Angiotensin AT1 Research Articles

Angiotensin AT2 Receptor is Anti-inflammatory and Reno-Protective in Lipopolysaccharide Mice Model: Role of IL-10.

Acute kidney injury (AKI) due to endotoxemic insult is predicted by the infiltration of neutrophils, monocytes and macrophages, and the release of pro-and anti-inflammatory cytokines to the site of injury. Earlier, we have demonstrated the role of angiotensin-II type 2 receptor (AT2R) stimulation in reno-protection in lipopolysaccharide (LPS)-induced inflammation and AKI in C57BL6/NHsd mice. Moreover, AT2R activation has been shown to increase the anti-inflammatory cytokine interleukin-10 (IL-10), its role in AT2R-mediated anti-inflammation and reno-protection is unknown. To address this question, in the present study mice were treated with the AT2R agonist C21 (0.3 mg/kg, intraperitoneally), LPS (5 mg/kg, intraperitoneally), or LPS with C21 pre-treatment with or without neutralizing IL-10 antibody. Treatment with C21 alone caused an increase in the plasma and kidney IL-10 levels, which peaks at 2-h, and returned to baseline at 6-h. The C21-induced IL-10 increase was blocked by the AT2R antagonist PD123319 suggesting AT2R’s involvement. LPS treatment caused a profound increase in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and the LPS-induced increase in these cytokines was attenuated by the C21 pre-treatment (1-h prior LPS) both in the plasma and kidney. Neutralizing IL-10 antibody treatment abrogated the C21-lowering of TNF-α and IL-6 in the kidney but not in the plasma. Similar results as related to the cytokines profiles in all the groups were also observed in the heart and spleen. The alteration in early cytokine profile prompted us to measure the markers of renal function (blood urea nitogen and urinary creatinine) in order to analyze the effect of IL-10 neutralization. However, it was too early to observe changes in renal function. Therefore, the renal function and injury markers were again measured at 24 h. Treatment with neutralizing IL-10 antibody attenuated the C21-mediated improvement in indices of the kidney function, but not the biomarkers of renal injury (kidney injury molecule-1 and neutrophil-gelatinase associated lipocalin). Collectively, our data suggest that the involvement of IL-10 in AT2R-mediated anti-inflammation and reno-protection against LPS is complex, mediating the renal cytokine profile and kidney filtration function, but not the plasma cytokine profile and renal injury markers.

ANGIOTENSIN AT2 RECEPTORS IN THE NUCLEUS OF THE SOLITARY TRACT REDUCE BLOOD PRESSURE VIA INHIBITION OF GABA SIGNALING

Objective: Sustained CNS administration of angiotensin AT2 receptor (AT2R) agonists lowers blood pressure (BP) in normal and hypertensive rodents, but the location(s) and mechanism(s) of action are unknown. One locus may be the intermediate solitary tract nucleus (intNTS), which contains a high density of AT2R located on GABA neurons, and it is well-known that increased GABA signaling in the intNTS contributes to increased BP and hypertension. Our studies have revealed that reductions in BP (∼8 mmHg) induced by 2-week intracerebroventricular (icv) infusion of the AT2R agonist C21 (7.5 ng/kg/h) in normotensive mice is associated with significant decreases in gene expression of GABA synthetic enzymes (GAD-1 & GAD-2) in the intNTS. From this, we developed the hypothesis that the depressor action of C21 is mediated through AT2R located on intNTS GABA neurons and a reduction of GABA signaling. Design and method: To test this, we engineered a novel mouse line that has Cre-recombinase expression directed to the AT2R gene (AT2R-Cre), and demonstrated that Cre recombinase activity mirrors AT2R mRNA expression within the NTS. Next, we used the Cre-LoxP system and virally (AAV)-mediated gene transfer to direct expression of the light-sensitive excitatory opsin channelrhodopsin-2 (ChR2) and/or eYFP specifically to neurons in the NTS that synthesize AT2R. Using these mice, we performed cardiovascular recordings during optogenetic manipulations (blue light) that selectively excite AT2R-expressing neurons in the intNTS. Results: These studies revealed that: (i) Optogenetic stimulation of the AT2R intNTS neurons expressing ChR2 in isoflurane anesthetized mice elicited significant increases in BP compared with control mice that express only eYFP; (ii) The AT2R-containing neurons that were activated by blue light stimulation are GABAergic, as indicated by increased expression and co-localization of c-Fos mRNA with GAD-1 mRNA and eYFP; (iii) Chronic icv infusion of C21 as above significantly blunted the light-induced increases in BP, an effect that was present across all patterns of optical stimulation. Conclusions: Collectively, these data indicate that AT2R-mediated decreases in BP involve a blunting of GABA signaling in the intNTS, and suggest that neurons expressing AT2R at this site may be an access point for cardiovascular control.

COMPOUND 21 IMPROVES VASCULAR FUNTION IN THE THORACIC AORTA FROM OBESE MICE: IMPLICATION OF AT2, MAS AND B2 RECEPTORS

Objective: The angiotensin AT2 Receptor (AT2R) is known to form heterodimers with either the Mas (MasR) or the bradykinin B2 receptor (B2R). Compound 21 (C21), a highly selective AT2R agonist, induces vascular relaxation through stimulation of nitric oxide (NO). Since AT2R is overexpressed in obese models, we hypothesize that C21 might prevent the development of vascular alterations associated with obesity. The aim of this study was to assess the effect of C21 on endothelial function in a model of diet-induced obesity and the potential crosstalk between AT2R, MasR and/or B2R in this response. Design and method: Eleven-week-old male C57BL6J mice (n = 20/group) were fed a standard (Chow) or a very high-fat diet (HF) for 6 weeks. Half of mice per group were simultaneously treated with C21 (1 mg/kg/day in drinking water, Chow-C21, HF-C21). Vascular reactivity experiments were performed in rings from thoracic aorta. NO production was determined in human endothelial cells (EA.hy926) using DAF-2DA (10-5 M). Results: Thoracic aortas from HF mice exhibited impaired relaxations to acetylcholine (Ach) compared to the Chow group. This alteration was prevented by treatment with C21 [Ach, ChowEmax = 85,6 ± 1,7%; HFEmax = 63,8 ± 3,0% vs HF-C21Emax = 86,0 ± 1,8%, P < 0.001]. In addition, NO availability calculated from the area under the Ach curve in presence and absence of L-NAME (NO synthase inhibitor) was significantly lower in HF mice and increased by C21 treatment [Chow = 237 arbitrary units (AU), HF = 168 AU vs HF-C21 = 239 AU; P < 0.001]. Aortic rings from HF mice exhibited stronger contractions to angiotensin II (Ang II, 10-9-10-6 M). PD123177 (AT2R antagonist, 10-7 M), A779 (MasR antagonist, 10-6 M) or HOE-140 (B2R antagonist, 10-7 M) significantly enhanced Ang II-induced contractions in Chow- but not in HF-rings. HF-C21 aortas exhibited similar responses to Chow rings. In EA.hy926 cells, C21 (10-6 M) significantly increased levels of phosphorylated eNOS and NO production. Both effects were abolished by PD123177, A779 or HOE-140, respectively. Conclusions: In conclusion, our data suggest that C21 improves endothelial dysfunction induced by HF-feeding due to an increase in endothelial NO availability via AT2R activation. In addition, both MasR and B2R are implicated in this response and seem necessary for mediating C21 effects.

Novel Interactions Involving the Mas Receptor Show Potential of the Renin-Angiotensin system in the Regulation of Microglia Activation: Altered Expression in Parkinsonism and Dyskinesia.

The renin–angiotensin system (RAS) not only plays an important role in controlling blood pressure but also participates in almost every process to maintain homeostasis in mammals. Interest has recently increased because SARS viruses use one RAS component (ACE2) as a target-cell receptor. The occurrence of RAS in the basal ganglia suggests that the system may be targeted to improve the therapy of neurodegenerative diseases. RAS-related data led to the hypothesis that RAS receptors may interact with each other. The aim of this paper was to find heteromers formed by Mas and angiotensin receptors and to address their functionality in neurons and microglia. Novel interactions were discovered by using resonance energy transfer techniques. The functionality of individual and interacting receptors was assayed by measuring levels of the second messengers cAMP and Ca2+ in transfected human embryonic kidney cells (HEK-293T) and primary cultures of striatal cells. Receptor complex expression was assayed by in situ proximity ligation assay. Functionality and expression were assayed in parallel in primary cultures of microglia treated or not with lipopolysaccharide and interferon-γ (IFN-γ). The proximity ligation assay was used to assess heteromer expression in parkinsonian and dyskinetic conditions. Complexes formed by Mas and the angiotensin AT1 or AT2 receptors were identified in both a heterologous expression system and in neural primary cultures. In the heterologous system, we showed that the three receptors—MasR, AT1R, and AT2R—can interact to form heterotrimers. The expression of receptor dimers (AT1R-MasR or AT2R-MasR) was higher in microglia than in neurons and was differentially affected upon microglial activation with lipopolysaccharide and IFN-γ. In all cases, agonist-induced signaling was reduced upon coactivation, and in some cases just by coexpression. Also, the blockade of signaling of two receptors in a complex by the action of a given (selective) receptor antagonist (cross-antagonism) was often observed. Differential expression of the complexes was observed in the striatum under parkinsonian conditions and especially in animals rendered dyskinetic by levodopa treatment. The negative modulation of calcium mobilization (mediated by AT1R activation), the multiplicity of possibilities on RAS affecting the MAPK pathway, and the disbalanced expression of heteromers in dyskinesia yield new insight into the operation of the RAS system, how it becomes unbalanced, and how a disbalanced RAS can be rebalanced. Furthermore, RAS components in activated microglia warrant attention in drug-development approaches to address neurodegeneration.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00986-4.

Interference with the renin-angiotensin system reduces the palatability of 0.3 M NaCl in sodium-deplete rats

The renin-angiotensin system (RAS) controls hypertonic NaCl intake driven by sodium appetite. Here we investigated whether the antagonism of RAS interferes with hedonic and aversive orofacial motor responses, or palatability, to intraoral infusion of 0.3 M NaCl (hNaCl). Adult rats were depleted of sodium by combined sc injection of furosemide and 24 h removal of ambient sodium. In experiment 1, losartan (AT1 angiotensin II receptor antagonist, intracerebroventricular, 200 μg/μl), produced a three-fold increase in aversive orofacial motor responses to hNaCl. Losartan also suppressed hNaCl intake recorded immediately thereafter. In experiment 2, each animal had repeated recordings of hNaCl intake and orofacial responses to hNaCl distributed for 180 min. Paired recordings of intake and orofacial responses occurred within five successive blocks after the recordings of only orofacial responses when the animals were still sodium deplete (block zero). Captopril (angiotensin converting enzyme blocker, intraperitoneal, 30 mg/kg) inhibited by 75% the hedonic orofacial responses to hNaCl in blocks zero and 1. The hedonic responses to captopril remained the same throughout blocks, but became similar to vehicle from blocks 2 to 5. There was no difference in aversive responses to 0.3 M NaCl between captopril and vehicle. Captopril produced a 70–100% inhibition of hNaCl intake in blocks 1 to 5. The results suggest that angiotensin II acts in the brain increasing the palatability of hypertonic sodium during the consummatory phase of sodium appetite.

The role of the renin-angiotensin system in skin physiology and pathophysiology.

Since its first description around the year 2000, the local renin-angiotensin system (RAS) in skin has been subject of an increasing number of studies with many additions over the last two to three years. A focus of research has been investigations on the role of cutaneous angiotensin receptors and locally synthesised angiotensin II in wound healing, in dermatoses associated with skin fibrosis and in melanoma. This review will provide an introduction into the RAS with emphasis on information relevant for the cutaneous RAS. It will discuss the role of the RAS in skin physiology, followed by a detailed review of the existing literature addressing the role of local angiotensin II and angiotensin AT1 and AT2 receptors in wound healing and in various skin diseases such as hypertrophic scars/keloids, scleroderma, dystrophic epidermolysis bullosa, Dupuytren's disease, squamous cell carcinoma, melanoma and psoriasis. In a final section, the potential relevance of drugs, which interfere with the RAS, for future therapy of dermatological disorders is discussed. Collectively, research about the RAS in skin can currently be described as an area, which has gained increasing attention by basic researchers, thus resulting in a multitude of preclinical studies pointing to the potential relevance of components of the RAS as drug targets in dermatological diseases. With a few small clinical studies already performed successfully for indications such as hypertrophic scars and keloids, it can be said that the skin RAS is now in the critical phase of translation from preclinical evidence to clinical use.

Angiotensin AT1 and AT2 receptor heteromer expression in the hemilesioned rat model of Parkinson\u2019s disease that increases with levodopa-induced dyskinesia

Background/aimsThe renin-angiotensin system (RAS) is altered in Parkinson’s disease (PD), a disease due to substantia nigra neurodegeneration and whose dopamine-replacement therapy, using the precursor levodopa, leads to dyskinesias as the main side effect. Angiotensin AT1 and AT2 receptors, mainly known for their role in regulating water homeostasis and blood pressure and able to form heterodimers (AT1/2Hets), are present in the central nervous system. We assessed the functionality and expression of AT1/2Hets in Parkinson disease (PD).MethodsImmunocytochemistry was used to analyze the colocalization between angiotensin receptors; bioluminescence resonance energy transfer was used to detect AT1/2Hets. Calcium and cAMP determination, MAPK activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify receptor expression in mouse primary cultures and in rat striatal sections.ResultsWe confirmed that AT1 and AT2 receptors form AT1/2Hets that are expressed in cells of the central nervous system. AT1/2Hets are novel functional units with particular signaling properties. Importantly, the coactivation of the two receptors in the heteromer reduces the signaling output of angiotensin. Remarkably, AT1/2Hets that are expressed in both striatal neurons and microglia make possible that candesartan, the antagonist of AT1, increases the effect of AT2 receptor agonists. In addition, the level of striatal expression increased in the unilateral 6-OH-dopamine lesioned rat PD model and was markedly higher in parkinsonian-like animals that did not become dyskinetic upon levodopa chronic administration if compared with expression in those that became dyskinetic.ConclusionThe results indicate that boosting the action of neuroprotective AT2 receptors using an AT1 receptor antagonist constitutes a promising therapeutic strategy in PD.

The renin-angiotensin system in cutaneous hypertrophic scar and keloid formation.

Hypertrophic scar and keloid are two types of fibroproliferative conditions that result from excessive extracellular matrix production. The underlying pathological mechanism is not entirely clear. Activation of the renin-angiotensin system (RAS) is associated with fibrosis in various organs. RAS components including angiotensin II (Ang II), angiotensin AT1 and AT2 receptors, and angiotensin-converting enzyme (ACE) are expressed in the skin and act independently from the plasma RAS. AT1 receptors, which are usually the dominating receptor subtype, promote fibrosis and scar formation, while AT2 receptors inhibit the aforementioned AT1 receptor-coupled effects. Elevated angiotensin II (Ang II) levels acting on the AT1 receptor contribute to skin scar formation through increased expression of inflammatory factors such as interleukin-6 (IL-6), angiogenic factors such as vascular endothelial growth factor (VEGF) and fibrinogenic factors such as transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), while at the same time suppressing the anti-fibrotic tissue inhibitors of matrix metalloproteinase (TIMPs). First, small clinical trials have provided evidence that inhibition of the ACE/Ang II/ AT1 receptor axis may be effective in the treatment of hypertrophic scars/keloids. This review provides a detailed overview of the current literature on the RAS in skin, wound healing and scar formation and discusses the translational potential of targeting this hormonal system for treatment and prevention of hypertrophic scars and keloids.

Renin angiotensin system inhibition attenuates adipocyte-breast cancer cell interactions

Obesity is a significant breast cancer (BC) risk factor and is associated with 20–40% increased risk in obese post-menopausal women compared to their lean counterparts. Several obesity-related metabolic dysregulations have been linked to BC risk, including overactivation of the renin-angiotensin system (RAS). Currently, RAS inhibitors including angiotensin converting enzyme inhibitor (ACEi) and AT1 receptor blockers (ARBs), are used as safe and effective anti-hypertensive therapies in BC patients. However, it is uncertain how inhibition of RAS in adipose tissue impacts obesity-BC crosstalk. We hypothesized that adipose RAS inhibition will reduce BC cell motility and inflammation. We determined (1) the direct effects of Ang II, ACEi (captopril; Cap) or ARB (telmisartan; Tel) on receptor positive MCF-7 and receptor triple negative MDA-MB-231 cells; and (2) the effects of conditioned media (CM) from human mesenchymal stem cells differentiated into adipocytes, which were pretreated with RAS inhibitors, on BC cells. We demonstrated that direct treatments of BC cells with Ang II, Cap or Tel did not alter inflammatory cytokines in either BC cell line. However, CM from Ang II-pretreated adipocytes significantly increased secretion of pro-inflammatory markers at protein level. RAS inhibitors reduced their secretion in MDA-MB-231, but not in MCF-7 cells. Additionally, CM from adipocytes treated with RAS inhibitors significantly reduced markers of inflammation, fat synthesis, and angiogenesis in both BC cell lines. Furthermore, CM from ACEi pretreated adipocytes reduced cell motility in both BC cell lines. Findings from our study indicate an important role of adipose RAS inhibition in adipocyte and BC cell crosstalk.

In silico studies on therapeutic agents for COVID-19: Drug repurposing approach

AimsThe severe acute respiratory syndrome coronavirus 2, better known as COVID-19 has become the current health concern to the entire world. Initially appeared in Wuhan, China around December 2019, it had spread to almost 187 countries due to its high contagious nature. Precautionary measures remain the sole obliging tactic to cease the person to person transmissions till any effective method of treatment or vaccine is developed. Amidst the pandemic, research and development of new molecule is labour-intensive and tedious process. Drug repurposing is the concept of identifying therapeutically potent molecule from the library of pre-existing molecules. Materials and methodsIn the present study, 61 molecules that are already being used in clinics or under clinical scrutiny as antiviral agents are surveyed via docking study. Docking study was performed using Maestro interface (Schrödinger Suite, LLC, NY). Key findingsOut of these 61 molecules, 37 molecules were found to interact with >2 protein structures of COVID-19. The docking results indicate that amongst the reported molecules, HIV protease inhibitors and RNA-dependent RNA polymerase inhibitors showed promising features of binding to COVID-19 enzyme. Along with these, Methisazone an inhibitor of protein synthesis, CGP42112A an angiotensin AT2 receptor agonist and ABT450 an inhibitor of the non-structural protein 3-4A might become convenient treatment option as well against COVID-19. SignificanceThe drug repurposing approach provide an insight about the therapeutics that might be helpful in treating corona virus disease.

Potential harmful effects of discontinuing ACE-inhibitors and ARBs in COVID-19 patients.

The discovery of angiotensin converting enzyme-2 (ACE-2) as the receptor for SARS- CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) has implicated the renin-angiotensin-aldosterone system in acute respiratory distress syndrome (ARDS) and respiratory failure in patients with coronavirus disease-19 (COVID-19). The angiotensin converting enzyme-1-angiotensin II-angiotensin AT1 receptor pathway contributes to the pathophysiology of ARDS, whereas activation of the ACE-2-angiotensin(1-7)-angiotensin AT2 receptor and the ACE-2-angiotensin(1-7)-Mas receptor pathways have been shown to be protective. Here we propose and discuss therapeutic considerations how to increase soluble ACE-2 in plasma in order for ACE-2 to capture and thereby inactivate SARS-CoV-2. This could be achieved by administering recombinant soluble ACE-2. We also discuss why and how ACEIs and ARBs provide cardiovascular, renal and also pulmonary protection in SARS-CoV-2- associated ARDS. Discontinuing these medications in COVID-19 patients may therefore potentially be harmful.

Substituting Angiotensin-(1-7) to Prevent Lung Damage in SARS-CoV-2 Infection?

Substituting Angiotensin-(1-7) to Prevent Lung Damage in SARS-CoV-2 Infection?

Effects of adjuvant-induced arthritis on the ventral prostate of rats treated with angiotensin AT1 receptor blocker

AimsTo analyze the prostatic compartments, extracellular matrix, microvascularization, transforming growth factor-beta (TGF-β) and angiotensin II receptors type 1 (AT1) levels, and histopathology of the ventral prostate in a rat model for rheumatoid arthritis, and to evaluate the effect of angiotensin II AT1 receptor blocker (ARB) in the disease. Main methodsFifteen male rats (90 days old) were divided into three groups (n = 5/group): control, adjuvant-induced arthritis without (AIA) or with AT1 receptor blocker (AIA + ARB). Animals were euthanized 60 days after immunization. The ventral prostate was collected, weighed, and processed for histological and immunohistochemical analysis. Key findingsOur results show that AIA increases production of the prostatic epithelium and extracellular matrix, accompanied by a reduction in the number of tissue capillaries. ARB treatment promotes decreased production of extracellular matrix and increased TGF-β and AT1 receptor immunostaining. SignificanceAIA may activate specific mechanisms that modify the prostatic environment; the use of ARB attenuates some altered prostate parameters in a rat model for arthritis.

Synergism between Angiotensin AT1 and AT2 Receptors function: Role of Angiotensin‐(1‐7) via AT1R in Modulating AT2R Agonist Response on Nitric Oxide in Kidney Cells

Angiotensin‐(1‐7) is evidently considered as an endogenous agonist for Mas receptor. Our recent studies show that MasR and AT2R physically interact and in response to their respective agonists Ang‐(1‐7) and C21 produce interdependent functional response in terms of nitric oxide (NO) generation and natriuresis. Moreover, recent studies suggest that Ang‐(1‐7) is cardio‐protective via AT1R stimulation. Considering the complex nature of Ang‐(1‐7) function, we tested as to how Ang‐(1‐7) affects AT2R function utilizing human kidney proximal tubule epithelial cells (HK‐2 cells). The agonists of MasR [(Ang‐(1‐7), AVE0991] or AT2R (C21), alone significantly stimulated NO formation at 10−6–10−5M (50–300%), but not at lower concentrations (10−12‐10−7M). Contrarily, Ang‐II alone reduced NO formation. In the interaction studies, the non‐responsive concentrations of Ang‐(1‐7) (10−12‐10−7M) synergized NO formation of C21 (10−12‐10−9M) or ang‐II (10−12‐10−9M) in HK‐2 cells (p<0.05 vs. either agonist alone). The synergy is observed only when the Ang‐(1‐7) was added to the HK‐2 cells 10 min prior to the C21/ang‐II, not when the agonists were added simultaneously. The synergy between Ang‐(1‐7) and C21/ang‐II was sensitive to AT2R antagonist (PD123319, 10−5M) and AT1R antagonist (candesartan, 10−5M), but not MasR antagonist (A779, 10−5M). Interestingly, pre‐stimulation with synthetic non‐peptide MasR selective agonist AVE0991 at any concentrations (10−10‐10−7M) did not show synergism with the AT2R agonist C21. Collectively, results show Ang‐(1‐7) at lower concentrations via AT1R, not MasR, synergies the AT2R function of NO formation and this phenomenon may have a physiological significance.Support or Funding InformationNIH R01 DK61578 and R01 DK117495

Measuring the Kinetics of G‐protein‐coupled Receptor Signaling and Biased Agonism.

Quantifying the time course of G‐protein‐coupled receptor signaling can reveal biological properties of GPCR function such as receptor desensitization, pathway selectivity, and spatiotemporally compartmentalized signaling. Applying signaling kinetics to modern receptor research and drug discovery requires assay modalities that can monitor signaling responses over time, and data analysis methods to extract the kinetic drug parameters. Here we describe a suite of fluorescent biosensors for monitoring multiple pathways of GPCR signaling (Gs, Gi, Gq and arrestin). These biosensors are genetically‐encoded and amenable to kinetic analysis, with continuous “real time” responses detected on plate readers or imaging systems. A kinetic data analysis framework is applied to the time course data using familiar equations on commonly‐used curve‐fitting software (e.g. GraphPad Prism). The analysis yields the initial rate of signaling (kTau), a biologically‐meaningful parameter of ligand efficacy at generating the signal. Biased agonism assessment is simplified using this approach, and demonstrated using arrestin recruitment and second messenger signaling via the AT1 angiotensin and GLP‐1 receptors. Bias is quantified as the ratio of the initial rates of signaling reported by each assay. This platform provides a straightforward method that investigators can use to quantify GPCR receptor pharmacology and biased signaling with the application of kinetic analysis to understanding receptor function and to the development of new therapeutics.Support or Funding InformationThis work was supported by grants from the National Institutes of HealthR44 GM125390‐02 and R44 DA050357‐01

Anomalous AMPK‐Regulated Angiotensin AT1R Expression and SIRT1‐Mediated Mitochondrial Biogenesis at RVLM in Hypertension Programming of Offspring to Maternal High Fructose Exposure

Tissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high‐fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP‐activated protein kinase (AMPK)‐regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)‐dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring. Increased systolic and diastolic blood pressure, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in rostral ventrolateral medulla (RVLM) where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator‐activated receptor g co‐activator a (PGC‐1a) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG‐CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD‐programmed increase in AT1R and decreases in SIRT1, PGC‐1a and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension. Together these results suggest that dysfunction of AMPK‐regulated AT1R expression and SIRT1‐mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and blood pressure in young offspring programmed by excessive maternal fructose consumption.Support or Funding InformationThis work was supported by research grants from the Chang Gung Medical Foundation to J.Y.H. Chan (CMRPG8C0053‐33 and OMRPG8G0011). Y.M. Chao was supported by a Postdoctoral Fellowship (MOST107‐2811‐B‐182A‐501) from the Ministry of Science and Technology, Taiwan.

Countermeasures to the impact of cosmic radiation exposure on myocardial mitochondrial function.

Space travel increases solar particle radiation exposure to astronauts and this is significantly elevated once travel moves beyond low Earth orbit. This includes γ radiation, as well as a combinations of high energy protons and heavy ions such as 56Fe, 28Si, and 16O.There are distinct differences in the biological response to low‐energy transfer (x‐rays) or high‐energy transfer (High‐LET) (56Fe) events. Airline flight crews are chronically exposed to High‐LET and higher incidents of cancer are reported for these individuals. Much of what we know about the consequences of high‐LET exposure comes from studies of nuclear workers and atom bomb survivors. For flight crews (astronauts), most estimates of galactic radiation exposure have a low fluence rate that is much lower that patients undergoing radiotherapy or the acute exposure of those that survived the A‐bomb. However, the cumulative radiation dose is likely to have long‐term deleterious effects on the health of astronauts. And this risk increases as flight duration is increased.The underlying mechanisms for these elevated risks are unknown, although direct DNA damage and elevated oxidant stress are highly suspected. Given the relatively low fluence rate of exposure it might be possible to manage these deleterious effects using small molecules currently available. To that end we have utilized the FDA approved drug library from MedChem Express (723 drugs) to perform a high throughput screen of cultured cells following exposure to cosmic radiation. The H9c2 cell and RBL‐2H3 mast cell lines were exposed to a total of 75 cGy using a simplified 5‐ion GCR protocol developed by NASA at the NSRL facility at the Brookhaven National Laboratory. Following GCR exposure cells were split onto 96 well test plates and treated for 7 days with vehicle or 10 μM drug. Media and drugs were changed on alternate days. At harvest cells were tested for viability (MTT), oxidant stress (DHE), cellular senescence (ONPG) and mitochondrial function (ATP). Using the non‐drug treated cells as controls, Z‐scores were calculated and a composite score was developed for each drug. The top 160 composite scores were retested following a similar protocol using1 μM of each drug. Within the 160 drugs, 33 are considered to have an anti‐inflammatory capacity. 5 of the eight 5‐HT antagonists considered useful were 5‐HT3 specific. 12 of 25 angiotensin converting enzyme inhibitors or AT1 antagonists were observed to be effective while no AT2 antagonists appeared useful. 10 of 46 drugs indentified as interacting with histamine receptors were deemed effective. 7 of 54 COX inhibitors deemed effective were COX2 but not COX1 inhibitors. Collectively these findings suggest that drugs interacting with GPCR/G coupled proteins that modulate inflammation and anti‐oxidant pathways may be useful in the management of cosmic radiation exposure of long duration flight crews.Support or Funding InformationSupported in part by NASA 80NSSC19K0436

Mechanisms Involved in Angiotensin Receptor Blockade Superiority over ACE‐inhibition in Attenuating Neuropathic Pain Induced in Rats

Although, the beneficial role of angiotensin‐converting enzyme inhibitors (ACEi) or AT1 receptor blockers (ARBs) in attenuating Neuropathic pain (NP) was previously reported, the exact underlying mechanisms as well as superiority of using central versus peripheral acting Renin angiotensin aldosterone system (RAAS) drugs in NP is not yet explored. We investigated the effect of 14 days treatment of centrally (telmisartan and ramipril) or peripherally (losartan and enalapril) ARBs and ACEi, respectively in attenuating peripheral NP induced by sciatic nerve chronic constriction injury (CCI) in rats compared to pregabalin, the standard treatment for NP. Behavioral changes, inflammatory markers (nuclear factor‐kappa B (NFкβ), tumor necrosis factor (TNF)‐α release, Cyclooxygenase‐2 (COX‐2), prostaglandin E2 (PGE2) and bradykinin), oxidative stress markers (NADPH oxidase and catalase), signal transducer and activator of transcription 3 (STAT‐3) activation, levels of phosphorylated P38 mitogen activated protein kinase (P38 MAPK), angiotensin converting enzyme (ACE), AT1 receptor (AT1R) and AT2 receptor (AT2R) as well as histopathological picture were assessed in brain stem and sciatic nerve. CCI resulted in an obvious painful behavior along with increased levels of inflammatory and oxidative stress markers, STAT3 activity as well as the levels of phosphorylated P38 MAPK, ACE, AT1R and AT2R and worsened histopathological picture in both the brainstem and sciatic nerve. Treatment of CCI rats with ARBs resulted in improving in animal behavior and all measured parameters and were more effective compared to ACE inhibitors. Centrally acting ARBs or ACEi were not superior compared to their peripherally acting drugs of the same category. These findings suggest ARBs (central or peripheral) as an effective treatment modality for NP.

Angiotensin AT2 receptors in the solitary tract nucleus lower blood pressure via inhibition of GABA signaling

Chronic CNS administration of angiotensin AT2 receptor (AT2R) agonists lowers blood pressure (BP) in normal and hypertensive rodents, but the location(s) and mechanism(s) of action are unknown. One locus may be the intermediate solitary tract nucleus (intNTS). Our previous studies demonstrated a high density of AT2R located on GABA neurons in the intNTS (de Kloet et al, Brain Struct. Func., 2016, 22:891–912), and decades of research have established increased GABA signaling in the intNTS as a pathophysiological mechanism contributing to increased BP and hypertension. These studies reveal that reductions in BP (~8 mmHg) induced by 2‐week intracerebroventricular (icv) infusion of the AT2R agonist C21 (7.5 ng/kg/h) in normotensive mice is associated with significant decreases in gene expression of GABA synthetic enzymes (GAD‐1 & GAD‐2) in the intNTS. Based on this, we developed the hypothesis that the depressor action of C21 is mediated through AT2R located on intNTS GABA neurons and a reduction of GABA signaling. To test this, we engineered a novel mouse line that has Cre‐recombinase expression directed to the AT2R gene (AT2R‐Cre), and demonstrated that Cre recombinase activity mirrors AT2R mRNA expression within the NTS. Next, we used the Cre‐LoxP system and virally (AAV)‐mediated gene transfer to direct expression of the light‐sensitive excitatory opsin channelrhodopsin‐2 (ChR2) and/or eYFP specifically to neurons in the intNTS that synthesize AT2R. Using these mice, we performed electrophysiological‐ or cardiovascular recordings during optogenetic manipulations (blue light) that selectively excite AT2R‐expressing neurons in the intNTS. For the electrophysiological studies, optical stimulation produced a strong inward current in voltage clamp (611.80 ± 78.55 pA; n=9), and robust frequency dependent firing of action potentials in current‐clamp, validating functional opsin expression within AT2R‐expressing neurons. Optical activation of the Chr2/eYFP‐containing AT2R‐expressing neurons in brain slices resulted in activation of adjacent (non‐eYFP‐labeled) neurons, as indicated by light‐evoked currents (n=31), an effect blocked by GABA receptor antagonists (PTX and CGP). Cardiovascular studies revealed that (i) Optogenetic stimulation of the AT2R intNTS neurons expressing ChR2 in isoflurane anesthetized mice elicited significant elevations in BP compared with control mice that express only eYFP; (ii) The AT2R‐containing neurons that were activated by light stimulation are GABAergic, as indicated by increased expression and co‐localization of c‐Fos mRNA with GAD‐1 mRNA and eYFP; (iii) Chronic icv infusion of C21 as above significantly blunted the light‐induced increases in BP, an effect that persisted across all patterns of optical stimulation. Collectively, these data indicate that AT2R‐mediated decreases in BP involve a blunting of GABA signaling in the intNTS, and suggest that neurons expressing AT2R in the intNTS may be an access point for cardiovascular control.Support or Funding InformationNIH grants HL‐125805 (AdK), HL‐145028 (AdK), HL‐136595 (EGK/CS)

Emerging Role of Angiotensin AT2 Receptor in Anti-Inflammation: An Update.

The hyperactive RAS and inflammation are closely associated. The angiotensin-II/AT1R axis of the RAS has been explored extensively for its role in inflammation and a plethora of pathological conditions. Understanding the role of AT2R in inflammation is an emerging area of research. The AT2R is expressed on a variety of immune and non-immune cells, which upon activation triggers the release of a host of cytokines and has multiple effects that coalesce to anti-inflammation and prevents maladaptive repair. The anti-inflammatory outcomes of AT2R activation are linked to its well-established signaling pathways involving formation of nitric oxide and activation of phosphatases. Collectively, these effects promote cell survival and tissue function. The consideration of AT2R as a therapeutic target requires further investigations.