Angiotensin AT2 Receptor Agonist Research Articles

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

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

The angiotensin AT2-receptor agonist compound 21 is an antagonist for the thromboxane TP-receptor – Implications for preclinical studies and future clinical use

Since the AT2-receptor (AT2R) agonist C21 has structural similarity to the AT1-receptor antagonists Irbesartan and Losartan, which are antagonists not only at the AT1R, but also at thromboxane TP-receptors, we tested the hypothesis that C21 has TP-receptor antagonistic properties as well. Isolated mouse mesenteric arteries from C57BL/6 J and AT2R-knockout mice (AT2R-/y) were mounted in wire myographs, contracted with either phenylephrine or the thromboxane A2 (TXA2) analogue U46619, and the relaxing effect of C21 (0.1 nM - 10 µM) was investigated. The effect of C21 on U46619-induced platelet aggregation was measured by an impedance aggregometer. Direct interaction of C21 with TP-receptors was determined by an β-arrestin biosensor assay. C21 caused significant, concentration-dependent relaxations in phenylephrine- and U46619-contracted mesenteric arteries from C57BL/6 J mice. The relaxing effect of C21 was absent in phenylephrine-contracted arteries from AT2R-/y mice, whereas it was unchanged in U46619-contracted arteries from AT2R-/y mice. C21 inhibited U46619-stimulated aggregation of human platelets, which was not inhibited by the AT2R-antagonist PD123319. C21 reduced U46619-induced recruitment of β-arrestin to human thromboxane TP-receptors with a calculated Ki of 3.74 µM. We conclude that in addition to AT2R-agonistic properties, C21 also acts as low-affinity TP-receptor antagonist, and that – depending on the constrictor – both mechanisms can be responsible for C21-induced vasorelaxation. Furthermore, by acting as a TP-receptor antagonist, C21 inhibits platelet aggregation. These findings are important for understanding potential off-target effects of C21 in the preclinical and clinical context and for the interpretation of C21-related myography data in assays with TXA2-analogues as constrictor.

Angiotensin AT2 Receptor Stimulation Alleviates Collagen-Induced Arthritis by Upregulation of Regulatory T Cell Numbers.

The angiotensin AT2 receptor (AT2R) is a main receptor of the protective arm of the renin-angiotensin system and exerts for instance anti-inflammatory effects. The impact of AT2R stimulation on autoimmune diseases such as rheumatoid arthritis (RA) is not yet known. We investigated the therapeutic potential of AT2R-stimulation with the selective non-peptide AT2R agonist Compound 21 (C21) in collagen-induced arthritis (CIA), an animal model for inflammatory arthritis. Arthritis was induced by immunization of DBA/1J mice with collagen type II (CII). Prophylactic and therapeutic C21 treatment alleviates arthritis severity and incidence in CIA. Joint histology revealed significantly less infiltrates of IL-1 beta and IL-17A expressing cells and a well-preserved articular cartilage in C21- treated mice. In CIA, the number of CD4+CD25+FoxP3+ regulatory T (Treg) cells significantly increased upon C21 treatment compared to vehicle. T cell differentiation experiments demonstrated increased expression of FoxP3 mRNA, whereas IL-17A, STAT3 and IFN-gamma mRNA expression were reduced upon C21 treatment. In accordance with the mRNA data, C21 upregulated the percentage of CD4+FoxP3+ cells in Treg polarizing cultures compared to medium-treated controls, whereas the percentage of CD4+IL-17A+ and CD4+IFN-gamma+ T cells was suppressed. To conclude, C21 exerts beneficial effects on T cell-mediated experimental arthritis. We found that C21-induced AT2R-stimulation promotes the expansion of CD4+ regulatory T cells and suppresses IL-17A production. Thus, AT2R-stimulation may represent an attractive treatment strategy for arthritis.

Abstract P250: A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist

The angiotensin AT2-receptor (AT2R) is a key component within the protective arm of the renin-angiotensin system (RAS), being involved in nitric oxide (NO) production and vasodilation. To this date, no quantitative high-throughput assay is available to identify AT2R agonists in vitro , which may be a reason for the low number of AT2R selective ligands in drug development programs. Objective: To design and validate a high-throughput method for detection of AT2R activation in vitro . Methods and Results: NO release was selected as readout for AT2R activation in AT2R transfected (CHO-AT2) and non-transfected (CHO-NT) CHO cells using DAF-FM (5x10-6 mol/L) as NO probe. Cells were seeded on 96-well plates and stimulated for 15 minutes with C21 or Ang II (established AT2R agonists, 10-6mol/L), Ang-(1-5) (molecule with unknown biological status, 10-6 mol/L) or Ang-(1-7) (Mas-receptor agonist, 10-7 mol/L). After fixation of cells, fluorescence signals were captured by fluorescence microscopy using an automated imaging system (ImageXpress Pico, Molecular Devices, San Jose, USA) and image analysis by ImageJ. In CHO-AT2, C21 (+34.78 ± 12.09%), Ang II (+28.76 ± 17.65%) and Ang-(1-5) (+78.00 ± 23.82%) increased NO release (one-way ANOVA, p < 0.05 vs control, at least 3 independent experiments), while Ang-(1-7) had no effect (+ 0.13 ± 4.74%). In CHO-NT, none of the compounds stimulated release of NO (C21 -4.91 ±10.24%; Ang II -4.79 ± 12.44%; Ang (1-5) -8.88 ± 18.16%; Ang-(1-7) -11.57 ± 19.95%) indicating that the responses in CHO-AT2 were AT2R specific. Conclusion: Measurement of NO release from AT2R transfected CHO cells by DAF-FM fluorescence in an automated way is suitable as high-throughput assay for the identification of AT2R-agonistic compounds in vitro , Application of the assay revealed that Ang-(1-5), which is commonly regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

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.

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

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

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.

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)

Angiotensin AT2 receptor–induced interleukin-10 attenuates neuromyelitis optica spectrum disorder–like pathology

Background: Neuromyelitis optica spectrum disorder (NMOSD) is a relapsing inflammatory central nervous system (CNS) disease for which there is no cure. Immunoglobulin G autoantibodies specific for the water channel aquaporin-4 are a serum biomarker, believed to induce complement-dependent astrocyte damage with secondary demyelination. Objective: To investigate the effect of angiotensin AT2 receptor (AT2R) stimulation on NMOSD-like pathology and its underlying mechanism. Methods: NMOSD-like pathology was induced in mice by intracerebral injection of immunoglobulin-G isolated from NMOSD patient serum, with complement. This mouse model produces the characteristic histological features of NMOSD. A specific AT2R agonist, Compound 21 (C21), was given intracerebrally at day 0 and by intrathecal injection at day 2. Results: Loss of aquaporin-4 and glial fibrillary acidic protein was attenuated by treatment with C21. Administration of C21 induced mRNA for interleukin-10 in the brain. NMOSD-like pathology was exacerbated in interleukin-10-deficient mice, suggesting a protective role. C21 treatment did not attenuate NMOSD-like pathology in interleukin-10-deficient mice, indicating that the protective effect of AT2R stimulation was dependent on interleukin-10. Conclusion: Our findings identify AT2R as a novel potential therapeutic target for the treatment of NMOSD. Interleukin-10 signaling is an essential part of the protective mechanism counteracting NMOSD pathology.

A16523 Mapping of the angiotensin AT2 receptor-coupled signalling network by time-resolved quantitative phosphoproteomics in human aortic endothelial cells identified HDAC-1 and p53 to be involved in AT2 receptor-mediated anti-proliferation

Objectives: The angiotensin AT2-receptor (AT2R) is a main component of the protective arm of the renin-angiotensin system. In order to get new and deeper insights into AT2R mediated signalling, this study mapped the AT2R-coupled phosphoproteome in human aortic endothelial cells (HAEC) by quantitative phosphoproteomics. Methods: HAEC were stimulated with the AT2R agonist C21 (1 μM) for 1, 3, 5 or 20 minutes. Following protein digestion and TMT peptide labelling, samples were subjected to TiO2−SIMAC phospho-peptide enrichment and LC−MS/MS. Data were analysed using Proteome Discoverer and the human sequence libraries SwissProt and UniProt. For confirmation of phosphoproteomics results, expression of p53 and phosphorylation of HDAC-1 were determined by Western Blot. Cellular translocation of HDAC-1 and p53 was assessed by immunofluorescence and activity of HDAC-1 by deacetylation assay. HAEC proliferation was determined by resazurin assay and apoptosis by caspase 3/7 assay. Results: AT2R stimulation of HAEC changed the phosphorylation status of 265 proteins at 471 sites (281 phosphorylations and 190 dephosphorylations). Gene Ontology and STRING analysis revealed the predominant phospho-modification of proteins involved in anti-proliferation, cell differentiation and apoptosis including HDAC-1, p53, BCL2 or HSP90. AT2R-mediated inhibition of HDAC-1 and activation of p53 were confirmed by additional experiments demonstrating changes in cellular localisation of HDAC-1 and p53, in total expression of p53, and in enzymatic activity and phosphorylation of HDAC1. Conclusion: This study mapped the AT2R coupled phospho-signalling network. It identified dephosphorylation and deactivation of HDAC-1 as well as nuclear importation and increased expression of p53 as novel, potential mediators of AT2R-coupled anti-proliferation.

AT2R (Angiotensin AT2 Receptor) Agonist, Compound 21, Prevents Abdominal Aortic Aneurysm Progression in the Rat.

The effects of the selective AT2R (angiotensin AT2 receptor) agonist, Compound 21 (C21), on abdominal aortic aneurysm formation were investigated in normotensive Wistar rats. Abdominal aortic aneurysm was induced by perfusion of isolated aortic segments with elastase. Treatment with C21 (0.03 mg/kg daily) was started after operation and continued for 14 days. Sham-operated animals and vehicle-treated animals after aneurysm induction served as controls. Aortic diameter, aortic wall distensibility, and pulse propagation velocity were measured via ultrasound. Hemodynamic parameters, aortic tissue protein expression, and serum cytokines were analyzed. On day 14 post aneurysm induction, aortic diameter of vehicle-treated animals was increased 1.6-fold compared with sham-operated rats (2.65±0.05 versus 1.70±0.06 mm; P<0.0001). C21 decreased aortic diameter in comparison to vehicle (1.9±0.06 versus 2.65±0.05; P<0.0001). Infrarenal blood velocity and aortic distensibility were reduced, whereas aortic wall stiffness was increased post aneurysm induction. These alterations were significantly ameliorated by treatment with C21 while blood pressure and cardiac contractility remained unchanged. Protein expression of IL-1β (interleukin-1β), NFκB (nuclear factor κB), MMP9 (matrix metalloproteinase 9), TGF-β1 (transforming growth factor-β1), and MLKL (mixed lineage kinase domain-like) in the aorta was significantly ( P<0.05) down-regulated in the C21 group compared with vehicle. Serum concentration of TGF-β1 was decreased by C21 in comparison to vehicle ( P<0.01). AT2R stimulation with C21 prevented extracellular matrix degradation, maintained vascular integrity of the aorta and prevented abdominal aortic aneurysm progression.

Abstract P240: Identifying the Angiotensin AT2-Receptor Coupled Phosphoproteome in Human Aortic Endothelial Cells by Time-Resolved, Quantitative Phosphoproteomics

Objectives: The angiotensin AT2-receptor (AT2R) is a main component of the protective arm of the renin-angiotensin system. In order to get new and deeper insights into AT2R mediated signalling, this study mapped the AT2R-coupled phosphoproteome in human aortic endothelial cells (HAEC) by quantitative phosphoproteomics. Methods: HAEC were stimulated with the AT2R agonist C21 (1 μM) for 1, 3, 5 or 20 minutes. Following protein digestion and TMT peptide labelling, samples were subjected to TiO2–SIMAC phospho-peptide enrichment and LC–MS/MS. Data were analysed using Proteome Discoverer and the human sequence libraries SwissProt and UniProt. For confirmation of phosphoproteomics results, expression of p53 and phosphorylation of HDAC-1 were determined by Western Blot. Cellular translocation of HDAC-1 and p53 was assessed by immunofluorescence and activity of HDAC-1 by deacetylation assay. HAEC proliferation was determined by resazurin assay and apoptosis by caspase 3/7 assay. Results: AT2R stimulation of HAEC changed the phosphorylation status of 265 proteins at 471 sites (281 phosphorylations and 190 dephosphorylations). Gene Ontology and STRING analysis revealed the predominant phospho-modification of proteins involved in anti-proliferation, cell differentiation and apoptosis including HDAC-1, p53, BCL2 or HSP90. AT2R-mediated inhibition of HDAC-1 and activation of p53 were confirmed by additional experiments demonstrating changes in cellular localisation of HDAC-1 and p53, in total expression of p53, and in enzymatic activity and phosphorylation of HDAC1. Conclusion: This study mapped the AT2R coupled phospho-signalling network. It identified dephosphorylation and deactivation of HDAC-1 as well as nuclear importation and increased expression of p53 as novel, potential mediators of AT2R-coupled anti-proliferation.

Angiotensin AT2 receptor agonist, compound 21, maintains vascular integrity and prevents abdominal aortic aneurysm progression in the rat

The effects of the selective angiotensin AT2 receptor agonist, compound 21 (C21), on abdominal aortic aneurysm (AAA) formation were investigated in normotensive Wistar rats.AAA was induced by perfusion of isolated aortic segments with elastase (Anidjar/Dobrin model). Treatment with C21 (0.03 and 0.3 mg/kg daily) was started after surgery and continued for 14 days. Sham operated animals and vehicle-treated animals after aneurysm induction (AI) served as controls. Aortic diameter and wall properties (distensibility, pulse propagation velocity) were measured infrarenally via ultrasound. Hemodynamic parameters, aortic tissue protein expression and serum cytokines were analysed.On day 14 post AI, aortic diameter of vehicle-treated animals was increased 1,6-fold compared to sham operated rats (p < 0.0001). C21 (0.03 mg/kg) decreased aortic diameter in comparison to vehicle (1.9mm ± 0.06 vs. 2.65mm ± 0.06; p < 0.0001). Infrarenal blood velocity and aortic distensibility were reduced, whereas aortic wall stiffness was increased post AI. These alterations were significantly ameliorated by treatment with C21 (p < 0.0001; p < 0.0001; p < 0.05). Blood pressure and cardiac contractility were not altered. Protein expression of IL1 beta, NF kappa B, MMP9, TGF-beta1 and MLKL in the aorta was significantly (p < 0.05) down-regulated in the C21 group compared with vehicle. In primary rat vascular smooth muscle cells, the release of MMP9, TGF-beta1 and MLKL was significantly diminished after C21 (1mM) treatment. Serum concentration of TGF-beta1 was also decreased by C21 in comparison to vehicle (p < 0.01).In conclusion, AT2 receptor stimulation with C21 prevented extracellular matrix degradation, maintained vascular integrity of the aorta and prevented AAA progression.

OP.7C.05] ANGIOTENSIN AT2 RECEPTOR AGONIST, COMPOUND 21, PREVENTS ABDOMINAL AORTIC ANEURYSM PROGRESSION AND THE DECREASE OF AORTIC WALL DISTENSIBILITY IN THE RAT

Objective: We studied the effects of selective AT2 receptor agonist, compound 21, (C21) treatment on experimental abdominal aortic aneurysm (AAA) formation in rats. Design and method: AAA was induced by perfusion of isolated aortic segments with elastase in normotensive Wistar rats. Treatment with C21 (0.03 mg/kg daily) was started after operation and continued for 14 days. Sham operated animals and vehicle-treated animals after aneurysm induction (AI) served as controls. Aortic diameter and blood velocities were measured infrarenally before and on days 7 and 14 post operation via ultrasound biomicroscopy. Analysis of pulse propagation velocity (PPV) and distensibility was performed using the Vevo Vasc® software. Hemodynamic parameters were measured via tail cuff and intraventricular Samba catheter. Aortic tissue expressions of MMP9, IL6, IL1beta, NFkappaB and MLKL as well as serum cytokines were analysed. Results: On day 14 post AI, infrarenal aortic diameter of AAA group was increased 1,55-fold compared to sham operated animals (2.65 mm ± 0.05, n = 8 vs. 1.7 mm ± 0.068, n = 6; p < 0.0001). C21 significantly decreased aortic diameter by 20% on day 7 post AI and by 28% on day 14 compared to vehicle-treated animals (1.9 mm ± 0.06, vs. 2.65 mm ± 0.06; p < 0.0001). Left ventricle parameters and blood pressure were not influenced by AI nor by additional treatment with C21. AI significantly reduced infrarenal blood velocity, distensibility and increased PPV. All these pathological effects were significantly ameliorated in C21 treated rats (p < 0, 0001; p < 0, 0001; p = 0, 0205; 2-wayANOVA). Treatment with C21 also reduced the expression of inflammatory markers IL1 beta, NF kappa B, protease MMP9 and MLKL, the marker of necroptotic cell death in the aortic media. Moreover, C21 reduced the increase of TGF-beta1 in serum (p = 0, 0055). Conclusions: The angiotensin AT2 receptor agonist, C21, prevents abdominal aortic aneurysm progression in the rat without affecting blood pressure or left ventricle parameters. C21 also prevents the decline of infrarenal aortic blood velocity, reduces the decrease of aortic wall distensibility at aneurysm site and the AAA-induced increase of inflammatory and necroptotic markers.

Abstract P237: The At2 Receptor Agonist, C21, Can Also Stimulate Mas and Mrgd Receptors

Background: It is well accepted that Compound 21 (C21) is a highly selective non-peptide angiotensin AT2 receptor agonist. C21 as well as angiotensin (Ang)-(1-7) have cardiovascular protective effects and are opponents of the often detrimental Ang II within the renin-angiotensin system. Since the chemical structure of C21 is similar to the Mas receptor specific non-peptidic agonist AVE0991, and we could recently show that the AT2 receptor blocker, PD123319, can also block the effects of Ang-(1-7) at its natural receptors, Mas and MrgD, we tested whether C21 is also not AT2-specific but can stimulate the two Ang-(1-7) receptors, too. Methods and Results: Using cAMP as readout in receptor-transfected HEK293 cells, we generated pharmacological proof that Mas (EC 50 = 1.995 x 10 -12 M) and MrgD (EC 50 = 2.958 x 10 -9 M) are functional receptors for C21, whereby the three receptor blockers, A779, D-Pro7-Ang-(1-7), and PD123319 showed receptor-specific effects towards C21 signalling. Furthermore, C21 elevated the cAMP concentration in primary cells such as mesangial cells (EC 50 = 1.12 x 10 -6 M). However, significant increase in cAMP levels, but not in PKA activity, was still detectable in mesangial cells isolated from AT2-deficient mice, but completely blunted in Mas/MrgD-double knockouts. Finally, in silico modelling was performed to illustrate the structural similarities and differences between C21, AVE0991, and Ang-(1-7). Conclusions: Our results identify C21 as not being a specific AT2 receptor agonist but also interacting with the two Ang-(1-7) receptors, Mas and MrgD. Therefore, the partial overlap in beneficial effects of Ang-(1-7) and C21 might be based on the stimulation of the same receptors under specific pathophysiological circumstances. This also enforces the revisit of such publications which concluded on AT2 function by only using C21.

Chronic angiotensin AT2R activation prevents high-fat diet-induced adiposity and obesity in female mice independent of estrogen

ObjectiveObesity is a known risk factor for various metabolic disorders and cardiovascular diseases. Recently we demonstrated that female angiotensin AT2 receptor (AT2R) knockout mice on high-fat diet (HFD) had higher body weight and adiposity with a parallel reduction in estrogen (17,β-estradiol/E2). The present study investigated whether the anti-adiposity effects of the AT2R are estrogen-dependent in female mice. MethodsFemale C57BL/6 ovary-intact (Ovi) mice were treated with an AT2R agonist (C21, 0.3mg/kg, daily i.p.). Ovariectomized (Ovx) mice, supplemented with E2 (5μg/day, pellets implanted subcutaneously), were treated with an AT2R agonist (C21, 0.3mg/kg, daily i.p.) or vehicle. After 4-days of pre-treatment with C21, Ovi and Ovx mice were placed on either normal diet (ND) or HFD while the C21 treatment continued for the next 10days. For a long-term study, Ovi mice were placed on HFD and treated with C21 for 12weeks. ResultsOvi mice fed the HFD had increased parametrial white adipose tissue (pWAT) weight, plasma free fatty acid and triglycerides compared to Ovi mice on ND. Ovariectomy alone caused similar changes in these parameters which were further increased by HFD-feeding. C21 treatment attenuated these HFD-induced changes in Ovi as well as Ovx mice. HFD also, increased the liver/body-weight ratio and decreased the liver expression of the β-oxidation enzyme, carnitine palmitoyltransferase 1 (CPT1-A). C21 treatment attenuated these changes as well. The long-term C21 treatment of Ovi mice lowered the HFD-induced body weight gain, increase in pWAT weight, parametrial adipocyte size and hyperinsulinemia induced by HFD. Finally, HFD drastically reduced urinary estrogen and the beneficial metabolic changes in response to C21-treatment occurred without significantly increasing urinary estrogen. ConclusionWe suggest that the pharmacological activation of AT2R by the agonist C21 reduces adiposity and body weight gain independent of estrogen in female mice. Improvement in fatty acid metabolism is a potential mechanism by which the AT2R exerts anti-adiposity effects.

Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist. HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS. FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules. Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor. Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%). FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p&lt;0.0001). Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.

Vascular effects of a tripeptide fragment of novokinine in hypertensive rats: Mechanism of the hypotensive action

BackgroundActivation of angiotensin AT2 receptors (AT2R) counteracts vasoconstrictor effects of AT1R stimulation and contributes to blood pressure control. We examined effects on mean arterial pressure (MAP) and renal hemodynamics of LKP, a tripeptide fragment of novokinine, an established AT2R agonist. MethodsEffects of intravenous LKP infusion and then superimposed losartan (AT1R antagonist) on MAP, total renal (RBF, Transonic probe) and renal medullary blood flows (laser-Doppler), and on renal excretion, were examined in anesthetized (1) Wistar rats with acute norepinephrine-induced hypertension, untreated or pretreated with AT2R antagonist (PD 123319) and (2) spontaneously hypertensive rats (SHR) maintained on standard or high-sodium (HS) diet. ResultsIn Wistar rats LKP decreased MAP (−4%, p<0.01) and increased renal medullary perfusion, these effects were abolished in rats pretreated with PD 123319 in which a post-LKP increase in MAP (+6%, p<0.02) occurred. LKP did not alter MAP in SHR; in those on HS diet RBF decreased (−14%, p<0.02), the response that was reverted by losartan. Addition of losartan always decreased or tended to decrease MAP. ConclusionsLKP lowered MAP in norepinephrine-induced hypertension, probably via activation of AT2R. At reduced availability of AT2R, as in SHR, LKP appeared to bind to different receptors, possibly AT1, and induced systemic or renal vasoconstriction. Compared to the parent novokinine, a smaller LKP molecule might be easier absorbed after oral application and more useful in therapy.

Abstract 620: Angiotensin AT2 Receptor Exerts an Anti-inflammatory Response in Lipopolysaccharide-activated THP-1 Macrophages

Macrophages have been shown to be an important contributor to the pathogenesis of hypertension and stroke. The angiotensin AT2 receptor (AT2R), which is expressed in macrophages, is known to promote vasodialation, natriuresis and lower inflammation. The goal of the present study was to explore the anti-inflammatory role of AT2R stimulation in human macrophage-like THP-1 cells activated by lipopolysaccharide (LPS). Phorbol 12-myristate 13-acetate (PMA) differentiated macrophage-like THP-1 cells were treated with AT2R agonist C21 (1 μmol/L) for 30 minutes prior to activation with LPS (1 μg/ml). Media and cells were collected after 24 hours and were analyzed for levels of pro- and anti-inflammatory cytokines and proteins. Pre-treatment with C21 resulted in a 4-fold increase (104.8±6.1 vs 406.7±52.3) in anti-inflammatory interleukin-10 (IL-10) production and a 5-fold decrease (3560±237 vs 588.8±15.94) in pro-inflammatory tumor necrosis factor-α (TNF-α) levels in the media in response to LPS. Predictably, LPS resulted in a 6-fold up-regulation of iNOS expression which was prevented with C21 pre-treatment. A modest decrease in the anti-inflammatory macrophage mannose receptor C type 2 (MRC2) expression was detected with LPS treatment. AT2R agonist pre-treatment, however, increased this receptor expression by ~70% after LPS activation. C21 alone also resulted in a 20% increase in MRC2 expression compared to untreated controls. The anti-inflammatory effect of AT2R activation was abolished in the presence of neutralizing IL-10 antibody (1 μg/ml), indicating a central role for IL-10 in mediating the beneficial response to C21 in LPS activated macrophages. Further, inhibition of nitric oxide (NO) by L-NAME prior to C21 pre-treatment also prevented the decrease in TNF-α and increase in IL-10 in response to AT2R agonist, which suggests that the anti-inflammatory response to C21 may be mediated via increase in NO production prior to LPS activation of macrophages. In conclusion, AT2R stimulation may potentially suppress the inflammatory response of macrophages to LPS by shifting the balance from pro- to anti-inflammatory cytokine production and may prove to be beneficial in the control of the inflammatory component of stroke and hypertension.