Angiotensin Type Research Articles

#4590 A PROSPECTIVE STUDY OF EPLERENONE IN THE TREATMENT OF PATIENTS WITH GLOMERULONEPHRITIS

Abstract Background and Aims Clinical evidence suggests that high aldosterone plasma levels contributes to progressive kidney disease. Although administration of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin type 1 receptor blockers (ARB) can delay kidney disease progression, plasma aldosterone levels may increase to a significant extent even after the initiation of such treatment. Thus, administration of mineralocorticoid receptor antagonists such as spironolactone, on top of treatment with ACEi or ARBs has been shown to reduce albuminuria and slow down kidney function deterioration. Nevertheless, there is scanty evidence on the effect of eplerenone in patients with kidney disease due to glomerulonephritis. Method In this prospective open-label study, we evaluated the effects of eplerenone in patients with biopsy proven glomerulonephritis already treated with ACEi or ARBs. Patients were divided in the active treatment arm, who received 25 mg of eplerenone daily besides ACEi or ARBs, and a control group that received only standard treatment. Protein excretion in 24 h urine, eGFR, blood pressure and serum K+ levels were measured at 3, 6 and 12 months after study initiation. Results Out of 83 patients who were screened, 62 patients were included in the final analysis. Twenty-six received eplerenone and 36 were treated with ACEi (25) or ARBs (9) alone (controls). After 1 year of treatment with eplerenone, proteinuria decreased from 1597 to 1145 mg/24 h while it did not alter in controls. Eplerenone reduced proteinuria especially in those with baseline proteinuria of >1000 mg/24 h. Kidney function remained stable in patients treated with eplerenone (from eGFR: 82.51±22.6 to 84.68±31.7 ml/min/1.73 m2, p = 0.8) and showed a non significant deterioration in controls (from eGFR: 68.39±26.2 to 66.63±29.8 ml/min/1.73 m2, p = 0.08). Systolic blood pressure was significantly reduced in the active treatment arm (from 128.1±17.5 to 126.3±10.93 mmHg, p = 0.03), whereas eplerenone did not increase serum K+ levels or had any other significant adverse effect. Conclusion Administration of eplerenone at a dose of 25 mg/day, on top of treatment with ACEi or ARBs, can further reduce proteinuria and systolic blood pressure in patients with chronic glomerulonephritis.

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>MR>>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<0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p<0.003), but also by G36 (p<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.

Cluster analysis of angiotensin biomarkers to identify antihypertensive drug treatment in population studies

BackgroundThe recent progress in molecular biology generates an increasing interest in investigating molecular biomarkers as markers of response to treatments. The present work is motivated by a study, where the objective was to explore the potential of the molecular biomarkers of renin-angiotensin-aldosterone system (RAAS) to identify the undertaken antihypertensive treatments in the general population. Population-based studies offer an opportunity to assess the effectiveness of treatments in real-world scenarios. However, lack of quality documentation, especially when electronic health record linkage is unavailable, leads to inaccurate reporting and classification bias.MethodWe present a machine learning clustering technique to determine the potential of measured RAAS biomarkers for the identification of undertaken treatments in the general population. The biomarkers were simultaneously determined through a novel mass-spectrometry analysis in 800 participants of the Cooperative Health Research In South Tyrol (CHRIS) study with documented antihypertensive treatments. We assessed the agreement, sensitivity and specificity of the resulting clusters against known treatment types. Through the lasso penalized regression, we identified clinical characteristics associated with the biomarkers, accounting for the effects of cluster and treatment classifications.ResultsWe identified three well-separated clusters: cluster 1 (n = 444) preferentially including individuals not receiving RAAS-targeting drugs; cluster 2 (n = 235) identifying angiotensin type 1 receptor blockers (ARB) users (weighted kappa κw = 74%; sensitivity = 73%; specificity = 83%); and cluster 3 (n = 121) well discriminating angiotensin-converting enzyme inhibitors (ACEi) users (κw = 81%; sensitivity = 55%; specificity = 90%). Individuals in clusters 2 and 3 had higher frequency of diabetes as well as higher fasting glucose and BMI levels. Age, sex and kidney function were strong predictors of the RAAS biomarkers independently of the cluster structure.ConclusionsUnsupervised clustering of angiotensin-based biomarkers is a viable technique to identify individuals on specific antihypertensive treatments, pointing to a potential application of the biomarkers as useful clinical diagnostic tools even outside of a controlled clinical setting.

The Protective Potential Role of ACE2 against COVID-19.

Due to the coronavirus disease 2019 (COVID-19), researchers all over the world have tried to find an appropriate therapeutic approach for the disease. The angiotensin-converting enzyme 2 (ACE2) has been shown as a necessary receptor to cell fusion, which is involved in infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is commonly crucial for all organs and systems. When ACE2 is downregulated via the SARS-CoV-2 spike protein, it results in the angiotensin II (Ang II)/angiotensin type 1 receptor axis overactivation. Ang II has harmful effects, which can be evidenced by dysfunctions in many organs experienced by COVID-19 patients. ACE2 is the SARS-CoV-2 receptor and has an extensive distribution; thus, some COVID-19 cases experience several symptoms and complications. We suggest strategy for the potential protective effect of ACE2 to the viral infection. The current review will provide data to develop new approaches for preventing and controlling the COVID-19 outbreak.

Irbesartan overcomes gemcitabine resistance in pancreatic cancer by suppressing stemness and iron metabolism via inhibition of the Hippo/YAP1/c-Jun axis

BackgroundChemoresistance is the main reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Thus, there is an urgent need to screen out new targets and compounds to reverse chemotherapeutic resistance.MethodsWe established a bio-bank of human PDAC organoid models, covering a representative range of PDAC tumor subtypes. We screened a library of 1304 FDA-approved compounds to identify candidates efficiently overcoming chemotherapy resistance. The effects of the compounds were evaluated with a CellTiter-Glo-3D assay, organoid apoptosis assay and in vivo patient-derived xenograft (PDX), patient-derived organoid (PDO) and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) genetically engineered mouse models. RNA-sequencing, genome editing, sphere formation assays, iron assays and luciferase assays were conducted to elucidate the mechanism.ResultsHigh-throughput drug screening of chemotherapy-resistant PDOs identified irbesartan, an angiotensin ‖ type 1 (AT1) receptor antagonist, which could synergistically enhance the ability of chemotherapy to kill PDAC cells. In vitro and in vivo validation using PDO, PDX and KPC mouse models showed that irbesartan efficiently sensitized PDAC tumors to chemotherapy. Mechanistically, we found that irbesartan decreased c-Jun expression by inhibiting the Hippo/YAP1 pathway and further overcame chemotherapy resistance in PDAC. We also explored c-Jun, a potential target of irbesartan, which can transcriptionally upregulate the expression of key genes involved in stemness maintenance (SOX9/SOX2/OCT4) and iron metabolism (FTH1/FTL/TFRC). More importantly, we observed that PDAC patients with high levels of c-Jun expression demonstrated poor responses to the current standard chemotherapy regimen (gemcitabine plus nab-paclitaxel). Moreover, patients with PDAC had significant survival benefits from treatment with irbesartan plus a standard chemotherapy regimen in two-center retrospective clinical cohorts and patients with high c-Jun expression exhibited a better response to combination chemotherapy.ConclusionsIrbesartan could be used in combination with chemotherapy to improve the therapeutic efficacy in PDAC patients with high levels of c-Jun expression. Irbesartan effectively inhibited chemotherapy resistance by suppressing the Hippo/YAP1/c-Jun/stemness/iron metabolism axis. Based on our findings, we are designing an investigator-initiated phase II clinical trial on the efficacy and safety of irbesartan plus a standard gemcitabine/nab-paclitaxel regimen in the treatment of patients with advanced III/IV staged PDAC and are hopeful that we will observe patient benefits.

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<0.01) and amplitude ( D=0.13, p<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<0.05) and sodium nitroprusside (SNP, max relaxation: 95±3% vs. 56±9%, p<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>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<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<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<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.

Chronic intermittent hypoxia induced increases in central angiotensin II release in the MnPO

The brain is sensitive to Angiotensin II (ANG II) and we have previously shown that ANG II is released by brain slices containing the median preoptic nucleus (MnPO) and subfornical organ (SFO) in an activity dependent manner. In addition, angiotensin II type 1 receptors and angiotensin converting enzyme 1 are upregulated in the MnPO following chronic intermittent hypoxia (CIH), an animal model of obstructive sleep apnea. Here, we investigate changes in ANG II release in the SFO to MnPO pathway following 7-day CIH treatment.Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions (0.4 μL) of AAV2-hSyn-ChR2(E123A)-eYFP-WPRE in the SFO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hrs) or Normoxia. The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For sniffer cell measurements, rats were anesthetized with isoflurane (2-3%) and sagittal slices (300 μm) containing the MnPO and SFO were cut using standard in vitro slice procedures. Sniffer cells consisted of Chinese Hamster Ovary cells transfected with a commercially available plasmid for the angiotensin type 1a (AT1a) receptor (Origene Tech.) and R-GECO (Addgene #32462) as previously described. Sniffer cells were placed on the median preoptic nucleus (MnPO) of sagittal in vitro brain slices. Both spontaneous and evoked (SFO stimulation) changes in fluorescent intensity of sniffer cells on the MnPO were measured.Sniffer cell recordings were made from 5 normoxia and 8 CIH treated rats. The frequency of spontaneous sniffer cell activity in slices from normoxic rats (n = 92) was comparable to rates previously reported in Sprague-Dawley rats. Spontaneous sniffer cell activity in slices from CIH treated rats (n = 156) was increased compared to normoxic treated rats (p < 0.05). Furthermore, bath application of TTX (1 uM) reduced, but did not abolish, spontaneous sniffer cell activity in slices from both normoxic (n = 95, p < 0.001) and CIH (n = 108, p < 0.001) treated rats. Incidence of optogenetic evoked responses in sniffer cells were limited in slices from both the normoxic and CIH treated rats and there were no differences detected in these evoked responses.The current study indicates that spontaneous ANG II release in the MnPO 1) is not entirely action potential dependent, 2) is enhanced following 7-days CIH treatment, and 3) the CIH induced increase is action potential dependent. While optogenetic stimulation of SFO increased sniffer cell activity, CIH had no effect evoked release of ANG II in these preliminary studies. Work supported by: P01 HL088052, R01 HL155977 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.

Role of the Renin-Angiotensin System Components in Renal Cell Carcinoma: A Literature Review.

The physiological aspects of renin-angiotensin system (RAS) components are described in this review. Additionally, we present the main results of studies that could indicate an association between alterations in these components and cancer, particularly renal cell carcinoma (RCC). The RAS undergoes a series of homeostatic and modulatory processes that extend to hypertrophy, hyperplasia, fibrosis, and remodeling, as well as angiogenesis, pro-inflammatory responses, cell differentiation, stem cell programming, and hematopoiesis. The link between cancer-related inflammation and RAS signaling converge in the response to tumor hypoxia and oxidative stress mechanisms, particularly with the angiotensin type 1 receptor leading to activation of transcription factors such as nuclear factor κB (NF-κB), as well as members of the signal transducer and activation of transcription (STAT) family and HIF1⍺. Dysregulation of the physiological actions of RAS in the microenvironment of inflammation and angiogenesis promotes tumor cell growth.

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

Angiotensin II constricts mouse iliac arteries: possible mechanism for aortic aneurysms.

Abdominal aortic aneurysms (AAA) result from maladaptive remodeling of the vascular wall and reduces structural integrity. Angiotensin II (AngII) infusion has become a standard laboratory model for studying AAA initiation and progression. We determined the different vasoactive responses of various mouse arteries to Ang II. Ex vivo isometric tension analysis was conducted on 18-week-old male C57BL/6 mice (n = 4) brachiocephalic arteries (BC), iliac arteries (IL), and abdominal (AA) and thoracic aorta (TA). Arterial rings were mounted between organ hooks, gently stretched and an AngII dose response was performed. Rings were placed in 4% paraformaldehyde for immunohistochemistry analysis to quantify peptide expression of angiotensin type 1 (AT1R) and 2 receptors (AT2R) in the endothelium, media, and adventitia. Results from this study demonstrated vasoconstriction responses in IL were significantly higher at all AngII doses when compared to BC, and TA and AA responses (maximum constriction-IL: 68.64 ± 5.47% vs. BC: 1.96 ± 1.00%; TA: 3.13 ± 0.16% and AA: 2.75 ± 1.77%, p < 0.0001). Expression of AT1R was highest in the endothelium of IL (p < 0.05) and in the media and (p < 0.05) adventitia (p < 0.05) of AA. In contrast, AT2R expression was highest in endothelium (p < 0.05), media (p < 0.01, p < 0.05) and adventitia of TA. These results suggest that mouse arteries display different vasoactive responses to AngII, and the exaggerated response in IL arteries may play a role during AAA development.

Abstract 1451: Cognition-mediated transcriptomic signature associated with androgen deprivation therapy in prostate cancer

Abstract Androgen deprivation therapy (ADT) is the mainstay treatment for non-metastatic and metastatic hormone-sensitive prostate cancer. Long-term ADT treatment results in adverse side-effects in patients including depression, memory loss, cognitive decline, and dementia. We aimed to identify critical molecules that could provide link between brain-related changes and pathological mechanisms responsible for cognitive decline in prostate cancer patients on ADT protocol. For these studies, gene expression data (GSE150807 and GSE151083) of human prostate cancer cells exposed to enzalutamide emulating ADT protocol was used to identify differential expression of genes at the transcript level. RNA sequencing analysis identified 9409 and 7757 differentially expressed genes (DEGs) in LNCaP and C4-2B enzalutamide exposed cells at p-value &amp;lt; 0.0005 and FDR&amp;lt; 0.05. In both cell lines, neuronal receptor signaling pathway was significantly overrepresented at 0.05 FDR-adjusted p-value (q-value). The genes associated with neuronal receptor signaling pathway belong to the ligand-gated ion channel and G-protein coupled receptors. Validation studies using brain glial cells treated with enzalutamide showed significant upregulation (p-value 0.001) of GABRB1, GABRA3, GABBR2 (GABA receptors/ligand-gated ion channel), BDNF (brain-derived neurotrophic factor), IFNB1 (interferon beta 1), NR3C1 (nuclear receptor subfamily 3 group c member/glucocorticoid receptor), ATGR1 (type-1 angiotensin II receptor), GRIN1 (glutamate ionotropic receptor NMDA type submit 1/ligated-gated ion channel), GRIN2D (glutamate ionotropic receptor NMDA type submit 2D/ligated-gated ion channel). In brain neuronal cells, enzalutamide exposure resulted in increased expression of NR3C1 whereas GABA/ligand-gated ion channel and others were downregulated and/or statistically non-significant. Further analysis using DisGenET (cytoscape) highlighted ATGR1, GABRB1, GRIN1, GRIN2D, and NR3C1 association with neuronal diseases including cognition disorder, forgetfulness, depressive mood, and others. The results of our study provide the first proof of evidence linking cognitive decline, depression, memory loss, and other neurological disorder more likely to develop in prostate cancer patients undergoing long-term androgen deprivation therapy. Citation Format: Shiv Shankar Verma, Eswar Shankar, Vaibhav Singh, Vijay Krishna, Sanjay Gupta. Cognition-mediated transcriptomic signature associated with androgen deprivation therapy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1451.

Cisplatin treatment reduces contraction to angiotensin II by altering expression of angiotensin II receptors: a pilot study.

The renin angiotensin system is a key regulator of blood pressure homeostasis. Angiotensin type 1 (AT1R) and 2 receptors (AT2R) have been investigated as targets for cisplatin-induced acute kidney injury; however, their therapeutic potential remains inconclusive. This pilot study aimed to determined the effect that acute cisplatin treatment had on angiotensin II (AngII)-induced contraction in blood vessels and expression profiles of AT1R and AT2R in mouse arteries and kidneys. Male C57BL/6 mice at 18week of age (n = 8) were treated with vehicle or bolus dose of cisplatin (12.5mg/kg). Thoracic aorta (TA), adnominal aorta (AA), brachiocephalic arteries (BC), iliac arteries (IL) and kidneys were collected for isometric tension and immunohistochemistry analysis. Cisplatin treatment reduced IL contraction to AngII at all doses (p < 0.01, p < 0.001, p < 0.0001); however, AngII did not induce contraction in TA, AA or BC in either treatment group. Following cisplatin treatment, AT1R expression was significantly upregulated in the media of TA (p < 0.0001) and AA (p < 0.0001), and in the endothelium (p < 0.05) media (p < 0.0001) and adventitia (p < 0.01) of IL. Cisplatin treatment significantly reduced AT2R expression in the endothelium (p < 0.05) and media (p < 0.05) of TA. In renal tubules, both AT1R (p < 0.01) and AT2R (p < 0.05) were increased following cisplatin treatment. Herein, we report that cisplatin reduces AngII-mediated contraction in IL and may be explained by an absence of normal counterregulatory expression of AT1R and AT2R, indicating other factors are involved.

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.

Molecular docking analysis of AGTR1 antagonists.

Cardiovascular diseases (CVDs) are the leading cause of death and morbidity globally. The renin-angiotensin system is an important regulatory system for maintaining cardiovascular and renal function. Therefore, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have emerged as first-line treatments for conditions such as hypertension and heart failure. Currently available synthetic medications used to treat various CVDs have been linked with various adverse effects. Therefore, this study focuses on targeting type-1 angiotensin II receptor (AGTR1) by natural compounds. The ZINC database natural compounds and standard AGTR1 inhibitors have been screened against the AGTR1 active site. The results showed that five compounds, namely ZINC85625504, ZINC62001623, ZINC70666587, ZINC06624086, and ZINC95486187, had similar binding energies to established AGTR1 inhibitors. These compounds were found to interact with crucial AGTR1 residues, indicating their potential as AGTR1 inhibitors. Moreover, the hit compounds demonstrated favorable drug-like characteristics and warrant further investigation for their potential use in managing CVD.

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.

Pre and postnatal exposure to 900 MHz electromagnetic fields induce inflammation and oxidative stress, and alter renin-angiotensin system components differently in male and female offsprings

AimsThis study was designed to investigate inflammation, oxidative stress and renin-angiotensin system components in brain and kidney tissues of female and male rats prenatally and/or postnatally exposed to 900 MHz electromagnetic field (EMF). It is aimed to evaluate the biological effects of 900 MHz EMF exposure due to the increase in mobile phone use and especially the more widespread use of the GSM 900 system. Main methodsMale and female Wistar albino offsprings were divided into four groups of control, prenatal, postnatal, and prenatal+postnatal exposed to 900 MHz EMF for 1 h/day (23 days during pregnancy for prenatal period, 40 days for postnatal period). The brain and kidney tissues were collected when they reached puberty. Key findingsIt was found that the total oxidant status, IL-2, IL-6, and TNF-α levels increased (p < 0.001) and the total antioxidant status levels decreased (p < 0.001) in all three EMF groups comparing to controls in both male and female brain and kidney tissues. The renin- angiotensin system components such as angiotensinogen, renin, angiotensin type 1 and type 2 receptors, and MAS1-like G protein-coupled receptor expression were higher (p < 0.001) in all three EMF exposure groups comparing to controls in both male and female brain and kidney tissues. Although there are some differences of the levels of proinflammatory markers, ROS components and RAS components in brain and kidney tissues between males and females, the common result of all groups was increase in oxidative stress, inflammation markers and angiotensin system components with exposure to 900 MHz EMF. SignificanceIn conclusion, our study suggested that the 900 MHz EMF can activate brain and kidney renin-angiotensin system, and this activation is maybe related to inflammation and oxidative stress in both male and female offsprings.

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.