Brain Angiotensin Research Articles

O98 NEW APPROACHES TARGETING THE RENIN-ANGIOTENSIN SYSTEM: BRAIN ACE2 ACTIVATION FOLLOWING BRAIN AMINOPEPTIDASE A BLOCKADE BY FIRIBASTAT IN SALT-DEPENDENT HYPERTENSION

Background and Objective: In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Ang II and Ang III have similar affinities for AT1 receptors and blocking brain AngIII formation by RB150 decreases arterial BP without inducing Ang II accumulation. Methods: We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. To answer this question, we use surgical animal models that could allow us to monitor awake blood pressure responses to intracerebroventricular (icv) injections and measure enzymatic activity. Results: We found that the intracerebroventricular administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Conclusions: Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Sex-Dependent Effects of Angiotensin Type 2 Receptor–Expressing Medial Prefrontal Cortex Interneurons in Fear Extinction Learning

BackgroundThe renin-angiotensin system has been identified as a potential therapeutic target for posttraumatic stress disorder, although its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC), but their function and mechanism in the mPFC remain unexplored. Therefore, we used a combination of imaging, cre/lox, and behavioral methods to investigate mPFC-AT2R–expressing neurons in fear and stess related behavior. MethodsTo characterize mPFC-AT2R–expressing neurons in the mPFC, AT2R-Cre/tdTomato male and female mice were used for immunohistochemistry. mPFC brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker were quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using a Cre-expressing adeno-associated virus. Mice then underwent Pavlovian auditory fear conditioning, elevated plus maze, and open field testing. ResultsImmunohistochemistry results revealed that AT2R was densely expressed throughout the mPFC and primarily expressed in somatostatin interneurons in a sex-dependent manner. Following fear conditioning, mPFC-AT2R Cre-lox deletion impaired extinction and increased exploratory behavior in female but not male mice, while locomotion was unaltered by mPFC-AT2R deletion in both sexes. ConclusionsThese results identify mPFC-AT2R+ neurons as a novel subgroup of somatostatin interneurons and reveal their role in regulating fear learning in a sex-dependent manner, potentially offering insights into novel therapeutic targets for posttraumatic stress disorder.

Blood pressure reduction through brain delivery of nanoparticles loaded with plasmid DNA encoding angiotensin receptor shRNA

Elevated brain angiotensin II activity plays a key role in the development of neurogenic hypertension. While blood pressure control in neurogenic hypertension has been successfully demonstrated by regulating central angiotensin II activity, current techniques involving cerebrovascular injections of potential therapeutic agents are not suitable for clinical translation. To address this gap, we present the synthesis of dual-functionalized liposomes functionalized with targeting ligand and cell penetrating peptide. Functionalized liposomes were synthesized using the thin film hydration technique and loaded with plasmid DNA encoding shRNA targeted towards Angiotensin II receptors (PEAS), via the post-insertion method. The synthesized liposomes had a cationic surface charge, an average size of 150 nm, and effectively entrapped over 89% of loaded PEAS. These liposomes loaded with PEAS demonstrated biocompatibility and efficient delivery to brain-derived cell lines, resulting in a remarkable reduction of over 70% in receptor expression within 7 days. To assess the therapeutic potential, spontaneously hypertensive rats were administered intravenous injections of functionalized liposomes loaded with PEAS, and the changes in mean arterial pressure were monitored for 45 days. Remarkably, this treatment led to a significant (p < 0.001) reduction in blood pressure of over 30 mmHg compared to saline-treated rats.

Sex-Dependent Effects of Angiotensin Type 2 Receptor Expressing Medial Prefrontal Cortex (mPFC) Interneurons in Fear Extinction Learning

Background: The renin-angiotensin system (RAS) has been identified as a potential therapeutic target for PTSD, though its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC). Their function and mechanism in the mPFC, however, remain unexplored. We therefore used a combination of neuroanatomical, chemogenetic, and behavioral methods to investigate mPFC-AT2R-expressing neuron involvement in fear learning. Methods: To characterize mPFC-AT2R-expressing neurons in the mPFC, AT2R-Cre/td-Tomato male and female brains were perfused and used for immunohistochemistry. Brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker was quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus. Mice then underwent Pavlovian auditory fear conditioning and anxiety-like and locomotor behavior testing using open field (OF) and elevated plus maze (EPM) tests. Results: IHC revealed that AT2R is expressed throughout the mPFC in males ( 208.6±48.8 cells/mm2) and females ( 139.0±59.8 cells/mm2) and has low co-expression on glutamatergic neurons ( TBR1 co-staining, males: 7.1%±1.8%; females: 13.2%±4.2%). Of the interneuron markers tested, AT2R is primarily expressed on somatostatin interneurons, and has higher colocalization in the mPFC of females than males ( males: 16.5%±1.2%, females: 31.8%±4.2%, p=0.02). Following fear conditioning and extinction, mPFC-AT2R deletion impaired extinction in female ( p=0.03) but not male ( p=0.76) mice. Locomotion in the OF was unaltered by mPFC-AT2R deletion in males or females ( total distance travelled; males: GFP 44.11m±5.92m, Cre 56.00m±10.29m, p=0.35; females: GFP 42.86m±4.85m, Cre 42.39m±4.97m, p=0.95), while AT2R-deleted females had increased exploratory behavior in the EPM ( open arm entries; males: GFP 12.38±1.02, Cre 13.22±1.54, p=0.66; females: GFP 12.50±0.96, Cre 20.00±2.81, p=0.02). Conclusion: These results lend support for mPFC-AT2R+ neurons as a novel somatostatin subgroup that influences fear extinction in a sex-dependent manner. This furthers underscores the role of mPFC in top-down regulation and a unique role for peptidergic (ie., angiotensin) mPFC regulation of fear and sex differences, and increases the understanding of circuitry and function of the brain RAS in disordered fear learning. This may lead to improved therapeutic treatments for PTSD. This research and PJM was supported by NIH1R01HL137103-01A1 and the American Heart Association 15CSA24340001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiotensin II involvement in the development and persistence of amphetamine-induced sensitization: Striatal dopamine reuptake implications.

Amphetamine (AMPH) exposure induces behavioural and neurochemical sensitization observed in rodents as hyperlocomotion and increased dopamine release in response to a subsequent dose. Brain Angiotensin II modulates dopaminergic neurotransmission through its AT1 receptors (AT1-R), positively regulating striatal dopamine synthesis and release. This work aims to evaluate the AT1-R role in the development and maintenance of AMPH-induced sensitization. Also, the AT1-R involvement in striatal dopamine reuptake was analysed. The sensitization protocol consisted of daily AMPH administration for 5days and tested 21 days after withdrawal. An AT1-R antagonist, candesartan, was administered before or after AMPH exposure to evaluate the participation of AT1-R in the development and maintenance of sensitization, respectively. Sensitization was evaluated by locomotor activity and c-Fos immunostaining. Changes in dopamine reuptake kinetics were evaluated 1day after AT1-R blockade withdrawal treatment, with or without the addition of AMPH in vitro. The social interaction test was performed as another behavioural output. Repeated AMPH exposure induced behavioural and neurochemical sensitization, which was prevented and reversed by candesartan. The AT1-R blockade increased the dopamine reuptake kinetics. Neither the AMPH administration nor the AT1-R blockade altered the performance of social interaction. Our results highlight the AT1-R's crucial role in AMPH sensitization. The enhancement of dopamine reuptake kinetics induced by the AT1-R blockade might attenuate the neuroadaptive changes that lead to AMPH sensitization and its self-perpetuation. Therefore, AT1-R is a prominent candidate as a target for pharmacological treatment of pathologies related to dopamine imbalance, including drug addiction and schizophrenia.

Meningeal interleukin-17-producing T cells mediate cognitive impairment in a mouse model of salt-sensitive hypertension.

Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.

Evaluation of prefrontal cortex expressing angiotensin type 2 receptors in fear-related behavior

Background: Evidence suggests that the renin-angiotensin system (RAS), a regulator of blood pressure and fluid homeostasis, is a potential therapeutic target for PTSD. Brain angiotensin II receptors are expressed across corticolimbic circuits such as the medial prefrontal cortex (mPFC), however, their role and mechanism are largely unknown. Given the importance of the mPFC in fear-related behavior, our studies sought to examine the role of mPFC-expressing angiotensin type 2 receptor (AT2R) neurons in fear learning in male and female mice. We proposed that activity of mPFC-AT2Rs, via inhibition of glutamatergic transmission, facilitates the extinction of cued fear. Methods: Using an AT2R-eGFP BAC reporter mouse, immunohistochemistry, retrograde tracing, and whole-cell patch-clamp recording were performed for neuroanatomical and ex-vivo analysis. To understand the role of mPFC-AT2Rs in fear learning, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus in AT2R-flox male and female mice (n=9-12 per group). Following viral integration (3 weeks), Pavlovian auditory fear conditioning (5x tone/shock pairings, 30 CS extinction) was used to examine the extinction of learned fear. Anxiety-like behavior and locomotion were also examined using the open field and elevated plus maze behavioral tests. Results: The immunofluorescence microscopy results identified AT2R-eGFP+ cells as predominantly glutamatergic ( 88.1% Tbr1, 11.9% GAD) and highly expressed in the prelimbic ( 120.6±13.25 cells/mm2) and infralimbic ( 163.7±17.30 cells/mm2) cortices of the mPFC, while retrograde labeling demonstrated that some mPFC-AT2R-eGFP+ neurons project to the basolateral amygdala (BLA). Ex-vivo activation using slice electrophysiology in mPFC-AT2R-eGFP+ neurons decreased the distribution of both frequency ( D=0.19, p&lt;0.01) and amplitude ( D=0.13, p&lt;0.01) of spontaneous excitatory postsynaptic currents (sEPSC). Selective mPFC-AT2R deletion impaired extinction learning as measured by enhanced percent freezing (day 2, F(1, 719)=5.12, p=0.02), and this effect on extinction occurred only in female mice. Generalized anxiety measures (% time in open arm, M: GFP 14.42%, Cre 13.58%, p=0.78; F: GFP 14.85%, Cre 21.66%, p=0.19) and locomotion ( total distance, M: GFP 44.11m, Cre 56.00m, p=0.35; F: GFP 42.86m, Cre 42.39m, p=0.95) were not affected by mPFC-AT2R deletion in males or females. Conclusions: These findings provide novel evidence for mPFC-AT2R+ neurons as a potential sex-dependent corticolimbic regulator of conditioned fear. We propose that mPFC AT2Rs contribute to extinction in females by decreasing excitatory glutamatergic output from the mPFC onto the amygdala. To further evaluate these results, future studies will utilize in vivo chemogenetic approaches to examine the role of this circuit in the physiology and extinction of learned fear. NIH 1R01HL137103-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Angiotensinergic neurotransmission in the bed nucleus of the stria terminalis is involved in cardiovascular responses to acute restraint stress in rats.

The brain angiotensin II acting via AT1 receptors is a prominent mechanism involved in physiological and behavioral responses during aversive situations. The AT2 receptor has also been implicated in stress responses, but its role was less explored. Despite these pieces of evidence, the brain sites related to control of the changes during aversive threats by the brain renin-angiotensin system (RAS) are poorly understood. The bed nucleus of the stria terminalis (BNST) is a limbic structure related to the cardiovascular responses by stress, and components of the RAS system were identified in this forebrain region. Therefore, we investigated the role of angiotensinergic neurotransmission present within the BNST acting via local AT1 and AT2 receptors in cardiovascular responses evoked by an acute session of restraint stress in rats. For this, rats were subjected to bilateral microinjection of either the angiotensin-converting enzyme inhibitor captopril, the selective AT1 receptor antagonist losartan, or the selective AT2 receptor antagonist PD123319 before they underwent the restraint stress session. We observed that BNST treatment with captopril reduced the decrease in tail skin temperature evoked by restraint stress, without affecting the pressor and tachycardic responses. Local AT2 receptor antagonism within the BNST reduced both the tachycardia and the drop in tail skin temperature during restraint. Bilateral microinjection of losartan into the BNST did not affect the restraint-evoked cardiovascular changes. Taken together, these data indicate an involvement of BNST angiotensinergic neurotransmission acting via local AT2 receptors in cardiovascular responses during stressful situations.

ATIP LEVELS ARE ASSOCIATED WITH LESS AMYLOID-Β BURDEN IN POSTMORTEM BRAINS OF OLDER ADULTS WITH ALZHEIMER’S DEMENTIA

Abstract Angiotensin system, aging, and Alzheimer's disease are tightly linked. Of the brain angiotensin receptors, the subtype 2 receptor (AT2R) is relatively less studied. Canonically, the AT2R functions through nitric oxide release, and its activation has been linked to vasodilatation and neurite outgrowth as well as anti-inflammation. How AT2R signals is not known, however, an AT2R-binding protein (ATIP) has been recently described. Controversies exist on the link between ATIP and AT2R functions. Here, we describe the development of the first non-antibody based ultra-sensitive and specific quantitative mass spectrometry assay for ATIP. Using a technique that permits targeted analysis of multiple peptides across multiple samples in a single mass spectrometry run, known as TOMAHAQ, we have identified specific human tryptic peptides that permit quantification of ATIP abundance. We have used this method to quantify ATIP in postmortem frontal cortex samples of older adults (n= 60) with Alzheimer’s dementia (AD). We correlated levels of ATIP to brain RAS receptors, and biomarkers of AD pathogenesis including oxidative stress, inflammation, mitochondrial dysfunction as well as amyloid-β, and tau burden. Our results show that ATIP expression (ANLKNPQIMYLEQELESLK sequence of ATIP) is positively correlated with neuronal nitric oxide synthase (nNOS) (p = 0.009, r = 0.337). Furthermore, expression of ATIP is negatively correlated with amyloid-β load in several brain regions including hippocampus (p= 0.014, r= -0.317), entorhinal cortex (p= 0.010, r= -0.331), frontal cortex (p= 0.023, r= -0.294), and overall (p= 0.004, r= -0.365). These results highlight a potential protective role for ATIP in Alzheimer’s disease.

Pharmaceutical Potential of Casein-Derived Tripeptide Met-Lys-Pro: Improvement in Cognitive Impairments and Suppression of Inflammation in APP/PS1 Mice.

Background:Tripeptide Met-Lys-Pro (MKP), a component of casein hydrolysates, has effective angiotensin-converting enzyme (ACE) inhibitory activity. Brain angiotensin II enzyme activates the NADPH oxidase complex via angiotensin II receptor type 1 (AT1) and enhances oxidative stress injury. ACE inhibitors improved cognitive function in Alzheimer’s disease (AD) mouse models and previous clinical trials. Thus, although undetermined, MKP may be effective against pathological amyloid-β (Aβ) accumulation-induced cognitive impairment.Objective:The current study aimed to investigate the potential of MKP as a pharmaceutical against AD by examining MKP’s effect on cognitive function and molecular changes in the brain using double transgenic (APP/PS1) mice.Methods:Experimental procedures were conducted in APP/PS1 mice (n = 38) with a C57BL/6 background. A novel object recognition test was used to evaluate recognition memory. ELISA was used to measure insoluble Aβ40, Aβ42, and TNF-α levels in brain tissue. Immunohistochemical analysis allowed the assessment of glial cell activation in MKP-treated APP/PS1 mice.Results:The novel object recognition test revealed that MKP-treated APP/PS1 mice showed significant improvement in recognition memory. ELISA of brain tissue showed that MKP significantly reduced insoluble Aβ40, Aβ42, and TNF-α levels. Immunohistochemical analysis indicated the suppression of the marker for microglia and reactive astrocytes in MKP-treated APP/PS1 mice.Conclusion:Based on these results, we consider that MKP could ameliorate pathological Aβ accumulation-induced cognitive impairment in APP/PS1 mice. Furthermore, our findings suggest that MKP potentially contributes to preventing cognitive decline in AD.

Central angiotensinergic mechanisms in female spontaneously hypertensive rats treated with estradiol

Estrogens reduce 0.3 M NaCl intake and palatability in a widely used model of essential hypertension, the spontaneously hypertensive rats (SHRs). Here we investigated whether the inhibitory effects of β-estradiol (E2, 10 μg/kg b.w. subcutaneously for 8 days) on water deprived partially-rehydrated (WD-PR) ovariectomized (OVX) adult female SHRs (fSHRs, n = 4–10/group) are related to interferences on brain angiotensin II AT1 receptors (AT1r). After WD-PR, E2 reduced 0.3 M NaCl intake (1.3 ± 0.6, vs. vehicle: 3.5 ± 1.2 ml/30 min), the number of hedonic responses to intraoral NaCl infusion (57 ± 11, vs. vehicle: 176 ± 32/min), and the relative angiotensin AT1r (Agtr1a) mRNA expression in the hypothalamus. Losartan (AT1r antagonist, 100 μg) intracerebroventricularly in OVX fSHRs treated with vehicle subcutaneously abolished 0.3 M NaCl intake (0.1 ± 0.1 ml/30 min) and only transiently reduced hedonic responses to intraoral NaCl. Losartan combined with E2 decreased the number of hedonic and increased the number of aversive responses to intraoral NaCl and abolished 0.3 M NaCl intake. E2 also reduced the pressor and dipsogenic responses to intracerebroventricular angiotensin II. The results suggest that AT1r activation increases palatability and induces NaCl intake in WD-PR fSHRs. E2 reduced hypothalamic Agtr1a mRNA expression, which may account for the effects of E2 on NaCl intake and palatability and intracerebroventricular angiotensin II-induced pressor and dipsogenic responses in OVX fSHRs. Future studies considering natural fluctuations in estrogen secretion might help to determine the degree of such interference in brain neuronal activity.

Higher Angiotensin II Type 1 Receptor Levels and Activity in the Postmortem Brains of Older Persons with Alzheimer's Dementia.

Aging is a key risk factor in Alzheimer's dementia (AD) development and progression. The primary dementia-protective benefits of angiotensin II subtype 1 receptor (AT1R) blockers are believed to arise from systemic effects on blood pressure. However, a brain-specific renin-angiotensin system (b-RAS) exists, which can be altered by AT1R blockers. Brain RAS acts mainly through 3 angiotensin receptors: AT1R, AT2R, and AT4R. Changes in these brain angiotensin receptors may accelerate the progression of AD. Using postmortem frontal cortex brain samples of age- and sex-matched cognitively normal individuals (n = 30) and AD patients (n = 30), we sought to dissect the b-RAS changes associated with AD and assess how these changes correlate with brain markers of oxidative stress, inflammation, and mitochondrial dysfunction as well as amyloid-β and paired helical filament tau pathologies. Our results show higher protein levels of the pro-inflammatory AT1R and phospho-ERK (pERK) in the brains of AD participants. Brain AT1R levels and pERK correlated with higher oxidative stress, lower cognitive performance, and higher tangle and amyloid-β scores. This study identifies molecular changes in b-RAS and offers insight into the role of b-RAS in AD-related brain pathology.

Angiotensin-(1-7) Analogue AVE0991 Modulates Astrocyte-Mediated Neuroinflammation via lncRNA SNHG14/miR-223-3p/NLRP3 Pathway and Offers Neuroprotection in a Transgenic Mouse Model of Alzheimer's Disease.

ObjectiveEmerging evidence suggests that brain angiotensin-(1–7) (Ang-(1–7)) deficiency contributes to the pathogenesis of Alzheimer’s disease (AD). Meanwhile, our previous studies revealed that restoration of brain Ang-(1–7) levels provided neuroprotection by inhibition of inflammatory responses during AD progress. However, the potential molecular mechanisms by which Ang-(1–7) modulates neuroinflammation remain unclear.Materials and MethodsAPP/PS1 mice were injected intraperitoneally with AVE0991 (a nonpeptide analogue of Ang-(1–7)) once a day for 30 consecutive days. Cognitive functions, neuronal and synaptic integrity, and inflammation-related markers were assessed. Since astrocytes played a crucial role in AD-related neuroinflammation whilst long noncoding RNAs (lncRNAs) were reported to participate in modulating inflammatory responses, astrocytes of APP/PS1 mice were isolated for high-throughput lncRNA sequencing to identify the most differentially expressed lncRNA following AVE0991 treatment. Afterward, the downstream pathways of this lncRNA in the anti-inflammatory action of AVE0991 were investigated using primary astrocytes.ResultsAVE0991 rescued spatial cognitive impairments and alleviated neuronal and synaptic damage in APP/PS1 mice. The levels of Aβ1-42 in the brain of APP/PS1 mice were not affected by AVE0991. By employing high-throughput lncRNA sequencing, our in vitro study demonstrated for the first time that AVE0991 suppressed astrocytic NLRP3 inflammasome-mediated neuroinflammation via a lncRNA SNHG14-dependent manner. SNHG14 acted as a sponge of miR-223-3p while NLRP3 represented a direct target of miR-223-3p in astrocytes. In addition, miR-223-3p participated in the AVE0991-induced suppression of astrocytic NLRP3 inflammasome.ConclusionOur results suggest that Ang-(1–7) analogue AVE0991 inhibits astrocyte-mediated neuroinflammation via SNHG14/miR-223-3p/NLRP3 pathway and offers neuroprotection in APP/PS1 mice. These findings reveal the underlying mechanisms by which Ang-(1–7) inhibits neuroinflammation under AD condition and uncover the potential of its nonpeptide analogue AVE0991 in AD treatment.

Sensory Afferent Renal Nerve Activated Gαi2 Subunit Proteins Mediate the Natriuretic, Sympathoinhibitory and Normotensive Responses to Peripheral Sodium Challenges.

We have previously reported that brain Gαi2 subunit proteins are required to maintain sodium homeostasis and are endogenously upregulated in the hypothalamic paraventricular nucleus (PVN) in response to increased dietary salt intake to maintain a salt resistant phenotype in rats. However, the origin of the signal that drives the endogenous activation and up-regulation of PVN Gαi2 subunit protein signal transduction pathways is unknown. By central oligodeoxynucleotide (ODN) administration we show that the pressor responses to central acute administration and central infusion of sodium chloride occur independently of brain Gαi2 protein pathways. In response to an acute volume expansion, we demonstrate, via the use of selective afferent renal denervation (ADNX) and anteroventral third ventricle (AV3V) lesions, that the sensory afferent renal nerves, but not the sodium sensitive AV3V region, are mechanistically involved in Gαi2 protein mediated natriuresis to an acute volume expansion [peak natriuresis (μeq/min) sham AV3V: 43 ± 4 vs. AV3V 45 ± 4 vs. AV3V + Gαi2 ODN 25 ± 4, p < 0.05; sham ADNX: 43 ± 4 vs. ADNX 23 ± 6, AV3V + Gαi2 ODN 25 ± 3, p < 0.05]. Furthermore, in response to chronically elevated dietary sodium intake, endogenous up-regulation of PVN specific Gαi2 proteins does not involve the AV3V region and is mediated by the sensory afferent renal nerves to counter the development of the salt sensitivity of blood pressure (MAP [mmHg] 4% NaCl; Sham ADNX 124 ± 4 vs. ADNX 145 ± 4, p < 0.05; Sham AV3V 125 ± 4 vs. AV3V 121 ± 5). Additionally, the development of the salt sensitivity of blood pressure following central ODN-mediated Gαi2 protein down-regulation occurs independently of the actions of the brain angiotensin II type 1 receptor. Collectively, our data suggest that in response to alterations in whole body sodium the peripheral sensory afferent renal nerves, but not the central AV3V sodium sensitive region, evoke the up-regulation and activation of PVN Gαi2 protein gated pathways to maintain a salt resistant phenotype. As such, both the sensory afferent renal nerves and PVN Gαi2 protein gated pathways, represent potential targets for the treatment of the salt sensitivity of blood pressure.

NRF2 Activation and Downstream Effects: Focus on Parkinson's Disease and Brain Angiotensin.

Reactive oxygen species (ROS) are signalling molecules used to regulate cellular metabolism and homeostasis. However, excessive ROS production causes oxidative stress, one of the main mechanisms associated with the origin and progression of neurodegenerative disorders such as Parkinson’s disease. NRF2 (Nuclear Factor-Erythroid 2 Like 2) is a transcription factor that orchestrates the cellular response to oxidative stress. The regulation of NRF2 signalling has been shown to be a promising strategy to modulate the progression of the neurodegeneration associated to Parkinson’s disease. The NRF2 pathway has been shown to be affected in patients with this disease, and activation of NRF2 has neuroprotective effects in preclinical models, demonstrating the therapeutic potential of this pathway. In this review, we highlight recent advances regarding the regulation of NRF2, including the effect of Angiotensin II as an endogenous signalling molecule able to regulate ROS production and oxidative stress in dopaminergic neurons. The genes regulated and the downstream effects of activation, with special focus on Kruppel Like Factor 9 (KLF9) transcription factor, provide clues about the mechanisms involved in the neurodegenerative process as well as future therapeutic approaches.

Fifty years of research on the brain renin-angiotensin system: what have we learned?

Although the existence of a brain renin-angiotensin system (RAS) had been proposed five decades ago, we still struggle to understand how it functions. The main reason for this is the virtual lack of renin at brain tissue sites. Moreover, although renin's substrate, angiotensinogen, appears to be synthesized locally in the brain, brain angiotensin (Ang) II disappeared after selective silencing of hepatic angiotensinogen. This implies that brain Ang generation depends on hepatic angiotensinogen after all. Rodrigues et al. (Clin Sci (Lond) (2021) 135:1353-1367) generated a transgenic mouse model overexpressing full-length rat angiotensinogen in astrocytes, and observed massively elevated brain Ang II levels, increased sympathetic nervous activity and vasopressin, and up-regulated erythropoiesis. Yet, blood pressure and kidney function remained unaltered, and surprisingly no other Ang metabolites occurred in the brain. Circulating renin was suppressed. This commentary critically discusses these findings, concluding that apparently in the brain, overexpressed angiotensinogen can be cleaved by an unidentified non-renin enzyme, yielding Ang II directly, which then binds to Ang receptors, allowing no metabolism by angiotensinases like ACE2 and aminopeptidase A. Future studies should now unravel the identity of this non-renin enzyme, and determine whether it also contributes to Ang II generation at brain tissue sites in wildtype animals. Such studies should also re-evaluate the concept that Ang-(1-7) and Ang III, generated by ACE2 and aminopeptidase A, respectively, have important functions in the brain.

Effects of firibastat in combination with enalapril and hydrochlorothiazide on blood pressure and vasopressin release in hypertensive DOCA-salt rats

In the brain, aminopeptidase A (APA) generates angiotensin III, one of the effector peptides of the brain renin-angiotensin system (RAS), exerting tonic stimulatory control over blood pressure (BP) in hypertensive rats. Oral administration of firibastat, an APA inhibitor prodrug, in hypertensive rats, inhibits brain APA activity, blocks brain angiotensin III formation and decreases BP. In this study, we evaluated the efficacy of firibastat in combination with enalapril, an angiotensin I-converting enzyme inhibitor, and hydrochlorothiazide (HCTZ), in conscious hypertensive deoxycorticosterone acetate (DOCA)-salt rats, which display high plasma arginine-vasopressin levels, low circulating renin levels and resistance to treatment by systemic RAS blockers. We determined mean arterial BP, heart rate, plasma arginine-vasopressin levels and renin activity in DOCA-salt rats orally treated with firibastat, enalapril or HCTZ administered alone or in combination. Acute oral firibastat administration (30 mg/kg) induced a significant decrease in BP, whereas enalapril (10 mg/kg) or HCTZ (10 mg/kg) administered alone induced no significant change in BP in conscious DOCA-salt rats. The BP decrease induced by acute and nine-day chronic tritherapy [Firibastat+Enalapril+HCTZ] was significantly greater than that observed after bitherapy [Enalapril+HCTZ]. Interestingly, the chronic administration of a combination of firibastat with [Enalapril+HCTZ] reduced plasma arginine-vasopressin levels by 62% relative to those measured in DOCA-salt rats receiving bitherapy. Our data show that tritherapy with firibastat, enalapril and HCTZ improves BP control and arginine-vasopressin release in an experimental salt-dependent model of hypertension, paving the way for the development of new treatments for patients with currently difficult-to-treat or resistant hypertension.

Physiology in Perspective: Insulin—100 Years of Physiological Discovery

Physiology in Perspective: Insulin—100 Years of Physiological Discovery

Brain ACE2 activation following brain aminopeptidase A blockade by firibastat in salt-dependent hypertension.

In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats without inducing angiotensin II accumulation. We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. We found that the intracerebroventricular (icv) administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Brain angiotensin system: a new promise in the management of epilepsy?

Epilepsy is a highly prevalent neurological disease and anti-epileptic drugs (AED) are almost the unique clinical treatment option. A disbalanced brain renin-angiotensin system (RAS) has been proposed in epilepsy and several reports have shown that angiotensin II (Ang II) receptor-1 (ATR1) activation is pro-inflammatory and pro-epileptogenic. In agreement, ATR1 blockage with the repurposed drug losartan has shown benefits in animal models of epilepsy. Processing of Ang II by ACE2 enzyme renders Ang-(1-7), a metabolite that activates the mitochondrial assembly (Mas) receptor (MasR) pathway. MasR activation presents beneficial effects, facilitating vasodilatation, increasing anti-inflammatory and antioxidative responses. In a recent paper published in Clinical Science, Gomes and colleagues (Clin. Sci. (Lond.) (2020) 134, 2263-2277) performed intracerebroventricular (icv) infusion of Ang-(1-7) in animals subjected to the pilocarpine model of epilepsy, starting after the first spontaneous motor seizure (SMS). They showed that this approach reduced the frequency of SMS, restored animal anxiety, increased exploration, and augmented the hippocampal expression of protective catalase enzyme and antiapoptotic protein B-cell lymphoma 2 (Bcl-2). Interestingly, but surprisingly, Gomes and colleagues showed that MasR expression and mTor activity were reduced in the hippocampus of the epileptic Ang-(1-7) treated animals. These results show that Ang-(1-7) administration could represent a new avenue for developing strategies for the management of epilepsy in clinical settings. However, future work is necessary to evaluate the levels of RAS metabolites and the activity of key enzymes in these experimental interventions to completely understand the therapeutic potential of the brain RAS manipulation in epilepsy.