Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro.

Angiotensin AT2-receptor (AT2R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT2R-agonists is very limited due to the lack of high-throughput assays for AT2R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT2R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT2R-transfected CHO cells (AT2R-CHO) or in non-transfected CHO cells (Flp-CHO) using the fluorescent probe DAF-FM diacetate. It is run in 96-well plates and fluorescence signals are semi-automatically quantified. The assay was tested for sensitivity (recognition of true positive results), selectivity (recognition of true negative results), and reliability (by calculating the repeatability coefficient (RC)). The high-throughput, semi-automated method was proven suitable, as the NO-releasing agents C21, CGP42112A, angiotensin-(1-7) and acetylcholine significantly increased NO release from HAEC. The assay is sensitive and selective, since the established AT2R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT2R-CHO cells, while the non-AT2R-agonists angiotensin-(1-7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT2R-agonists, of which 39 met our requirements for reliability (RC ≤ 20% different from RC for C21). Our newly developed high-throughput method for detection of AT2R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT2R-agonists in future drug development programs.

Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties.

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.

Sodium Intake and Disease: Another Relationship to Consider.

Sodium (Na+) is crucial for numerous homeostatic processes in the body and, consequentially, its levels are tightly regulated by multiple organ systems. Sodium is acquired from the diet, commonly in the form of NaCl (table salt), and substances that contain sodium taste salty and are innately palatable at concentrations that are advantageous to physiological homeostasis. The importance of sodium homeostasis is reflected by sodium appetite, an "all-hands-on-deck" response involving the brain, multiple peripheral organ systems, and endocrine factors, to increase sodium intake and replenish sodium levels in times of depletion. Visceral sensory information and endocrine signals are integrated by the brain to regulate sodium intake. Dysregulation of the systems involved can lead to sodium overconsumption, which numerous studies have considered causal for the development of diseases, such as hypertension. The purpose here is to consider the inverse-how disease impacts sodium intake, with a focus on stress-related and cardiometabolic diseases. Our proposition is that such diseases contribute to an increase in sodium intake, potentially eliciting a vicious cycle toward disease exacerbation. First, we describe the mechanism(s) that regulate each of these processes independently. Then, we highlight the points of overlap and integration of these processes. We propose that the analogous neural circuitry involved in regulating sodium intake and blood pressure, at least in part, underlies the reciprocal relationship between neural control of these functions. Finally, we conclude with a discussion on how stress-related and cardiometabolic diseases influence these circuitries to alter the consumption of sodium.

Quantitative Phosphoproteomics of the Angiotensin AT2-Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis.

BACKGROUND: Angiotensin AT2-receptor signaling is atypical for a G-protein coupled receptor and incompletely understood. To obtain novel insights into AT2-receptor signaling, we mapped changes in the phosphorylation status of the entire proteome of human aortic endothelial cells in response to AT2-receptor stimulation. METHODS: Phosphorylation status of human aortic endothelial cells after stimulation with C21 (1 µM; 0, 1, 3, 5, 20 minutes) was determined utilizing time-resolved quantitative phosphoproteomics. Specific changes in protein phosphorylation and acetylation were confirmed by Western Blotting. Functional tests included resazurin assay for cell proliferation, and caspase 3/7 luminescence assay or FACS analysis of annexin V expression for apoptosis. RESULTS: AT2-receptor stimulation significantly altered the phosphorylation status of 172 proteins (46% phosphorylations, 54% dephosphorylations). Bioinformatic analysis revealed a cluster of phospho-modified proteins involved in antiproliferation and apoptosis. Among these proteins, HDAC1 (histone-deacetylase-1) was dephosphorylated at serine421/423 involving serine/threonine phosphatases. Resulting HDAC1 inhibition led to p53 acetylation and activation. AT2-receptor stimulation induced antiproliferation and apoptosis, which were absent when cells were co-incubated with the p53 inhibitor pifithrin-α, thus indicating p53-dependence of these AT2-receptor mediated functions. CONCLUSIONS: Contrary to the prevailing view that AT2-receptor signaling largely involves phosphatases, our study revealed significant involvement of kinases. HDAC1 inhibition and resulting p53 activation were identified as novel, AT2-receptor coupled signaling mechanisms. Furthermore, the study created an openly available dataset of AT2-receptor induced phospho-modified proteins, which has the potential to be the basis for further discoveries of currently unknown, AT2-receptor coupled signaling mechanisms.

The Angiotensin AT2 Receptor: From a Binding Site to a Novel Therapeutic Target.

Discovered more than 30 years ago, the angiotensin AT2 receptor (AT2R) has evolved from a binding site with unknown function to a firmly established major effector within the protective arm of the renin-angiotensin system (RAS) and a target for new drugs in development. The AT2R represents an endogenous protective mechanism that can be manipulated in the majority of preclinical models to alleviate lung, renal, cardiovascular, metabolic, cutaneous, and neural diseases as well as cancer. This article is a comprehensive review summarizing our current knowledge of the AT2R, from its discovery to its position within the RAS and its overall functions. This is followed by an in-depth look at the characteristics of the AT2R, including its structure, intracellular signaling, homo- and heterodimerization, and expression. AT2R-selective ligands, from endogenous peptides to synthetic peptides and nonpeptide molecules that are used as research tools, are discussed. Finally, we summarize the known physiological roles of the AT2R and its abundant protective effects in multiple experimental disease models and expound on AT2R ligands that are undergoing development for clinical use. The present review highlights the controversial aspects and gaps in our knowledge of this receptor and illuminates future perspectives for AT2R research. SIGNIFICANCE STATEMENT: The angiotensin AT2 receptor (AT2R) is now regarded as a fully functional and important component of the renin-angiotensin system, with the potential of exerting protective actions in a variety of diseases. This review provides an in-depth view of the AT2R, which has progressed from being an enigma to becoming a therapeutic target.

Angiotensin receptors - affinitiy and beyond.

This commentary on the article "Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors" by Sanja Bosnyak et al. (Clini. Sci. (Lond.) (2011) 121(7): 297-303. https://doi.org/10.1042/CS20110036) summarises the main findings of the study, followed by a discussion of the findings and their relevance for various aspects of the biology of receptors of the renin-angiotensin system in the context of the current state of knowledge.

Targeting angiotensin type-2 receptors located on pressor neurons in the nucleus of the solitary tract to relieve hypertension in mice.

AIMS: These studies evaluate whether angiotensin type-2 receptors (AT2Rs) that are expressed on γ-aminobutyric acid (GABA) neurons in the nucleus of the solitary tract (NTS) represent a novel endogenous blood pressure-lowering mechanism. METHODS AND RESULTS: Experiments combined advanced genetic and neuroanatomical techniques, pharmacology, electrophysiology, and optogenetics in mice to define the structure and cardiovascular-related function of NTS neurons that contain AT2R. Using mice with Cre-recombinase directed to the AT2R gene, we discovered that optogenetic stimulation of AT2R-expressing neurons in the NTS increases GABA release and blood pressure. To evaluate the role of the receptor, per se, in cardiovascular regulation, we chronically delivered C21, a selective AT2R agonist, into the brains of normotensive mice and found that central AT2R activation reduces GABA-related gene expression and blunts the pressor responses induced by optogenetic excitation of NTS AT2R neurons. Next, using in situ hybridization, we found that the levels of Agtr2 mRNAs in GABAergic NTS neurons rise during experimentally induced hypertension, and we hypothesized that this increased expression may be exploited to ameliorate the disease. Consistent with this, final experiments revealed that central administration of C21 attenuates hypertension, an effect that is abolished in mice lacking AT2R in GABAergic NTS neurons. CONCLUSION: These studies unveil novel hindbrain circuits that maintain arterial blood pressure, and reveal a specific population of AT2R that can be engaged to alleviate hypertension. The implication is that these discrete receptors may serve as an access point for activating an endogenous depressor circuit.

Correcting the imbalanced protective RAS in COVID-19 with angiotensin AT2-receptor agonists.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is responsible for the global corona virus disease 2019 (COVID-19) pandemic enters host cells via a mechanism that includes binding to angiotensin converting enzyme (ACE) 2 (ACE2). Membrane-bound ACE2 is depleted as a result of this entry mechanism. The consequence is that the protective renin-angiotensin system (RAS), of which ACE2 is an essential component, is compromised through lack of production of the protective peptides angiotensin-(1-7) and angiotensin-(1-9), and therefore decreased stimulation of Mas (receptor Mas) and angiotensin AT2-receptors (AT2Rs), while angiotensin AT1-receptors (AT1Rs) are overstimulated due to less degradation of angiotensin II (Ang II) by ACE2. The protective RAS has numerous beneficial actions, including anti-inflammatory, anti-coagulative, anti-fibrotic effects along with endothelial and neural protection; opposite to the deleterious effects caused by heightened stimulation of angiotensin AT1R. Given that patients with severe COVID-19 exhibit an excessive immune response, endothelial dysfunction, increased clotting, thromboses and stroke, enhancing the activity of the protective RAS is likely beneficial. In this article, we discuss the evidence for a dysfunctional protective RAS in COVID and develop a rationale that the protective RAS imbalance in COVID-19 may be corrected by using AT2R agonists. We further review preclinical studies with AT2R agonists which suggest that AT2R stimulation may be therapeutically effective to treat COVID-19-induced disorders of various organ systems such as lung, vasculature, or the brain. Finally, we provide information on the design of a clinical trial in which patients with COVID-19 were treated with the AT2R agonist Compound 21 (C21). This trial has been completed, but results have not yet been reported.

Brain Angiotensin Type-1 and Type-2 Receptors in Physiological and Hypertensive Conditions: Focus on Neuroinflammation.

PURPOSE OF REVIEW: To review recent data that suggest opposing effects of brain angiotensin type-1 (AT1R) and type-2 (AT2R) receptors on blood pressure (BP). Here, we discuss recent studies that suggest pro-hypertensive and pro-inflammatory actions of AT1R and anti-hypertensive and anti-inflammatory actions of AT2R. Further, we propose mechanisms for the interplay between brain angiotensin receptors and neuroinflammation in hypertension. RECENT FINDINGS: The renin-angiotensin system (RAS) plays an important role in regulating cardiovascular physiology. This includes brain AT1R and AT2R, both of which are expressed in or adjacent to brain regions that control BP. Activation of AT1R within those brain regions mediate increases in BP and cause neuroinflammation, which augments the BP increase in hypertension. The fact that AT1R and AT2R have opposing actions on BP suggests that AT1R and AT2R may have similar opposing actions on neuroinflammation. However, the mechanisms by which brain AT1R and AT2R mediate neuroinflammatory responses remain unclear. The interplay between brain angiotensin receptor subtypes and neuroinflammation exacerbates or protects against hypertension.

The Renin-Angiotensin System in Hypertension, a Constantly Renewing Classic: Focus on the Angiotensin AT2-Receptor.

It is common knowledge that the renin-angiotensin system (RAS), in particular angiotensin II acting through the angiotensin AT1-receptor (AT1R), is pivotal for the regulation of blood pressure (BP) and extracellular volume. More recent findings have revealed that the RAS is far more complex than initially thought and that it harbours additional mediators and receptors, which are able to counteract and thereby fine-tune AT1R-mediated actions. This review will focus on the angiotensin AT2-receptor (AT2R), which is one of the "counter-regulatory" receptors within the RAS. It will review and discuss data related to the role of the AT2R in regulation of BP and focus on the following 3 questions: Do peripheral AT2R have an impact on BP regulation, and, if so, does this effect become apparent only under certain conditions? Are central nervous system AT2R involved in regulation of BP, and, if so, which brain areas are involved and what are the mechanisms? Does dysfunction of AT2R contribute to the pathogenesis of hypertension in preeclampsia?

Angiotensin Type 2 Receptors: Painful, or Not?

Pain in response to various types of acute injury can be a protective stimulus to prevent the organism from using the injured part and allow tissue repair and healing. On the other hand, neuropathic pain, defined as 'pain caused by a lesion or disease of the somatosensory nervous system', is a debilitating pathology. The TRPA1 neurons in the Dorsal Root Ganglion (DRG) respond to reactive oxygen species (ROS) and induce pain. In acute nerve injury and inflammation, macrophages infiltrating the site of injury undergo an oxidative burst, and generate ROS that promote tissue repair and induce pain via TRPA1. The latter discourages using the injured limb, with a lack of movement helping wound healing. In chronic inflammation caused by diabetes, cancer etc., ROS levels increase systemically and modulate TRPA1 neuronal functions and cause debilitating neuropathic pain. It is important to distinguish between drug targets that elicit protective vs. debilitating pain when developing effective drugs for neuropathic pain. In this context, the connection of the Angiotensin type 2 receptor (AT2R) to neuropathic pain presents an interesting dilemma. Several lines of evidence show that AT2R activation promotes anti-inflammatory and anti-nociceptive signaling, tissue repair, and suppresses ROS in chronic inflammatory models. Conversely, some studies suggest that AT2R antagonists are anti-nociceptive and therefore AT2R is a drug target for neuropathic pain. However, AT2R expression in nociceptive neurons is lacking, indicating that neuronal AT2R is not involved in neuropathic pain. It is also important to consider that Novartis terminated their phase II clinical trial (EMPHENE) to validate that AT2R antagonist EMA401 mitigates post-herpetic neuralgia. This trial, conducted in Australia, United Kingdom, and a number of European and Asian countries in 2019, was discontinued due to pre-clinical drug toxicity data. Moreover, early data from the trial did not show statistically significant positive outcomes. These facts suggest that may AT2R not be the proper drug target for neuropathic pain in humans and its inhibition can be harmful.

Brain angiotensin type-1 and type-2 receptors: cellular locations under normal and hypertensive conditions.

Brain angiotensin-II (Ang-II) type-1 receptors (AT1Rs), which exert profound effects on normal cardiovascular, fluid, and metabolic homeostasis, are overactivated in and contribute to chronic sympathoexcitation and hypertension. Accumulating evidence indicates that the activation of Ang-II type-2 receptors (AT2Rs) in the brain exerts effects that are opposite to those of AT1Rs, lowering blood pressure, and reducing hypertension. Thus, it would be interesting to understand the relative cellular localization of AT1R and AT2R in the brain under normal conditions and whether this localization changes during hypertension. Here, we developed a novel AT1aR-tdTomato reporter mouse strain in which the location of brain AT1aR was largely consistent with that determined in the previous studies. This AT1aR-tdTomato reporter mouse strain was crossed with our previously described AT2R-eGFP reporter mouse strain to yield a novel dual AT1aR/AT2R reporter mouse strain, which allowed us to determine that AT1aR and AT2R are primarily localized to different populations of neurons in brain regions controlling cardiovascular, fluid, and metabolic homeostasis. Using the individual AT1aR-tdTomato reporter mice, we also demonstrated that during hypertension induced by the administration of deoxycorticosterone acetate-salt, there was no shift in the expression of AT1aR from neurons to microglia or astrocytes in the paraventricular nucleus, a brain area important for sympathetic regulation. Using AT2R-eGFP reporter mice under similar hypertensive conditions, we demonstrated that the same was true of AT2R expression in the nucleus of the solitary tract (NTS), an area critical for baroreflex control. Collectively, these findings provided a novel means to assess the colocalization of AT1R and AT2R in the brain and a novel view of their cellular localization in hypertension.

Angiotensin receptor expression revealed by reporter mice and beneficial effects of AT2R agonist in retinal cells.

The renin-angiotensin system (RAS) plays a vital role in cardiovascular physiology and body homeostasis. In addition to circulating RAS, a local RAS exists in the retina. Dysfunction of local RAS, resulting in increased levels of Angiotensin II (Ang II) and activation of AT1R-mediated signaling pathways, contributes to tissue pathophysiology and end-organ damage. Activation of AT2R on other hand is known to counteract the effects of AT1R activation and produce anti-inflammatory and anti-oxidative effects. We examined the expression of angiotensin receptors in the retina by using transgenic dual reporter mice and by real-time RT-PCR. We further evaluated the effects of C21, a selective agonist of AT2R, in reducing Ang II, lipopolysaccharide (LPS) and hydrogen peroxide induced oxidative stress and inflammatory responses in cultured human ARPE-19 cells. We showed that both AT1Ra and AT2R positive cells are detected in different cell types of the eye, including the RPE/choroid complex, ciliary body/iris, and neural retina. AT1Ra is more abundantly expressed than AT2R in mouse retina, consistent with previous reports. In the neural retina, AT1Ra are also detected in photoreceptors whereas AT2R are mostly expressed in the inner retinal neurons and RGCs. In cultured human RPE cells, activation of AT2R with C21 significantly blocked Ang II, LPS and hydrogen peroxide -induced NF-κB activation and inflammatory cytokine expression; Ang II and hydrogen peroxide-induced reactive oxygen species (ROS) production and MG132-induced apoptosis, comparable to the effects of Angiotensin-(1-7) (Ang-(1-7)), another protective component of the RAS, although C21 is more potent in reducing some of the effects induced by Ang II, whereas Ang-(1-7) is more effective in reducing some of the LPS and hydrogen peroxide-induced effects. These results suggest that activation of AT2R may represent a new therapeutic approach for retinal diseases.

Impaired Autonomic Nervous System-Microbiome Circuit in Hypertension.

Hypertension affects an estimated 103 million Americans, yet gaps in knowledge continue to limit its successful management. Rapidly emerging evidence is linking gut dysbiosis to many disorders and diseases including hypertension. The evolution of the -omics techniques has allowed determination of the abundance and potential function of gut bacterial species by next-generation bacterial sequencing, whereas metabolomics techniques report shifts in bacterial metabolites in the systemic circulation of hypertensive patients and rodent models of hypertension. The gut microbiome and host have evolved to exist in balance and cooperation, and there is extensive crosstalk between the 2 to maintain this balance, including during regulation of blood pressure. However, an understanding of the mechanisms of dysfunctional host-microbiome interactions in hypertension is still lacking. Here, we synthesize some of our recent data with published reports and present concepts and a rationale for our emerging hypothesis of a dysfunctional gut-brain axis in hypertension. Hopefully, this new information will improve the understanding of hypertension and help to address some of these knowledge gaps.

Anti-fibrotic mechanisms of angiotensin AT2 -receptor stimulation.

The angiotensin AT2 -receptor is a main receptor of the protective arm of the renin-angiotensin system. Understanding of this unconventional G-protein coupled receptor has significantly advanced during the past decade, largely because of the availability of a selective non-peptide AT2 -receptor agonist, which allowed the conduct of a multitude of studies in animal disease models. This article reviews such preclinical studies that in their entirety provide strong evidence for an anti-fibrotic effect mediated by activation of the AT2 -receptor. Prevention of the development of fibrosis by AT2 -receptor stimulation has been demonstrated in lungs, heart, blood vessels, kidney, pancreas and skin. In lungs, AT2 -receptor stimulation was even able to reverse existing fibrosis. The article further discusses intracellular signalling mechanisms mediating the AT2 -receptor-coupled anti-fibrotic effect, including activation of phosphatases and subsequent interference with pro-fibrotic signalling pathways, induction of matrix-metalloproteinases and hetero-dimerization with the AT1 -receptor, the TGF-ßRII-receptor or the RXFP1-receptor for relaxin. Knowledge of the anti-fibrotic effects of the AT2 -receptor is of particular relevance because drugs targeting this receptor have entered clinical development for indications involving fibrotic diseases.

Microglial Cells Impact Gut Microbiota and Gut Pathology in Angiotensin II-Induced Hypertension.

RATIONALE: Increased microglial activation and neuroinflammation within autonomic brain regions have been implicated in sustained hypertension, and their inhibition by minocycline-an anti-inflammatory antibiotic-produces beneficial effects. These observations led us to propose a dysfunctional brain-gut communication hypothesis for hypertension. However, it has been difficult to reconcile whether an anti-inflammatory or antimicrobial action is the primary beneficial effect of minocycline in hypertension. Accordingly, we utilized chemically modified tetracycline-3 (CMT-3)-a derivative of tetracycline that has potent anti-inflammatory activity-to address this question. OBJECTIVE: Test the hypothesis that central administration of CMT-3 would inhibit microglial activation, attenuate neuroinflammation, alter selective gut microbial communities, protect the gut wall from developing hypertension-associated pathology, and attenuate hypertension. METHODS AND RESULTS: Rats were implanted with radiotelemetry devices for recording mean arterial pressure. Ang II (angiotensin II) was infused subcutaneously using osmotic mini-pumps to induce hypertension. Another osmotic mini-pump was surgically implanted to infuse CMT-3 intracerebroventricularly. Intracerebroventricular CMT- 3 infusion was also investigated in SHR (spontaneously hypertensive rats). Physiological, pathological, immunohistological parameters, and fecal microbiota were analyzed. Intracerebroventricular CMT-3 significantly inhibited Ang II-induced increases in number of microglia, their activation, and proinflammatory cytokines in the paraventricular nucleus of hypothalamus. Further, intracerebroventricular CMT-3 attenuated increased mean arterial pressure, normalized sympathetic activity, and left ventricular hypertrophy in Ang II rats, as well as in the SHR. Finally, CMT-3 beneficially restored certain gut microbial communities altered by Ang II and attenuated pathological alterations in gut wall. CONCLUSIONS: These observations demonstrate that inhibition of microglial activation alone was sufficient to induce significant antihypertensive effects. This was associated with unique changes in gut microbial communities and profound attenuation of gut pathology. They suggest, for the first time, a link between microglia and certain microbial communities that may have implications for treatment of hypertension.

Importance of AT1 and AT2 receptors in the nucleus of the solitary tract in cardiovascular responses induced by a high-fat diet.

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

Butyrate regulates inflammatory cytokine expression without affecting oxidative respiration in primary astrocytes from spontaneously hypertensive rats.

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

ACE2 activator diminazene aceturate reduces adiposity but preserves lean mass in young and old rats.

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