Angiotensin Type 1 Receptor Research Articles

Abstract 039: B-arrestin Defficiency In The Subfornical Organ Alters Blood Pressure Regulation

The activation of the angiotensin type 1 receptor (AT 1 R) in the brain through the canonical Gαq-mediated signaling results in dipsogenic and pressor responses to angiotensin (AngII). Non-canonical or β-arrestin-mediated signaling of AT 1 R activation is hypothesized to counterbalance maladaptive G protein signaling during disease. Recently, we found that global genetic deletion of β-arrestin2 (ARRB2) resulted in an exacerbated increases in blood pressure (BP) in response to deoxycorticosterone acetate-salt treatment, suggesting a protective role for ARRB2 in hypertension. We hypothesized that ARRB2 activation specifically within the brain contributes to BP regulation. First, global male and female ARRB2-deficient (ARRB2-KO) mice were used to evaluate acute BP responses to intracerebroventricular (ICV) infusion of AngII (1μg). Although no differences at baseline BP were observed, ARRB2-KO mice exhibited greater maximal BP after AngII (144±17 vs. 177±24 mmHg; n=14 and 19, p<0.05). Next, we examined the role of ARRB2 within the brain via ICV injection of adeno-associated virus (AAV)-CRE into ARRB2 FLOX mice. Infection with ICV AAV-CRE primarily targeted the subfornical organ with minimal off-targets. In preliminary studies, these mice exhibited higher BP in response to AngII (179±5 vs. 187±6 mmHg; n=4 p=0.09). Thus, deletion of ARRB2 within the subfornical organ exacerbates the pressor response to Ang II. Finally, we evaluated BP responses to ICV infusion of a β-arrestin biased agonist specific for the AT 1 R (TRV120027 0.5 ug/h) in spontaneously hypertensive BPH/2J and normotensive BPN/3J mice. BPH/2J mice exhibited significantly higher baseline BP compared to BPN/3J (124±5 vs. 153±3 mmHg; n=4 p<0.05), and greater reductions in BP with ICV infusion of TRV120027 (-4±3 vs. -9±1 mmHg; n=4 p<0.05), suggesting that the beneficial BP-lowering effects of β-arrestin activation are more robust during hypertension. Overall, these data indicate that ARRB2 in the subfornical organ is protective against increases in BP. Stimulation of the AT 1 R β-arrestin axis in the brain may represent a novel strategy to treat hypertension.

Sex-dependent effects of angiotensin II type 1 receptor blocker on molecular and behavioral changes induced by single prolonged stress

Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that not only entails alterations in fear behavior and anxiety but also includes neuroendocrine dysfunctions involving the hypothalamic pituitary adrenal (HPA) axis and the renin-angiotensin system. Recent preclinical studies demonstrate that activation of the angiotensin type 1 receptor (AT1R) in the paraventricular region of the hypothalamus (PVR) promotes anxiety-like behaviors and enables microglia proliferation. An increase in microglia and anxiety-like behavior also occurs in the PTSD animal model single-prolonged stress (SPS). In the present study, we tested whether AT1Rs contribute to the effects of SPS on behavior and microglia in brain structures important for HPA axis regulation and fear behavior. To test this, male and female animals were exposed to SPS and then given the oral AT1R antagonist candesartan beginning one week later. Candesartan did not alter auditory fear conditioning or extinction in SPS-exposed male or female animals. However, we found that the male animals exposed to SPS showed increased anxiety-like behavior, which was reversed by candesartan. In contrast, neither SPS nor candesartan altered anxiety-like behavior in the female animals. At the molecular level, SPS increased the cellular expression of AT1Rs in the PVR of male animals and candesartan reversed this effect, whereas AT1Rs in the PVR of females were unaltered by either SPS or candesartan. Iba1-expressing microglia increased in the PVR after SPS exposure and was reversed by candesartan in both sexes suggesting that SPS stimulates AT1Rs to increase microglia in the PVR. Collectively, these results suggest that the contribution of AT1Rs to the molecular and behavioral effects of SPS is sex-dependent.

Investigational Drugs for the Treatment of Postherpetic Neuralgia: Systematic Review of Randomized Controlled Trials.

The pharmacological treatment of postherpetic neuralgia (PHN) is unsatisfactory, and there is a clinical need for new approaches. Several drugs under advanced clinical development are addressed in this review. A systematic literature search was conducted in three electronic databases (Medline, Web of Science, Scopus) and in the ClinicalTrials.gov register from 1 January 2016 to 1 June 2023 to identify Phase II, III and IV clinical trials evaluating drugs for the treatment of PHN. A total of 18 clinical trials were selected evaluating 15 molecules with pharmacological actions on nine different molecular targets: Angiotensin Type 2 Receptor (AT2R) antagonism (olodanrigan), Voltage-Gated Calcium Channel (VGCC) α2δ subunit inhibition (crisugabalin, mirogabalin and pregabalin), Voltage-Gated Sodium Channel (VGSC) blockade (funapide and lidocaine), Cyclooxygenase-1 (COX-1) inhibition (TRK-700), Adaptor-Associated Kinase 1 (AAK1) inhibition (LX9211), Lanthionine Synthetase C-Like Protein (LANCL) activation (LAT8881), N-Methyl-D-Aspartate (NMDA) receptor antagonism (esketamine), mu opioid receptor agonism (tramadol, oxycodone and hydromorphone) and Nerve Growth Factor (NGF) inhibition (fulranumab). In brief, there are several drugs in advanced clinical development for treating PHN with some of them reporting promising results. AT2R antagonism, AAK1 inhibition, LANCL activation and NGF inhibition are considered first-in-class analgesics. Hopefully, these trials will result in a better clinical management of PHN.

Diabetes-induced stimulation of the renin-angiotensin system in the rat brain cortex

Cerebrovascular disease is a threat to people with diabetes and hypertension. Diabetes can damage the brain by stimulating the renin-angiotensin system (RAS), leading to neurological deficits and brain strokes. Diabetes-induced components of the RAS, including angiotensin-converting enzyme (ACE), angiotensin-II (Ang-II), and angiotensin type 1 receptor (AT1R), have been linked to various neurological disorders in the brain. In this study, we investigated how diabetes and high blood pressure affected the regulation of these major RAS components in the frontal cortex of the rat brain. We dissected, homogenized, and processed the brain cortex tissues of control, streptozotocin-induced diabetic, spontaneously hypertensive (SHR), and streptozotocin-induced SHR rats for biochemical and Western blot analyses. We found that systolic blood pressure was elevated in SHR rats, but there was no significant difference between SHR and diabetic-SHR rats. In contrast to SHR rats, the heartbeat of diabetic SHR rats was low. Western blot analysis showed that the frontal cortexes of the brain expressed angiotensinogen, AT1R, and MAS receptor. There were no significant differences in angiotensinogen levels across the rat groups. However, the AT1R level was increased in diabetic and hypertensive rats compared to controls, whereas the MAS receptor was downregulated (p < 0.05). These findings suggest that RAS overactivation caused by diabetes may have negative consequences for the brain's cortex, leading to neurodegeneration and cognitive impairment.

Manipulation of components of the renin angiotensin system in renal proximal tubules fails to alter atherosclerosis in hypercholesterolemic mice.

Whole body manipulation of the renin-angiotensin system (RAS) consistently exerts profound effects on experimental atherosclerosis development. A deficit in the literature has been a lack of attention to the effects of sex. Also, based on data with gene-deleted mice, the site of RAS activity that influences lesion formation is at an unknown distant location. Since angiotensin (AngII) concentrations are high in kidney and the major components of the RAS are present in renal proximal tubule cells (PTCs), this study evaluated the role of the RAS in PTCs in atherosclerosis development. Mice with an LDL receptor -/- background were fed Western diet to induce hypercholesterolemia and atherosclerosis. We first demonstrated the role of AT1 receptor antagonism on atherosclerosis in both sexes. Losartan, an AngII type 1 (AT1) receptor blocker, had greater blood pressure-lowering effects in females than males, but equivalent effects between sexes in reducing atherosclerotic lesion size. To determine the roles of renal AT1a receptor and angiotensin-converting enzyme (ACE), either component was deleted in PTCs after weaning using a tamoxifen-inducible Cre expressed under the control of an Ndrg1 promoter. Despite profound deletion of AT1a receptor or ACE in PTCs, the absence of either protein did not influence development of atherosclerosis in either sex. Conversely, mice expressing human angiotensinogen and renin in PTCs or expressing human angiotensinogen in liver but human renin in PTCs did not change atherosclerotic lesion size in male mice. Whole-body AT1R inhibition reduced atherosclerosis equivalently in both male and female mice; however, PTC-specific manipulation of the RAS components had no effects on hypercholesterolemia-induced atherosclerosis.

Possible role of LCZ696 in atherosclerosis: new inroads and perspective.

LCZ696 blocks both angiotensin receptor type 1 (ATR1) and neprilysin (NEP), which are intricate in the degradation of natriuretic peptides (NPs) and other endogenous peptides. It has been shown NEP inhibitors and LCZ696 could be effectively in the management of atherosclerosis (AS). However, the underlying mechanism of LCZ696 in AS is needed to be clarified entirely. Hence, this review is directed to reconnoiter the mechanistic role of LCZ696 in AS. The anti-inflammatory role of LCZ696 is related to the inhibition of transforming growth factor beta (TGF-β)-activated kinase 1 (TAK) and nod-like receptor pyrin 3 receptor (NLRP3) inflammasome. Moreover, LCZ696, via inhibition of pro-inflammatory cytokines, oxidative stress, apoptosis and endothelial dysfunction can attenuate the development and progression of AS. In conclusion, LCZ696 could be effective in the management of AS through modulation of inflammatory and oxidative signaling. Preclinical and clinical studies are recommended in this regard.

Nonatherosclerotic Renovascular Hypertension.

Renovascular hypertension (RVH) is a secondary form of high blood pressure resulting from impaired blood flow to the kidneys with subsequent activation of the renin-angiotensin-aldosterone system. Often, this occurs due to abnormally small, narrowed, or blocked blood vessels supplying one or both kidneys (ie: renal artery occlusive disease) and is correctable. Juxtaglomerular cells release renin in response to decreased pressure, which in turn catalyzes the cleavage of circulating angiotensinogen synthesized by the liver to the decapeptide angiotensin I. Angiotensin-converting enzyme then cleaves angiotensin I to form the octapeptide angiotensin II, a potent vasopressor and the primary effector of renin-induced hypertension. The effects of angiotensin II are mediated by signaling downstream of its receptors. Angiotensin receptor type 1 is a G-protein-coupled receptor that activates vasoconstrictor and mitogenic signaling pathways resulting in peripheral arteriolar vasoconstriction and increased renal tubular reabsorption of sodium and water which promotes intravascular volume expansion. Angiotensin II stimulates the adrenal cortical release of aldosterone, which promotes renal tubular sodium reabsorption, resulting in volume expansion. Angiotensin II acts on glial cells and regions of the brain responsible for blood pressure regulation increasing renal sympathetic activation. Angiotensin II simulates the release of vasopressin from the pituitary which stimulates thirst and water reabsorption from the kidney to expand the intravascular volume and cause peripheral vasoconstriction (increased sympathetic tone). All of these mechanisms coalesce to increase arterial pressure by way of arteriolar constriction, enhanced cardiac output, and the retention of sodium and water.

Interactions between Angiotensin Type-1 Antagonists, Statins, and ROCK Inhibitors in a Rat Model of L-DOPA-Induced Dyskinesia.

Statins have been proposed for L-DOPA-induced dyskinesia (LID) treatment. Statin anti-dyskinetic effects were related to the inhibition of the Ras-ERK pathway. However, the mechanisms responsible for the anti-LID effect are unclear. Changes in cholesterol homeostasis and oxidative stress- and inflammation-related mechanisms such as angiotensin II and Rho-kinase (ROCK) inhibition may be involved. The nigra and striatum of dyskinetic rats showed increased levels of cholesterol, ROCK, and the inflammatory marker IL-1β, which were reduced by the angiotensin type-1 receptor (AT1) antagonist candesartan, simvastatin, and the ROCK inhibitor fasudil. As observed for LID, angiotensin II-induced, via AT1, increased levels of cholesterol and ROCK in the rat nigra and striatum. In cultured dopaminergic neurons, angiotensin II increased cholesterol biosynthesis and cholesterol efflux without changes in cholesterol uptake. In astrocytes, angiotensin induced an increase in cholesterol uptake, decrease in biosynthesis, and no change in cholesterol efflux, suggesting a neuronal accumulation of cholesterol that is reduced via transfer to astrocytes. Our data suggest mutual interactions between angiotensin/AT1, cholesterol, and ROCK pathways in LID, which are attenuated by the corresponding inhibitors. Interestingly, these three drugs have also been suggested as neuroprotective treatments against Parkinson's disease. Therefore, they may reduce dyskinesia and the progression of the disease using common mechanisms.

The ACE2 activator diminazene aceturate ameliorates colitis by repairing the gut-vascular barrier in mice.

Alleviating vascular barrier injury improves colitis. Angiotensin converting enzyme 2/angiotensin 1-7/Mas receptor (ACE2/Ang1-7/MasR) axis-related drugs have various biological properties, such as inhibition of inflammation and fibrosis, but their role in improving the gut-vascular barrier (GVB) has rarely been reported. This study aims to investigate the effects of diminazene aceturate (DIZE), an ACE2 activator, on vascular barrier damage in colitis. Mice were randomly divided into three groups: control, dextran sulfate sodium salt (DSS), and DIZE+DSS. Mice in the DSS group drank DSS for 8days starting on day 4. Mice in the DIZE+DSS group were pregavaged with DIZE for 3days and then drank DSS for 8days while continuing to be gavaged with DIZE for 4days. Mice were euthanized and samples were collected on the last day. Injury to colonic structure and colonic microvasculature was assessed by visual observation and appropriate staining. DSS-induced colonic and microvascular pathological damage in mice was substantially reversed by DIZE treatment. Molecular pathways were investigated by Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme linked immunosorbent assay (ELISA). DSS treatment upregulated angiotensin converting enzyme (ACE), angiotensin type 1 receptor (AT1R) protein, pro-inflammatory cytokines and inhibited tight junction-related protein expression. DIZE treatment activated ACE2/MasR protein expression and reversed epithelial barrier damage and inflammatory infiltration during DSS injury. In addition, DIZE treatment inhibited vascular endothelial growth factor A/vascular endothelial growth factor receptor 2/proto-oncogene tyrosine-protein kinase Src (VEGFA/VEGFR2/Src) pathway activation and restored vascular adhesion-linker protein vascular endothelial cadherin (VE-cadherin) expression during DSS injury. In conclusion, DIZE treatment ameliorated colitis, which was associated with balancing the two axes of the renin-angiotensin system (RAS) and repairing the GVB injury.

Ang-(1-7) attenuates podocyte injury induced by high glucose in vitro.

The incidence of diabetic nephropathy (DN) is gradually increasing worldwide. Podocyte injury, such as podocyte apoptosis and loss of the slit diaphragm (SD)-specific markers are early pathogenic features of DN. The cultured mouse podocytes were separated into a high glucose-treated (HG, 30mM) group to mimic DN in vitro, a low glucose-treated (LG, 5mM) group as a control and HG+ angiotensin-(1-7)(Ang-(1-7)) and HG+Ang-(1-7) + D-Ala7-Ang-(1-7) (A779, Ang-(1-7)/Mas receptor antagonist) experimental groups. The Cell Counting Kit-8 (CCK-8) method and flow cytometry was used to detect podocyte activity and podocyte apoptosis respectively. The expression of angiotensin type 1 receptor (AT1R), Mas receptor (MasR) and podocyte-specific markers were examined by q-PCR and Western blot, respectively. The results showed that the decrease in podocyte activity; the increase in podocyte apoptosis; the decreased mRNA and protein expression of nephrin, podocin, WT-1 and MasR; and the upregulated expression of AT1R induced by HG could be reversed by Ang-(1-7). However, these effects were blocked by A779. The possible mechanisms of the Ang-(1-7)-mediated effect depended on MasR. In addition, the protective effect of Ang-(1-7) on podocyte activity was dose-dependent and most obvious at 10 µM. A779 had the greatest antagonistic action against Ang-(1-7) at a concentration of 10 μM. This study reveals that binding of Ang-(1-7) to its specific receptor MasR may counteract the effects of Ang II mediated by AT1R to significantly attenuate podocyte injury induced by high glucose. Ang-(1-7)/MasR targeting in podocytes may be a therapeutic approach to attenuate renal injury in DN.

AUTOREACTIVE T CELLS TO ANGIOTENSIN II TYPE 1 RECEPTOR IN SALT-DEPENDENT HYPERTENSION DUE TO PRIMARY ALDOSTERONISM

Objective: Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but its pathogenic role is still unknown. Primary Aldosteronism (PA), the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary hypertension and by high titer of autoantibodies against the angiotensin type-1 receptor (AT1R). As T cells are broadly recognised as HMOD-mediators we investigated: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of aldosterone receptors on T lymphocytes. Design and Methods: The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number and immunoblotting from healthy donors and PA patients. PBMCs from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFN? release with flow cytometry. Results: PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR&gt;MR&gt;&gt;GPER. Aldosterone significantly increased IFN? release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-? production by CD8+ T cells by acting via the MR, as it was prevented (p&lt;0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p&lt;0.003), but also by G36 (p&lt;0.02), and mimicked by G1. Conclusion: These results showed the presence of AT1R-autoreactive T cells in patients with PA and demonstrates that, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs.

Chronically hypertensive transgenic mice expressing human AT1R haplotype-I exhibit increased susceptibility to Francisella tularensis.

Age-related illnesses, including hypertension and accompanying metabolic disorders, compromise immunity and exacerbate infection-associated fatalities. Renin-angiotensin system (RAS) is the key mechanism that controls blood pressure. Upregulation of RAS through angiotensin receptor type 1 (AT1R), a G-protein coupled receptor, contributes to the pathophysiological consequences leading to vascular remodeling, hypertension, and end-organ damage. Genetic variations that increase the expression of human AT1R may cause the above pathological outcomes associated with hypertension. Previously we have shown that our chronically hypertensive transgenic (TG) mice containing the haplotype-I variant (Hap-I, hypertensive genotype) of human AT1R (hAT1R) gene are more prone to develop the metabolic syndrome-related disorders as compared to the TG mice containing the haplotype-II variant (Hap-II, normotensive genotype). Since aging and an increased risk of hypertension can impact multiple organ systems in a complex manner, including susceptibility to various infections, the current study investigated the susceptibility and potential effect of acute bacterial infection using a Gram-negative intracellular bacterial pathogen, Francisella tularensis in our hAT1R TG mice. Our results show that compared to Hap-II, F. tularensis-infected aged Hap-I TG mice have significantly higher mortality post-infection, higher bacterial load and lung pathology, elevated inflammatory cytokines and altered gene expression profile favoring hypertension and inflammation. Consistent with worsened phenotype in aged Hap-I mice post-Francisella infection, gene expression profiles from their lungs revealed significantly altered expression of more than 1,400 genes. Furthermore, bioinformatics analysis identified genes associated with RAS and IFN-γ pathways regulating blood pressure and inflammation. These studies demonstrate that haplotype-dependent over-expression of the hAT1R gene leads to enhanced susceptibility and lethality due to F. tularensis LVS infection, which gets aggravated in aged animals. Clinically, these findings will help in exploring the role of AT1R-induced hypertension and enhanced susceptibility to infection-related respiratory diseases.

Role of Angiotensin II in Cardiovascular Diseases: Introducing Bisartans as a Novel Therapy for Coronavirus 2019.

Cardiovascular diseases (CVDs) are the main contributors to global morbidity and mortality. Major pathogenic phenotypes of CVDs include the development of endothelial dysfunction, oxidative stress, and hyper-inflammatory responses. These phenotypes have been found to overlap with the pathophysiological complications of coronavirus disease 2019 (COVID-19). CVDs have been identified as major risk factors for severe and fatal COVID-19 states. The renin-angiotensin system (RAS) is an important regulatory system in cardiovascular homeostasis. However, its dysregulation is observed in CVDs, where upregulation of angiotensin type 1 receptor (AT1R) signaling via angiotensin II (AngII) leads to the AngII-dependent pathogenic development of CVDs. Additionally, the interaction between the spike protein of severe acute respiratory syndrome coronavirus 2 with angiotensin-converting enzyme 2 leads to the downregulation of the latter, resulting in the dysregulation of the RAS. This dysregulation favors AngII/AT1R toxic signaling pathways, providing a mechanical link between cardiovascular pathology and COVID-19. Therefore, inhibiting AngII/AT1R signaling through angiotensin receptor blockers (ARBs) has been indicated as a promising therapeutic approach to the treatment of COVID-19. Herein, we review the role of AngII in CVDs and its upregulation in COVID-19. We also provide a future direction for the potential implication of a novel class of ARBs called bisartans, which are speculated to contain multifunctional targeting towards COVID-19.

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.

Regulation of Renin-Angiotensin System Activity and Blood Pressure by Vascular Smooth Muscle Cullin-3

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) in smooth muscle cells (SMCs) cause vascular dysfunction, arterial stiffness, and severe hypertension. We hypothesized that conditional deletion of CUL3 in SMCs causes severe hypertension through a renin-angiotensin system (RAS)-dependent mechanism. Mice carrying a conditional allele of CUL3 were bred to mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). Tamoxifen was administered to generate smooth muscle CUL3 knockout (S-CUL3KO). Primary aortic SMCs were used to elucidate the contribution of CUL3 to intracellular angiotensin II-mediated calcium flux. Calcium response in SMCs was analyzed via ratiometric confocal fluorescent microscopyS-CUL3KO mice demonstrated hypertension following three weeks of tamoxifen, mean arterial pressure (MAP) was 108±1 vs. 140±1 mmHg (ISM-CRE vs. S-CUL3KO). Aorta from S-CUL3KO mice exhibited impaired vasorelaxation to acetylcholine (ACh, max relaxation: 84±4% vs. 16±9%, p&lt;0.05) and sodium nitroprusside (SNP, max relaxation: 95±3% vs. 56±9%, p&lt;0.05). Captopril administration (7 days) markedly reduced MAP to 84±0.3 mmHg and improved vasorelaxation in response to both ACh (72±6%) and SNP (91±4%) in S-CUL3KO mice, suggesting RAS-dependency of the hypertension. Additionally, candesartan treatment (7 days) substantially decreased MAP from 138±1 to 96±3 mmHg suggesting that hypertension in S-CUL3KO group is angiotensin type-1 receptor mediated. Q-PCR and RNAScope analyses demonstrated no difference in renin expression in the kidney between the S-CUL3KO and control mice suggesting a blunting of the baroreceptor mechanism regulating renin mRNA expression. Equilibrium angiotensin II plasma levels in both groups were not different (2517±352 and 2379±549 pmol/L, p&gt;0.05). Preliminary data demonstrated that acute application of 10 μM of angiotensin II to S-CUL3KO SMCs resulted in a sustained increase in intracellular calcium which was abolished by a preincubation with 2 μM of candesartan.Our data suggest that CUL3 may play a vital role in baroreceptor mechanism in the kidney and regulate angiotensin II response in SMCs which are essential for responding to changes in perfusion pressure. Ultimately, understanding CUL3 target proteins may benefit the development of novel treatments for hypertension. R35 HL144807 (to CDS). 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.

B-Arrestin2 Deficiency in the Brain Alters Blood Pressure Regulation

Low-renin hypertension is a common subset of hypertensive patients, which is associated with salt sensitivity. Deoxycorticosterone (DOCA)-salt hypertension, a prototypical model of low-renin hypertension, requires activation of the angiotensin type-1 receptor (AT 1 R) in specific brain areas. G protein-mediated signaling of the AT 1 R within the brain is known to induce dipsogenic and pressor responses to Ang II stimulation. Non-canonical or b-arrestin-mediated signaling is thought to counterbalance the maladaptive G-protein (Gαq) signaling during disease. Recently, we found that global deletion of β-arrestin2 (ARRB2) exhibited an exacerbated increase in blood pressure (BP) in response to DOCA-salt compared to WT suggesting a protective role for ARRB2. However, the role of the non-canonical AT1R/β-arrestin pathway within the brain is understudied. Consequently, we hypothesized that b-Arrestin activation within the brain contributes to BP regulation. Global male and female β-arrestin1 ( Arrb1)- and β-arrestin2 ( Arrb2)-KO mice were employed to evaluate acute BP response upon treatment with intracerebroventricular (ICV) infusion of AngII (1ug). Age- and sex-matched C57BL/6 mice served as controls. Mice were simultaneously instrumented with radiotelemeters and ICV-cannula, BP was continuously recorded before and after AngII infusion in awake animals. At baseline, Arrb2-KO mice showed a slight increase in BP WT (WT=134.0±10.7 vs Arrb2-KO=143.7±17.2 mmHg; n=14 and 19, respectively). Further, Arrb2-KO mice exhibited significantly higher BP in response to AngII (WT=143.7±17.2 vs Arrb2-KO=176.9±24.2 mmHg; n=14 and 19, respectively p&lt;0.05). These findings suggest that loss of ARRB2 exacerbates the pressor response to AngII within the brain. Additionally, we evaluated BP upon activation β-arrestin using TRV120027 (TRV27), a β-arrestin biased agonist specific for the AT 1 R. We used the BPH2/J genetic mouse model of low renin hypertension. Mice were subjected to continuous infusion of TRV27 (6ug/h), and telemetric BP was continuously recorded. BPN/2J mice were used as the normotensive controls. At baseline, BPH2/J exhibited significantly higher BP when compared to normotensive controls (BPN=123.9±5.3 vs. BPH=153.4±3.4 mmHg; n=4 p&lt;0.05). Further, chronic delivery of ICV-TRV showed a higher magnitude of BP reduction in the BPH2/J group compared to controls (BPN=-3.8±3.0 vs. BPH=-8.8±1.3 mmHg; n=4 p&lt;0.05), suggesting that the effects of b-arrestin activation are predominant under hypertensive states. Overall, endogenous ARRB2 counterbalances the deleterious effects of AT 1 R overactivation in the brain. The stimulation of the AT 1 R β-arrestin axis in the brain using biased AT 1 R agonist that penetrate the brain-blood barrier might be a potential strategy to treat low renin hypertension. HL084207 and HL144807 to CDS, and 22PRE898004 to NMM 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.

Computational investigation of the impact of potential AT2R polymorphism on small molecule binding

For more than a century, the renin-angiotensin system (RAS) has been acknowledged for playing a crucial part in the physiological control of arterial pressure, as well as sodium and fluid balance. It is now generally acknowledged that one of the receptor of RAS system i.e. angiotensin type 2 receptor (AT2R) functions as a repair system during pathophysiologic circumstances and performs a significant protective role. Efforts have been made previously to design suitable agonist and antagonist molecules to potentially modulate AT2R. One of the agonists and antagonists, named C21 and EMA401, has been studied in a number of pathological conditions. Additionally, a wide panel of single nucleotide polymorphisms (SNPs) has been reported for AT2R, which might potentially affect the efficacy of these molecules. Therefore, computational investigations have been carried out to analyze all the SNPs (1151) reported in NCBI to find potential SNPs affecting the active site of AT2R, as this domain is still unexplored. Structures of these polymorphic forms were modeled, and in silico drug interaction studies with C21 and EMA401 were carried out. The two mutants (rs868939201 and rs1042852794) that significantly affect the binding affinity as that of the wild type were subjected to molecular dynamics simulations. Our analysis of native and mutant AT2R and their complexes with C21 and EMA401 indicated that the occurrence of these mutations affects the conformation of the protein and has affected the binding of these ligand molecules. The study’s findings will aid in the development of better, more versatile medications in the near future, and also in vitro and in vivo studies might be planned in accordance with recent findings. Communicated by Ramaswamy H. Sarma

440 Role of angiotensin II in SARS-CoV-2 pathophysiology in a hamster model of COVID-19

OBJECTIVES/GOALS: Hamsters develop COVID-19 similarly to people because the SARS-CoV-2 spike protein binds with high affinity to hamster ACE2 resulting in host cell entry and replication. Our goal was to establish a hamster model that mirrors the lung and brain pathophysiology observed in COVID-19. METHODS/STUDY POPULATION: Hamsters infected with SARS CoV-2 are sacrificed on day 1 and day 6 postinfection. Lung histopathology scoring model was implemented for assessment all pathological relevant changes in the lungs of infected animals on tissue sections stained with hematoxylin and eosin. To quantify the extent and severity of lung pathology, two scoring systems were used: the first evaluated all relevant changes in the lungs of the infected animals and the second evaluated only the pathology associated with the pulmonary vasculature. Percentage of airway affected, airway severity, bronchiolar epithelial hyperplasia, alveoli affected, alveolar severity, type II pneumocyte hyperplasia and vessels affected were analyzed. Total airway score plus total lung alveolar score give lung histopathology score. RESULTS/ANTICIPATED RESULTS: Compared to the control hamster, the hamsters day 1 postinfection, exhibited a higher total airway score [9.00 ± 1.35 vs. 0.25 ± 0.1; p DISCUSSION/SIGNIFICANCE: Establishing this outstanding small animal model of COVID-19 will facilitate studies investigating diagnostics, prognosis and response to treatment in COVID-19 disease. These studies will provide insights that will complement on-going clinical trials on angiotensin type 1 receptor (AT1R) blockers (ARBs) in COVID-19.

Angiotensin-(1-9) attenuates adriamycin-induced cardiomyopathy in rats via the angiotensin type 2 receptor.

Adriamycin (ADR) causes irreversible damage to the heart, leading to ADR-induced cardiomyopathy (ACM). Angiotensin-(1-9) [Ang-(1-9)] is a peptide from the counter-regulatory renin-angiotensin system, but the effects on ACM is unclear. Our study was aimed to explore the effects and underlying molecular mechanisms of Ang-(1-9) against ACM in Wistar rats. Rats were injected intraperitoneally with ADR via six equal doses (each containing 2.5mg/kg) within a period of 2 weeks to induce ACM. After 2weeks of ADR treatment, the rats were treated with Ang-(1-9) (200ng/kg/min) or angiotensin type 2 receptor (AT2R) antagonist PD123319 (100ng/kg/min) for 4weeks. Although Ang-(1-9) treatment did not influence blood pressure, it significantly improved left ventricular function and remodeling in ADR-treated rats, by inhibiting collagen deposition, the expression of TGF-β1, inflammatory response, cardiomyocyte apoptosis and oxidative stress. Moreover, Ang-(1-9) reduced ERK1/2 and P38 MAPK phosphorylation. The therapeutic effects of Ang-(1-9) were blocked by the AT2R antagonist PD123319, which also offset the down-regulation protein expression of pERK1/2 and pP38 MAPK induced by Ang-(1-9). These data suggest that Ang-(1-9) improved left ventricular function and remodeling in ADR-treated rats by an AT2R/ ERK1/2 and P38 MAPK-dependent mechanism. Thus, the Ang-(1-9)/AT2R axis may provide a novel and promising target to the prevention and treatment of ACM.

The intrarenal renin-angiotensin system in hypertension: insights from mathematical modelling.

The renin-angiotensin system (RAS) plays a pivotal role in the maintenance of volume homeostasis and blood pressure. In addition to the well-studied systemic RAS, local RAS have been documented in various tissues, including the kidney. Given the role of the intrarenal RAS in the pathogenesis of hypertension, a role established via various pharmacologic and genetic studies, substantial efforts have been made to unravel the processes that govern intrarenal RAS activity. In particular, several mechanisms have been proposed to explain the rise in intrarenal angiotensin II (Ang II) that accompanies Ang II infusion, including increased angiotensin type 1 receptor (AT1R)-mediated uptake of Ang II and enhanced intrarenal Ang II production. However, experimentally isolating their contribution to the intrarenal accumulation of Ang II in Ang II-induced hypertension is challenging, given that they are fundamentally connected. Computational modelling is advantageous because the feedback underlying each mechanism can be removed and the effect on intrarenal Ang II can be studied. In this work, the mechanisms governing the intrarenal accumulation of Ang II during Ang II infusion experiments are delineated and the role of the intrarenal RAS in Ang II-induced hypertension is studied. To accomplish this, a compartmental ODE model of the systemic and intrarenal RAS is developed and Ang II infusion experiments are simulated. Simulations indicate that AT1R-mediated uptake of Ang II is the primary mechanism by which Ang II accumulates in the kidney during Ang II infusion. Enhanced local Ang II production is unnecessary. The results demonstrate the role of the intrarenal RAS in the pathogenesis of Ang II-induced hypertension and consequently, clinical hypertension associated with an overactive RAS.