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 of the hypothalamus (PVN), 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.
Previously we demonstrated that central administration of angiotensin-(1-7) [Ang-(1-7)] into rats elicits significant cerebroprotection against ischemic stroke elicited by endothelin-1 induced middle cerebral artery occlusion. Ang-(1-7), acting via its receptor Mas, reduced cerebral infarct size, and rats exhibited improved performance on neurological exams. These beneficial actions of Ang-(1-7) were not due to inhibition of the effects of endothelin-1 on cerebral vasoconstriction or effects on cerebral blood flow, and so we considered other potential mechanisms. Here we investigated the possibility that the Ang-(1-7)-induced cerebroprotection involves an anti-inflammatory effect, since stroke-induced cerebral damage includes an excessive intracerebral inflammatory response. Our quantitative RT-PCR analyses revealed that central Ang-(1-7) treatment attenuates the increased expression of mRNAs for inducible nitric oxide synthase (iNOS), several pro-inflammatory cytokines and cluster of differentiation molecule 11b (microglial marker) within the cerebral cortex following endothelin-1 induced stroke. Western blotting confirmed similar changes in iNOS protein expression in the cerebral cortex. In support of these observations, immunostaining revealed the presence of immunoreactive Mas on activated microglia within the cerebral cortical infarct zone, and in vitro experiments demonstrated that lipopolysaccharide-induced increases in nitric oxide production in glial cultures are attenuated by Ang-(1-7) acting via Mas. Collectively these findings demonstrate an anti-inflammatory action of Ang-(1-7) in the brain, and suggest that the cerebroprotective action of this peptide in ischemic stroke may involve effects on nitric oxide generation by microglia.
Over-activation of brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme (ACE2) inhibits RAS activity by converting angiotensin II, the effector peptide of RAS, to angiotensin-(1-7), which activates Mas receptors (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ~62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA.
The angiotensin converting enzyme 2/angiotensin-(1-7)/Mas axis represents a promising target for inducing stroke neuroprotection. Here, 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. Amongst 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 4h post stroke, but rebounded to reach higher than baseline levels 3d post-stroke. Treatment following 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 following stroke suggest that it may be a favorable therapeutic target. Indeed, significant neuroprotection resulted from post-stroke 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.
Obesity is a widespread health concern that is associated with an increased prevalence of hypertension and cardiovascular disease. Both obesity and hypertension have independently been associated with increased levels of inflammatory cytokines and immune cells within specific brain regions, as well as increased activity of the renin-angiotensin system (RAS). To test the hypothesis that high-fat diet (HFD) induced obesity leads to an angiotensin-II (Ang-II)-dependent increase in inflammatory cells within specific forebrain regions that are important for cardiovascular regulation, we first assessed microglial activation, astrocyte activation, inflammation and RAS component gene expression within selected metabolic and cardiovascular control centers of the forebrain in adult male C57BL/6 mice given either a HFD or a low-fat diet (LFD) for 8 weeks. Subsequently, we assessed the necessity of the paraventricular nucleus of the hypothalamus (PVN) angiotensin type-1a (AT1a) receptor for these responses by using the Cre/lox system in mice to selectively delete the AT1a receptor from the PVN. These studies reveal that in addition to the arcuate nucleus of the hypothalamus (ARC), the PVN and the subfornical organ (SFO), two brain regions that are known to regulate blood pressure and energy balance, also initiate proinflammatory responses after the consumption of a diet high in fat. They further indicate that some, but not all, of these responses are reversed upon deletion of AT1a specifically within the PVN.
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