pressure in AAV-ChR2-eYFP mice relative to AAV-eYFP controls. This effect was rapid in its onset (34 ± 9 vs. -2 ± 4 mmHg at 5 min, p < 0.05) but persisted for the entire 50 min of cardiovascular recording (45 ± 11 vs. -3 ± 7 mmHg at 50 min, p < 0.05). Intriguingly, the optogenetic stimulation also resulted in 62% increase in Fos induction in AVP neurons within the PVN relative to AAV-eYFP controls (86 ± 5 vs. 52 ± 2 %; p

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.

Background: Compound 21 (C21), a selective small-molecule angiotensin type 2 receptor (AT2R) agonist, has been proven in multiple preclinical studies to reduce infarct size and ameliorate neurological deficits, when administered after ischemic stroke via intracerebroventricular or intraperitoneal routes. However, C21 poorly penetrates the blood brain barrier (BBB). In this study, we used the novel and non-invasive approach of intranasal trans-olfactory (INTO) application, in order to bypass the BBB and deliver C21 directly into the brain. The therapeutic efficacy of INTO application of C21 was assessed in a model of transient middle cerebral artery occlusion (MCAO). Methods: (i) Male SD rats (12 weeks old) underwent ischemic stroke by endothelin-1-induced MCAO. They were randomly divided into two treatment groups, either receiving 0.9% saline or C21 (1.5 ug/kg) at 1.5, 4, 24 and 48 h post-stroke, using a rat intranasal catheter device (Impel Neuropharma, Seattle, WA) for INTO application. All rats underwent blinded neurological assessments at 4, 24 and 72 h after stroke, and immediately after the 72 h tests, were euthanized and cerebral infarct volumes were assessed by TTC staining. (ii) Male SD rats (12 weeks old) underwent implantation of a telemetry transducer (DSI, St. Paul, MN) into the abdominal aorta for measurement of blood pressure, heart rate and locomotor activity after INTO C21 (1.5 ug/kg) vs. 0.9% saline at baseline and post-ischemic stroke. Results: (i) Post-stroke INTO delivery of C21 (1.5 ug/kg) elicited a significant lowering of % cerebral infarct volume (25.4 ± 4.7; n=9) compared with saline-treated rats (48.4 ± 4.4; n=21) [p Conclusions: Our results demonstrate, that INTO delivery of C21 exerts protective effects after ischemic stroke. These studies suggest INTO administration as potential future route of application of C21 to stroke patients.

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

Stress elicits neuroendocrine, autonomic, and behavioral responses that mitigate homeostatic imbalance and ensure survival. However, chronic engagement of such responses promotes psychological, cardiovascular, and metabolic impairments. In recent years, the renin-angiotensin system has emerged as a key mediator of stress responding and its related pathologies, but the neuronal circuits that orchestrate these interactions are not known. These studies combine the use of the Cre-recombinase/loxP system in mice with optogenetics to structurally and functionally characterize angiotensin type-1a receptor-containing neurons of the paraventricular nucleus of the hypothalamus, the goal being to determine the extent of their involvement in the regulation of stress responses. Initial studies use neuroanatomical techniques to reveal that angiotensin type-1a receptors are localized predominantly to the parvocellular neurosecretory neurons of the paraventricular nucleus of the hypothalamus. These neurons are almost exclusively glutamatergic and send dense projections to the exterior portion of the median eminence. Furthermore, these neurons largely express corticotrophin-releasing hormone or thyrotropin-releasing hormone and do not express arginine vasopressin or oxytocin. Functionally, optogenetic stimulation of these neurons promotes the activation of the hypothalamic-pituitary–adrenal and hypothalamic-pituitary–thyroid axes, as well as a rise in systolic blood pressure. When these neurons are optogenetically inhibited, the activity of these neuroendocrine axes are suppressed and anxiety-like behavior in the elevated plus maze is dampened. Collectively, these studies implicate this neuronal population in the integration and coordination of the physiological responses to stress and may therefore serve as a potential target for therapeutic intervention for stress-related pathology.SIGNIFICANCE STATEMENTChronic stress leads to an array of physiological responses that ultimately rouse psychological, cardiovascular, and metabolic impairments. As a consequence, there is an urgent need for the development of novel therapeutic approaches to prevent or dampen deleterious aspects of “stress.” While the renin-angiotensin system has received some attention in this regard, the neural mechanisms by which this endocrine system may impact stress-related pathologies and consequently serve as targets for therapeutic intervention are not clear. The present studies provide substantial insight in this regard. That is, they reveal that a distinct population of angiotensin-sensitive neurons is integral to the coordination of stress responses. The implication is that this neuronal phenotype may serve as a target for stress-related disease.

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.

Viral vectors have been utilized extensively to introduce genetic material into the central nervous system. In order to investigate gene functions in cardiovascular control regions of rat brain, we applied WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) enhanced-adenoviral (Ad) and adeno-assoicated virus (AAV) type 2 vectors to mediate neuronal gene delivery to the paraventricular nucleus of the hypothalamus, the nucleus tractus solitarius and the rostral ventrolateral medulla, three important cardiovascular control regions known to express renin-angiotensin system (RAS) genes. Ad or AAV2 harboring an enhanced green fluorescent protein (EGFP) reporter gene or the angiotensin type 2 receptor gene were microinjected into these brain regions in adult rats. Our results demonstrated that both AAV2 and Ad vectors elicited long-term neuronal transduction in these regions. Interestingly, we found that the WPRE caused expression of GFP driven by the synapsin1 promoter in pure glial cultures or co-cultures of neurons and glia derived from rat hypothalamus and brainstem. However, in rat paraventricular nucleus WPRE did not cause expression of GFP in glia. This demonstrates the potential use of these vectors in studies of physiological functions of certain genes in the cardiovascular control regions of the brain.

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.

Introduction: Abundant preclinical studies show efficacy for therapies targeted to the protective axis of the renin-angiotensin system in stroke. The expression and activity of angiotensin converting enzyme 2 (ACE2), a cardio and neuro-protective carboxypeptidase in this axis, have recently been shown to be dynamically altered during stroke in animal models. Hypothesis: The aim of this study was to characterize the previously unexplored changes in activity of ACE2, and other RAS enzymes, in the serum of patients experiencing acute ischemic stroke. Methods: Blood samples were obtained from patients with acute ischemic stroke (n=20) at presentation and again at three days post-stroke and enzyme activity levels in isolated serum were analyzed by fluorometric assay. These were compared to enzyme activity levels in samples obtained from a cohort of patients presenting with transient ischemic attack or other clinical presentations mimicking stroke and from healthy control participants (n=20). Results: Ischemic stroke resulted in significantly lower levels of serum ACE2 activity at an average of 3.5 hours after stroke as compared to both transient ischemic attack/stroke mimic and healthy control participant levels, followed three days later by an increase back to control levels. ACE activity was also significantly decreased following stroke, but without increases at three days. Renin enzyme activity during stroke did not significantly differ from controls at either time point. Serum ACE2 activity was negatively correlated with systolic blood pressure at presentation among stroke patients, while activity levels from a separate cohort of healthy young adults were positively correlated. Multiple regression analyses indicated that ACE2 activity levels were significantly correlated with a diagnosis of ischemic stroke, with those in the lowest tertile of all participants were at 7.7 times increased odds of having had a stroke. Conclusions: The characterization of the dynamic and novel changes in serum ACE2 activity in human stroke, which concur with preclinical studies, along with the demonstration of correlations with systolic blood pressure, provides new insight for the development of therapies that target this protective system in ischemic stroke.

Background: The renin angiotensin system is a promising target for stroke neuroprotection and therapy through activation of angiotensin type II receptors (AT2Rs). Compound 21 (C21), a selective non-peptide AT2R agonist, has been shown to exhibit neuroprotection and improve stroke outcomes in preclinical studies. Stimulation of AT2Rs is believed to counteract the negative effects of angiotensin type 1 receptor and provide distinctive beneficial anti-inflammatory and neurotropic effects. We hypothesized that C21 given after stroke through peripheral injections would have sustained neuroprotective effects in aged rats. Methods: Aged adult male SD rats (18-20 months) underwent ischemic stroke by monofilament middle cerebral artery occlusion (MCAO) and were randomly divided into two groups that received intraperitoneal (IP) injections of either 0.9% NaCl or 0.03mg/kg C21 at reperfusion (90 min), 24h, and 48h after stroke. All animals received blinded neurological exams at 4h, 24h, 72h, 7d, 14d, and 21d post-stroke. Infarct size was assessed by magnetic resonance imaging at 21 days. Results: Post-stroke treatment with C21 significantly improved neurological function, as evidenced by neurological testing using Rotarod and somatosensory dysfunction exams. At 7d and 14d after stroke, C21-treated rats had significantly increased Rotarod times versus saline-treated rats, and at 21d, the somatosensory function was significantly improved as measured by time to removal of paw adhesive. Infarct volume tended to be non-significantly decreased by C21 treatment at 21d post-stroke. Conclusions: Our findings indicate that targeting the renin-angiotensin system, specifically by stimulation of AT2Rs with C21, improves neurological function in aged rats with stroke over a sustained period of 21 days. These findings encourage further research into the renin-angiotensin system and specifically AT2Rs, and offers hope for effective alternatives for treating stroke.

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.

Introduction: The expression and activity of angiotensin converting enzyme 2 (ACE2), a cardio and neuro-protective carboxypeptidase, has recently been shown to be dynamically altered during and after stroke in animal models. Hypothesis: The aim of this study was to characterize the previously unexplored changes in activity of ACE2 in the serum of patients experiencing acute ischemic stroke. Methods: Serum samples were obtained from patients presenting with acute ischemic stroke (n=20) and again at three days post-stroke and enzyme activity levels were analyzed by fluorometric assay and compared to levels from non-stroke control patients (n=20). Results: Ischemic stroke resulted in a significant decrease in serum ACE2 at an average of 3.5 hours after stroke, which was followed by a rebound increase by three days later (Fig 1). ACE activity was also significantly decreased following stroke, but without rebound increases. Tumor necrosis factor-alpha converting enzyme activity was not significantly different from control. ACE2 activity was negatively correlated with systolic blood pressure (SBP) among stroke patients (Fig 2), while activity was positively correlated with SBP among a cohort of healthy young adults (n=16). Conclusions: The characterization of the dynamic and novel changes in serum ACE2 activity in human stroke, which follow observations from preclinical studies, along with the demonstration of a negative association with SBP, provides new insight for the development of therapies that target this protective system in the context of ischemic stroke. Data are mean ± SEM. RFU: relative fluorescence units; *p

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.

The effects of the halogenated aromatic amino acid 3,5-dibromo-D: -tyrosine (3,5-DBr-D: -Tyr) were studied in rat models of stroke and epileptic seizures caused by middle cerebral artery occlusion (MCAo) through respective intracerebral injection of endothelin-1 (ET-1) and intraperitoneal (i.p.) injection of pentylenetetrazole (PTZ). 3,5-DBr-D: -Tyr was administered as three bolus injections (30 or 90 mg/kg, i.p.) starting at 30, 90, and 180 min after ET-1 administration or as a single bolus (30 mg/kg, i.p.) 15 min prior to PTZ administration. Neurological deficits and infarct volume were estimated 3 days after ET-1 administration and seizure score was assessed during the first 20 min after PTZ administration. The safety of 3,5-DBr-D: -Tyr was evaluated in control animals using telemetry to measure cardiovascular parameters and immunostaining to assess the level of activated caspase-3. 3,5-DBr-D: -Tyr significantly improved neurological function and reduced infarct volume in the brain even when the treatment was initiated 3 h after the onset of MCAo. 3,5-DBr-D: -Tyr significantly depressed PTZ-induced seizures. 3,5-DBr-D: -Tyr did not cause significant changes in arterial blood pressure, heart rate and spontaneous locomotor activity, nor did it increase the number of activated caspase-3 positive cells in the brain. We conclude that 3,5-DBr-D: -Tyr, by alleviating the deleterious effects of MCAo and PTZ in rats with no obvious intrinsic effects on cardiovascular parameters and neurodegeneration, exhibits promising potential as a novel therapeutic direction for stroke and seizures.

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.

Angiotensin II (Ang II) type 1 receptor blocking drugs have been shown to inhibit the growth of prostate cancer cells and delay the development of prostate cancer. Functional Ang II type 2 receptors (AT2R) are present in these cells and inhibit growth induced by epidermal growth factor. The present studies report apoptosis of prostate cancer cells induced by AT2R overexpression. A recombinant adenoviral vector expressing AT2R (Ad-G-AT2R-EGFP) was transduced into prostate cancer cells, including androgen-independent (DU145 and PC3) and androgen-dependent cell lines (LNCaP). Following AT2R transduction, apoptosis was analyzed by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining and caspase-3 activity assays. The results indicate that increased expression of AT2R alone induced apoptosis in the prostate cancer lines, an effect that did not require Ang II. AT2R overexpression in DU145 cells induced inhibition of proliferation, a significant reduction of S-phase cells, and an enrichment of G1-phase cells. The data also indicate that overexpression of AT2R led to apoptosis via an extrinsic cell death signaling pathway that is dependent on activation of p38 mitogen-activated protein kinase, caspase-8, and caspase-3. Finally, the apoptosis induced by AT2R overexpression is partially dependent on the activation of p53, but not on p21. The observations presented here suggest that the ability of increased AT2R expression to induce apoptosis in prostate cancer cells may have potential therapeutic implications for this disease, and suggest that AT2R is a promising novel target gene for prostate cancer gene therapy. [Mol Cancer Ther 2009;8(12):3255–65]

Background and purpose: Abnormal glutamatergic activity is implicated in neurologic and neuropsychiatric disorders. Selective glutamate receptor antagonists were highly effective in animal models of stroke and seizures but failed in further clinical development because of serious side effects, including an almost complete set of symptoms of schizophrenia. Therefore, the novel polyvalent glutamatergic agent 3,5-dibromo-L-phenylalanine (3,5-DBr-L-Phe) was studied in rat models of stroke, seizures and sensorimotor gating deficit. Experimental approach: 3,5-DBr-L-Phe was administered intraperitoneally as three boluses after intracerebral injection of endothelin-1 (ET-1) adjacent to the middle cerebral artery to cause brain injury (a model of stroke). 3,5-DBr-L-Phe was also given as a single bolus prior to pentylenetetrazole (PTZ) injection to induce seizures or prior to the administration of the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) to cause disruption of prepulse inhibition (PPI) of startle (sensorimotor gating deficit). Key results: Brain damage caused by ET-1 was reduced by 52%, which is comparable with the effects of MK-801 in this model as reported by others. 3,5-DBr-L-Phe significantly reduced seizures induced by PTZ without the significant effects on arterial blood pressure and heart rate normally caused by NMDA antagonists. 3,5-DBr-L-Phe prevented the disruption of PPI measured 3 days after the administration of ET-1. 3,5-DBr-L-Phe also eliminated sensorimotor gating deficit caused by MK-801. Conclusion and implications: The pharmacological profile of 3,5-DBr-L-Phe might be beneficial not only for developing a therapy for the neurological and cognitive symptoms of stroke and seizures but also for some neuropsychiatric disorders.

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.

Endogenous levels of angiotensin II (Ang II) are increased in the cortex and hypothalamus following stroke, and Ang II type 1 receptor blockers (ARBs) have been shown to attenuate the deleterious effects in animal stroke models using middle cerebral artery (MCA) intraluminal occlusion procedures. However, the endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO) model of cerebral ischaemia is thought to more closely mimic the temporal events of an embolic stroke. This method provides rapid occlusion of the MCA and a gradual reperfusion that lasts for 16–22 h. The aim of the present study was to evaluate whether systemic administration of an ARB prior to ET-1-induced MCAO would provide cerebroprotection during this model of ischaemic stroke. Injection of 3 μl of 80 μm ET-1 adjacent to the MCA resulted in complete occlusion of the vessel that resolved over a period of 30–40 min. Following ET-1-induced MCAO, rats had significant neurological impairment, as well as an infarct that consisted of 30% of the ipsilateral grey matter. Systemic pretreatment with 0.2 mg kg−1 day−1 candesartan for 7 days attenuated both the infarct size and the neurological deficits caused by ET-1-induced MCAO without altering blood pressure. This study confirms the cerebroprotective properties of ARBs during ischaemic stroke and validates the ET-1-induced MCAO model for examination of the role of the brain renin–angiotensin system in ischaemic stroke.

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.

It is well known that in fMLP-stimulated neutrophils, phosphatidyl inositol 3,4,5-trisphosphate [PI(3,4,5)P3] localizes at the leading edge of the cells. However, no effort has been made to study the PI 4,5-bisphosphate [PI(4,5)P2] distribution in these cells. In fact, it has been suggested that PI(4,5)P2 is unlikely to localize, as its basal level is orders of magnitude higher than that of PI(3,4,5)P3. We developed an optimized immunostaining protocol for studying the endogenous distribution of PI(4,5)P2 in neutrophil-like HL-60 cells. We show that PI(4,5)P2 localizes sharply at the leading edge with an intensity gradient similar to that for PI(3,4,5)P3. The enzymes for the production of PI(4,5)P2, namely, PI5KIα and PI5KIγ, were also found to localize at the leading edge, further supporting our finding that PI(4,5)P2 localizes at the leading edge. These results imply that complementary regulation of PI3K and phosphate and tensin homolog (PTEN) is not the sole or dominant mechanism of PI(3,4,5)P3 polarization in HL-60 cells.

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.

The chronic neurogenic hypertension that involves increased effects of angiotensin II (Ang II) via its type 1 receptor (AT1R) within brain cardiovascular control centers is associated with induction of microglial activation and neuroinflammation in the paraventricular nucleus of the hypothalamus (PVN). However, whether Ang II exerts direct effects via AT1R located on microglia is not established. Therefore, the objective of this study was to determine the cellular localization of AT1R in the PVN. Naïve twelve-week old normotensive (Sprague Dawley, SD; Wistar Kyoto, WKY) rats and spontaneously hypertensive rats (SHR) were euthanized and perfused with 4% paraformaldehyde. Brains were removed and sectioned coronally at the level of the PVN. Sections throughout the entire PVN underwent RNAscope fluorescence in situ hybridization to determine AT1aR mRNA expression, combined with immunohistochemistry using microglia (Ionized calcium binding adaptor molecule 1; Iba1)-, neuron (HuC/D)-, or astrocyte (Glial fibrillary acidic protein; GFAP)-specific markers. The results, obtained from at least 3 SD and 3 WKY rats indicate strong co-localization of AT1aR transcripts with neurons in the neuroendocrine and parvocellular PVN regions, as expected. By contrast, there was no detectable co-localization of AT1aR mRNA with either microglia or astrocytes throughout the PVN of these rats. Further, qRT-PCR revealed that while both AT1aR- and AT1bR mRNAs were detectable in SD rat hypothalamus (1.00±0.14; 0.40±0.12; n=7), neither transcript was detectable in microglia cultured from the hypothalamus of SD rats (n=6). The pattern of AT1aR mRNA expression in the PVN of SHR, a hypertensive model that exhibits over activity of Ang II/AT1R actions at the PVN, was similar to that observed in the SD and WKY rats, i.e. strong co-localization with HuC/D-positive cells, and no detectable co-localization with either Iba1 or GFAP-positive cells. Collectively, these findings indicate that AT1R are localized to neurons, not glia, in the PVN of normotensive rats or SHR in situ . Further, they suggest that it is unlikely that Ang II exerts direct effects at microglia in the PVN, and that induction of microglial activation at this site following Ang II infusion is likely an indirect action.

Pulmonary fibrosis (PF) is a fatal disease characterized by scarring of the lung tissue. A dysregulated renin angiotensin system (RAS) has been implicated in the pathology of PF. Recent reports suggest a beneficial role for the angiotensin type II receptor (AT2R) in cardiopulmonary disorders; however, the effects of AT2R in PF have not been studied. We have taken advantage of a recently identified AT2R agonist, Compound 21 (C21), to evaluate its actions in the bleomycin (BLM) model of PF. PF was induced by a single intra-tracheal instillation of 2.5mg/kg of BLM. Following BLM administration, a subset of animals was treated with C21 (0.03mg/kg, ip, daily) for 2 weeks (prevention protocol). In a subsequent study, C21 was administered after three days of BLM injection (reversal protocol). For both prevention and reversal protocols, hemodynamic parameters were measured, and tissues collected after 15 days of BLM injection. BLM caused significant increase in right ventricular systolic pressure (RVSP) and induced maladaptive cardiac remodeling. However, C21 treatment considerably reduced BLM-induced elevation in RVSP (Con: 29+1; BLM: 47+2; BLM+C21(P): 37+2; BLM+C21(R): 41+3 mmHg), attenuated ventricular hypertrophy (Con: 0.28+0.01; BLM: 0.41+0.01; BLM+C21(P): 0.35+0.01; BLM+C21(R): 0.37+0.02) and improved right heart function in both prevention (P) and reversal (R) protocols. Histopathological assessment of hematoxylin and eosin stained lung sections also showed decreased collagen accumulation in the C21 treated group. Collectively, our results suggest that the AT2R agonist, C21 exerts potent antifibrotic effects and may hold promise for patients with PF.

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...

Background: Recent studies show that pharmacological induction of the angiotensin converting enzyme 2/angiotensin-(1-7)/mas [ACE2-Ang-(1-7)-Mas] axis, a protective pathway of the renin angiotensin system, elicits neuroprotection in ischemic stroke. However, endogenous levels and activity of the components of this axis in the brain and serum following stroke are not well established. Here, we assessed the post-stroke activity and expression of ACE2 in rat cerebral cortex and serum after ischemic stroke in rats, in the absence or presence of an ACE2 activator. Methods: Sprague Dawley rats underwent sham surgery or endothelin-1-induced middle cerebral artery occlusion (ET-1 MCAO). Activity of ACE2 was analyzed within serum and cerebral cortical tissue samples using a fluorometric assay, and mRNA levels were assessed by qRT-PCR. In an additional experiment, rats received daily intraperitoneal administration of diminazene aceturate (DIZE), a putative ACE2 activator, or vehicle after ET-1 MCAO. Data are normalized to corresponding control values and expressed as means ± SEM with a significance of p Results: ACE2 activity levels were significantly increased in ischemic brain cortex at 4, 12, and 24 h after a stroke (4h: 237.1±46.1%; 12h: 212.4±12.8%; 24h: 191.6±19.1%) versus rats with sham strokes. Paradoxically, there was a significant decrease in ACE2 mRNA levels in the ischemic cortex at 24h (0.71±0.1) compared to shams (1.0±0.08). After decreasing in activity at 4h after stroke, serum ACE2 activity was increased at 24h in stroked rats (96.08±9.4%) versus shams (70.80±7.1%). Post-stroke treatment with DIZE (7.5 mg/kg) resulted in significantly increased ACE2 activity in serum (213.7±49.8%) versus controls, two days following stroke. Conclusions: Activity of the protective enzyme ACE2 is increased in rat cerebral cortex following stroke, with a rebound increase in serum activity. Post-stroke treatment with an ACE2 activator resulted in significantly increased ACE2 activity in serum. These results suggest that stroke therapeutics designed to target the ACE2/Ang-(1-7)/Mas axis may act in synergy with endogenous changes in the acute post-stroke setting, lending promise to their further study as potential neuroprotective agents.