Agonist C21 Research Articles

Abstract MP32: Megalin and lysosomal disruption in the kidney epithelial cells of high salt diet fed rats: protective role of angiotensin II type 2 receptor

Earlier we have reported that 2-days high salt diet (HSD) feeding caused proteinuria in obese rats and the AT 2 R agonist treatment prevented proteinuria. Since HSD feeding for 2 days and the AT 2 R activation did not affect blood pressure, GFR, glomerular structure, and inflammation, the mechanism responsible for proteinuria remains unknown. Megalin is a proximal tubule epithelial cells endocytic receptor responsible for filtered protein reabsorption. The absorbed proteins are directed to lysosomes for degradation. Lysosomes is a nutrient sensing subcellular compartment and a critical part of the endocytic machinery and cellular trafficking. We hypothesized that high salt intake caused lysosomal stress, which negatively impacted megalin-protein cellular trafficking and thus caused proteinuria. Therefore, we evaluated the markers of lysosomal stress and megalin expression in the proximal tubules of HSD fed obese Zucker rats. Urinary excretion of N-acetyl-glucosaminidase (NAG), which is a marker of lysosomal injury and oxidative stress, was significantly increased in HSD fed rats compared with normal salt diet (NSD: 16.6 vs HSD: 40.5 RFU/hr/µg Cr). This increase was associated with an increase in another oxidative stress marker H 2 O 2 in the urine (NSD: 0.37 vs HSD: 1.72 ΔRFU/min). Confocal analysis revealed that megalin was localized in the lysosomes in HSD fed rats, as megalin was co-localized with the lysosomal marker LAMP-1 in the proximal tubules. Moreover, in HSD fed rats there was a remarkable increase (2-fold) in the lysosomal size (fig) and number per tubule (NSD: 18 vs HSD: 48). The AT 2 R agonist C21 (0.3 mg/Kg/day, i.p.) treatment of HSD-fed rats restored surface megalin expression, reduced urinary NAG and H 2 O 2 and the lysosomal number and size. Our in vitro studies performed in OK cells (proximal tubule epithelial cells) treated with high NaCl (100 mM) without/with the AT 2 R agonist C21 (1µM) also revealed that compared to NSD, high NaCl reduced megalin surface expression and C21 treatment prevented it. We conclude that high salt intake causes lysosomal stress. Particularly, large lysosomal size potentially is affecting endocytic machinery, including impairing megalin-protein dissociation and megalin recycling, thus causing proteinuria.

Angiotensin II Type 2 Receptor Inhibits M1 Polarization and Apoptosis of Alveolar Macrophage and Protects Against Mechanical Ventilation-Induced Lung Injury.

Angiotensin II (Ang II) is associated with macrophage polarization and apoptosis, but the role of the angiotensin type 2 receptor (AT2R) in these processes remains controversial. However, the effect of AT2Rs on alveolar macrophages and mechanical ventilation-induced lung injury has not been determined.Mechanical ventilation-induced lung injury in Sprague‒Dawley (SD) rats and LPS-stimulated rat alveolar macrophages (NR8383) were used to determine the effects of AT2Rs, selective AT2R agonists and selective AT1Rs or AT2R antagonists. Macrophage polarization, apoptosis, and related signaling pathways were assessed via western blotting, QPCR and flow cytometry.AT2R expression was decreased in LPS-stimulated rat alveolar macrophages (NR8383). Administration of the AT2R agonist CGP-42112 was associated with an increase in AT2R expression and M2 polarization, but no effect was observed upon administration of the AT2R antagonist PD123319 or the AT1R antagonist valsartan. In mechanical ventilation-induced lung injury in Sprague‒Dawley (SD) rats, the administration of the AT2R agonist C21 was associated with attenuation of the pathological damage score, lung wet/dry weight, cell count and protein content in BALF. C21 can significantly reduce proinflammatory factor TNF-α, IL-1β levels, increase anti-inflammatory factor IL-4, IL-10 levels in BALF, compared with the model group (p < 0.01). Similarly, compared with those at the same time points, the M1/M2 ratios in alveolar macrophages and apoptosis in peritoneal macrophages at 4h, 6h and 8h in the mechanical ventilation models were lower after C21 administration.These findings indicated that the expression of AT2Rs in alveolar macrophages mediates M1 macrophage polarization and apoptosis and that AT2Rs play a protective role in mediating mechanical ventilation-induced lung injury.

Angiotensin-II type 2 receptor-mediated renoprotection is independent of receptor Mas in obese Zucker rats fed high-sodium diet.

The consumption of a high-sodium diet (HSD) is injurious and known to elevate blood pressure (BP), especially in obesity. Acute infusion studies depict a functional interdependency between angiotensin-II type 2 receptor (AT2R) and receptor Mas (MasR). Hence, we hypothesize that the subacute blockade of MasR should reverse AT2R-mediated renoprotection in obese Zucker rats (OZRs). Male OZRs were fed an HSD (for 14days) and treated with the AT2R agonist C21 (100ng/min) without or with a MasR antagonist A779 (1,000ng/min). The indices of oxidative stress, proteinuria, kidney injury, and BP were measured before and after, along with the terminal measurements of an array of inflammatory and kidney injury markers. The HSD significantly decreased the estimated glomerular filtration rate and urinary osmolality and increased thirst, diuresis, natriuresis, kaliuresis, plasma creatinine, urinary excretion of H2O2, proteinuria, renal expression and urinary excretion of kidney injury markers (NGAL and KIM-1), and BP indexes. The HSD feeding showed early changes in the renal expression of CRP, ICAM-1, and galectin-1. The C21 treatment prevented these pathological changes. The MasR antagonist A779 attenuated C21-mediated effects on the urinary excretion and renal expression of NGAL and oxidative stress in the absence of inflammation and BP changes. Overall, we conclude that the subacute functional interactions between AT2R and MasR are weak or transient and that the beneficial effects of AT2R activation are independent of the MasR blockade in the kidney of male obese rats fed an HSD.

Acute In Vivo Administration of Compound 21 Stimulates Akt and ERK1/2 Phosphorylation in Mouse Heart and Adipose Tissue

The angiotensin II type 2 (AT2) receptor has a role in promoting insulin sensitivity. However, the mechanisms underlying the AT2 receptor-induced facilitation of insulin are still not completely understood. Therefore, we investigated whether acute in vivo administration of AT2 receptor agonist compound 21 (C21) could activate insulin signaling molecules in insulin-target tissues. We report that, in male C57BL/6 mice, an acute (5 min, 0.25 mg/kg; i.v.) injection of C21 induces the phosphorylation of Akt and ERK1/2 at activating residues (Ser473 and Thr202/Tyr204, respectively) in both epididymal white adipose tissue (WAT) and heart tissue. In WAT, the extent of phosphorylation (p) of Akt and ERK1/2 induced by C21 was approximately 65% of the level detected after a bolus injection of a dose of insulin known to induce maximal activation of the insulin receptor (IR). In the heart, C21 stimulated p-Akt to a lesser extent than in WAT and stimulated p-ERK1/2 to similar levels to those attained by insulin administration. C21 did not modify p-IR levels in either tissue. We conclude that in vivo injection of the AT2 receptor agonist C21 activates Akt and ERK1/2 through a mechanism that does not involve the IR, indicating the participation of these enzymes in AT2R-mediated signaling.

Angiotensin II type 2 receptor agonist attenuates LPS-induced acute lung injury through modulating THP-1-derived macrophage reprogramming.

Acute respiratory distress syndrome (ARDS) is a devastating respiratory disorder, characterized by overwhelming inflammation in the alveoli without effective pharmacological treatment. We aimed to investigate the effect and mechanism of angiotensin II type 2 receptor (AT2R) agonist, Compound 21 (C21), on the lipopolysaccharide (LPS)-induced acute lung injury (ALI) model. The protective effect of C21 was evaluated via enzyme-linked immunosorbent assay (ELISA), Western blot (WB), real-time PCR, and fluorescence microscopy in LPS-challenged THP1-derived macrophages. Besides, the in vivo efficacy of C21 was assessed using cell counting, ELISA, protein quantification, hematoxylin-eosin (H&E) staining, and WB in an LPS-induced ALI mouse model. The results showed thatC21 significantly inhibited the secretion of pro-inflammatory cytokines (CCL-2, IL-6), overproduction of intracellular ROS, and activation of inflammatory pathways (NF-κB/NLRP3, p38/MAPK) in THP-1 cell-derived macrophages stimulated by LPS. In in vivo study, intraperitoneal injection of C21 could reduce airway leukocytes accumulation and chemokine/cytokine (keratinocyte chemoattractant (KC), IL-6) generation, as well as alleviate diffuse alveolar damage induced by LPS. Conclusively,the AT2R agonist C21 significantly inhibited LPS-stimulated excess inflammatory responses and oxidative stress in macrophages. Meanwhile, C21 could effectively alleviate acute inflammation and tissue damage in the lungs of ALI mice challenged by LPS. The results of this study bring new hope for the early treatment of ALI/ARDS.

Effects of the Oral Angiotensin II Type 2 Receptor Agonist C21 in Sugen-Hypoxia Induced Pulmonary Hypertension in Rats.

Substantial evidence supports the involvement of the renin-angiotensin system in pulmonary hypertension (PH), and the angiotensin II type 2 receptor (AT2R) is known to exert tissue protective actions. The effect of the selective AT2R agonist C21 (also known as Compound 21 or buloxibutid) was evaluated in the rat Sugen-hypoxia PH model. After a single injection of Sugen 5416 and hypoxia for 21 days, C21 (2 or 20 mg/kg) or vehicle was administered perorally twice daily from Day 21 to Day 55. On Day 56, hemodynamic assessments were performed, and lung and heart tissue were prepared for quantification of cardiac and vascular remodeling and fibrosis. Treatment with C21 20 mg/kg improved cardiac output and stroke volume and decreased right ventricular hypertrophy (all p < 0.05). Treatment with C21 2 mg/kg significantly decreased vessel wall and muscular layer thickness and increased the luminal opening in vessels >100 μm (all p < 0.05). There were no significant differences between the two C21 doses on any parameter, and post hoc analyses comparing the merged C21 groups with the vehicle group showed that C21 treatment reduced vascular remodeling (reduced endothelial proliferation and thickening of the vascular wall) in vessels of all sizes; moreover, the diastolic pulmonary artery pressure and right ventricular pressure were reduced along with reduction of right ventricular hypertrophy. Sugen 5416 and hypoxia increased pulmonary collagen deposition, which was counteracted by C21 20 mg/kg. In conclusion, the effects of C21 on vascular remodeling, hemodynamic alterations, and fibrosis suggest that AT2R agonists may have a role in Group 1 and 3 PH treatment.

Angiotensin II type 2 receptor activation preserves megalin in the kidney and prevents proteinuria in high salt diet fed rats

Proteinuria is a risk factor for and consequence of kidney injury. Angiotensin II type 2 receptor (AT2R) is an emerging reno-protective target and is anti-proteinuric under pathological conditions, including high salt-fed obese animals. However, the mechanisms remain unknown, particularly whether the anti-proteinuric activity of AT2R is independent of its anti-hypertensive and anti-inflammatory effects. In the present study, obese Zucker rats were fed high sodium (4%) diet (HSD) for 48 h, a time in which blood pressure does not change. HSD caused proteinuria without affecting glomerular slit diaphragm proteins (nephrin and podocin), glomerular filtration rate, inflammatory and fibrotic markers (TNFα, IL-6, and TGF-β), ruling out glomerular injury, inflammation and fibrosis but indicating tubular mechanisms of proteinuria. At cellular and molecular levels, we observed a glycogen synthase kinase (GSK)-3β-mediated megalin phosphorylation, and its subsequent endocytosis and lysosomal degradation in HSD-fed rat kidneys. Megalin is a major proximal tubular endocytic protein transporter. The AT2R agonist C21 (0.3 mg/kg/day, i.p.) administration prevented proteinuria and rescued megalin surface expression potentially by activating Akt-mediated phosphorylation and inactivation of GSK-3β in HSD-fed rat kidneys. Overall, AT2R has a direct anti-proteinuric activity, potentially via megalin regulation, and is suggested as a novel target to limit kidney injury.

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.

PS-BPB04-6: PROOF OF BIOLOGICAL ACTIVITY AND EXPLORATION OF EARLY SIGNALING EVENTS OF ANGIOTENSIN-(1–5)

Introduction: Recombinant human ACE2 increases the circulating levels of angiotensin-(1–5) [Ang-(1–5)], a peptide thus far regarded as biologically inactive. Since ACE2 is a central component of the protective RAS, we hypothesized that Ang-(1–5) is a new biologically active peptide within this hormonal system. Objective: To investigate biological activity and signaling mechanisms of Ang-(1–5). Methods: In order to show a biological effect and to test whether Ang-(1–5) signals through the AT2-receptor (AT2R), nitric oxide (NO) release was measured by DAF-FM fluorescence in AT2R transfected (AT2R-CHO) or non-transfected (NT-CHO) CHO cells, treated with Ang-(1–5) or C21 (AT2R agonist, positive control) (0.1 nM to 10 μM) for 15 minutes. Vehicle (cell media) treated cells served as negative control. To investigate Ang-(1–5) signaling patterns, human aortic endothelial cells (HAEC) were treated with vehicle or Ang-(1–5) (1 μM) for 1, 3, 5 or 20 minutes. Proteins were harvested, digested and labeled with TMTpro-16plex. Phosphopeptide enrichment was carried out by TiO2. Samples were subjected to LC-MS/MS analysis and the resulting mass spectra were searched against the human SwissProt database. Results: Ang-(1–5) induced a concentration-dependent increase in NO production in AT2-CHO cells (Emax: 65.60 ± 14.02%), thus proving its biological activity. Ang-(1–5) had 69% higher efficacy than the established AT2R agonist C21 (Emax: 38.76 ± 10.24%). Ang-(1–5) seems to signal through the AT2R, because effects on NO release were absent in NT-CHO cells. Treatment of HAEC with Ang-(1–5) significantly modified the phosphorylation status of 831 proteins at 1799 residues. The majority of residues (1079) were dephosphorylated while 729 residues were phosphorylated. Changes in protein phosphorylation in response to Ang-(1–5) occurred at all investigated time points, most of them after 20 minutes. Functional bioinformatic analysis revealed a cluster of proteins involved in cell cycle and cell division regulation. Conclusion: This study provides evidence that Ang-(1–5) is an endogenous AT2R agonist with high efficacy towards the AT2R. The early signaling phosphorylation pattern resembles those of other protective RAS agonists, such as C21 and Ang-(1–7).

Activation of angiotensin II type 2 receptor attenuates lung injury of collagen-induced arthritis by alleviating endothelial cell injury and promoting Ly6Clo monocyte transition

As one of the most frequent extra-articular manifestations of rheumatoid arthritis (RA), interstitial lung disease (ILD) is still challenging due to unrevealed pathophysiological mechanism. To address this question, in the present study, we used the classical collagen-induced arthritis (CIA) mouse model to determine the related-immune mechanism of lung injury and possible pharmacological treatment for RA-ILD. At the peak of arthritis, we found CIA mice developed apparent lung injury, characterized by interstitial thickening, inflammatory cell infiltration, and lymphocyte follicle formation. Additionally, the endothelial injury occurred as the number of endothelial cells (ECs) and their CD31 expression decreased. Along with those, monocytes, predominantly Ly6Chi monocytes with pro-inflammatory phenotype, were also increased. While in the remission period of arthritis, ECs gradually increased with retrieved CD31 expression, leading to decreased infiltrating monocytes, but boosted Ly6Clo population. Ly6Clo monocytes were prone to locate around damaged ECs, promoted ECs proliferation and vascular tube formation, and lessened the expression of adhesion molecules. In addition, we evaluated angiotensin II type 2 receptor (Agtr2), which has been demonstrated to be protective against lung injury, could be beneficial in RA-ILD. We found elevated Agtr2 in CIA lung tissue, and activation of Agtr2, within its specific agonist C21, alleviated the pulmonary inflammation in vivo, reduced ECs injury, and promoted monocytes conversion from Ly6Chi to Ly6Clo monocytes in vitro. Our data reveal a potential pathological mechanism of RA-ILD that involves ECs damage and inflammatory monocytes infiltration and provide a potential drug target, Agtr2, for RA-ILD treatment.

Angiotensin Type 2 Receptor Pharmacological Agonist Relieves Neurocognitive Deficits via Reducing Neuroinflammation and Microglial Engulfment of Dendritic Spines.

Mechanically ventilated patients sufferingcritical illness are at high risk of developing neurocognitive impairments. Angiotensin type 2 receptor (AGTR2) has been demonstrated to be anti-inflammatory and neuroprotective. The present study thus aimed to investigate whether AGTR2 can alleviate cerebral dysfunction in mice subjected to cochallenge with lipopolysaccharide (LPS) and mechanical ventilation (MV), and to reveal the underlying mechanism. We utilized a mice model that received a single injection of LPS (1mg/kg, intraperitoneally) followed 2h later by MV (10ml/kg, lasting for 2h). Pretreatment with the AGTR2 pharmacological agonist C21 (0.03, 0.3, and 3mg/kg, intraperitoneally, once daily, lasting for 10days). Locomotor activity and behavioral deficits were evaluated 24h post-MV by open-field and fear-condition tests. Brain hippocampus and prefrontal cortex tissues were collected for immunofluorescence staining and western blotting to evaluate the resulting impacts on microglia, including morphological traits, functional markers, synaptic engulfment, superoxide production, and signaling molecules. Compared with vehicle-control, pre-administrated C21 reduced the branch endpoints and length of microglia processes in a dose-dependent manner in mice subjected to LPS/MV. The neuroprotective effect of AGTR2 was behaviorally confirmed by the improvement of memory decline in LPS/MV-treated mice following C21 pretreatment. In addition to morphological alterations, C21 reduced microglial functional markers and reduced microglial-dendrite contact and microglial engulfment of synaptic protein markers. In terms of the underlying molecular mechanism, AGTR2 stimulation by C21 leads to activation of protein phosphatase 2A, which subsequently mitigates microglial PKCδ and NF-κB activation, and inhibites NOX2-derived ROS production. The AGTR2 agonist C21 alleviates behavioral deficits in those mice subjected to LPS/MV, via mechanisms that involve reactive microglia and abnormal synaptic plasticity in NOX2-derived ROS and the PKCδ-NFκB pathway.

Investigation of the effect of the angiotensin II type 2 receptor (AT2R) agonist C21 on plasma NT-proBNP, a diagnostic biomarker, in subjects hospitalised with COVID-19

Abstract The prohormone N-terminal pro-B-type natriuretic peptide (NT-proBNP) is released from stretched cardiac myocytes and is a diagnostic biomarker for heart failure and cardiac dysfunction as well as pulmonary embolism and pneumonia that are frequent complications to severe Coronavirus Disease 2019 (COVID-19). NT-proBNP is frequently elevated in COVID-19. In a recent publication, it was demonstrated that NT-proBNP was strongly associated with mortality in patients with COVID-19, and further investigation of its usefulness as a prognostic tool to predict disease outcomes in COVID-19 was suggested (1). In the recently completed phase 2 trial (angiotensin II type 2 receptor agonist COVID-19 trial [ATTRACT]; NCT04452435) in subjects hospitalised with COVID-19, it was investigated whether treatment with the AT2R agonist C21 for 7 days affected the release of the plasma biomarker NT-proBNP. ATTRACT was a randomised, double-blind, placebo-controlled, phase 2 trial that investigated the safety and efficacy of C21 treatment (100 mg twice daily) for 7 days in hospitalised subjects with COVID-19, not requiring mechanical ventilation. The results of the trial demonstrated that treatment with C21 on top of standard of care (vast majority of patients received glucocorticoids) significantly reduced the proportion of subjects requiring supplemental oxygen at Day 14, indicating faster recovery with C21 treatment compared to placebo. Blood samples for exploratory analysis were taken before and after 7 days of treatment with C21 or placebo. Plasma NT-proBNP was markedly elevated in both treatment groups before treatment, with average values of 357 and 438 pg/mL in the placebo and C21 groups, respectively, as compared to normal levels of approximately &amp;lt;100 pg/mL. After 7 days of treatment, the C21 group experienced a dramatic reduction in plasma NT-proBNP (by 259 pg/mL) as compared to the placebo group (63 pg/mL) (p=0.02). The results show that short-term C21 treatment decreased the release of NT-proBNP in subjects hospitalised with COVID-19. Further investigations are needed to elucidate whether this is related to effects on COVID-19-induced pulmonary damage or direct protective effects on the heart. We are currently conducting a global phase 3 trial (VP-C21–008) further investigating the effect of C21 in subjects hospitalised with COVID-19 including determination of NT-proBNP. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Vicore Pharma AB

Angiotensin II type-2 receptor activation in alveolar macrophages mediates protection against cigarette smoke-induced chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally. Cumulative evidence has implicated renin-angiotensin system (RAS) in the pathogenesis of COPD. Alveolar macrophages (AMs) are the first line immune defense in the respiratory system and play a critical role in the lung homeostasis. This study aimed to investigate the role of AMs in contributing to the protective effects of angiotensin II type-2 receptor (AT2R) activation in cigarette smoke (CS)-induced COPD. The AM polarization, phagocytosis and metabolism, and the underlying biochemical mechanisms of compound 21 (C21), a selective and potent non-peptide small molecule AT2R agonist, were evaluated in a two-week CS-induced COPD mouse model. C21 restored AM phagocytosis ability, reversing CS-induced AM phagocytosis impairment. CS exposure polarized AMs towards M1 phenotype, whereas, C21 skewed the CS-exposed AMs towards M2 phenotype. C21 reprogrammed CS-exposed AM metabolism from a high glycolysis-driven process to support inflammation energy demand to a high mitochondrial respiration process to limit inflammation. Besides, C21 upregulated AT2R and Mas receptor levels in CS-exposed AMs, favoring the anti-inflammatory Ang II/AT2R axis and Ang 1–7/Mas axis in the RAS. C21 restored the normal levels of sirtuin 1 (SIRT1) and MAPK phosphatase 1 (MKP1) in CS-exposed AMs, leading to the reduction of phospho-p38, phospho-ERK and p65 subunit of NF-κB levels in CS-exposed AMs. We report here for the first time that AT2R agonist C21 acts by boosting the protective functions of AMs against CS-induced COPD, and our results support the development of AT2R agonist for the treatment of COPD.

Abstract 116: Proof Of Biological Activity And Exploration Of Early Signaling Events Of Angiotensin-(1-5)

Introduction: Recombinant human ACE2 increases the circulating levels of angiotensin-(1-5) [Ang-(1-5)], a peptide thus far regarded as biologically inactive. Since ACE2 is a central component of the protective RAS, we hypothesized that Ang-(1-5) is a new biologically active peptide within this hormonal system. Objective: To investigate biological activity and signaling mechanisms of Ang-(1-5). Methods: In order to show a biological effect and to test whether Ang-(1-5) signals through the AT 2 -receptor (AT 2 R), nitric oxide (NO) release was measured by DAF-FM fluorescence in AT 2 R transfected (AT 2 R-CHO) or non-transfected (NT-CHO) CHO cells, treated with Ang-(1-5) or C21 (AT 2 R agonist, positive control) (0.1nM to 10μM) for 15 minutes. Vehicle (cell media) treated cells served as negative control. To investigate Ang-(1-5) signaling patterns, human aortic endothelial cells (HAEC) were treated with vehicle or Ang-(1-5) (1μM) for 1, 3, 5 or 20 minutes. Proteins were harvested, digested and labeled with TMTpro-16plex. Phosphopeptide enrichment was carried out by TiO 2 . Samples were subjected to LC-MS/MS analysis and the resulting mass spectra were searched against the human SwissProt database. Results: Ang-(1-5) induced a concentration-dependent increase in NO production in AT 2 -CHO cells (E max : 65.60 ± 14.02%), thus proving its biological activity. Ang-(1-5) had 69% higher efficacy than the established AT 2 R agonist C21 (E max : 38.76 ± 10.24%). Ang-(1-5) seems to signal through the AT 2 R, because effects on NO release were absent in NT-CHO cells. Treatment of HAEC with Ang-(1-5) significantly modified the phosphorylation status of 831 proteins at 1799 residues. The majority of residues (1079) were dephosphorylated while 729 residues were phosphorylated. Changes in protein phosphorylation in response to Ang-(1-5) occurred at all investigated time points, most of them after 20 minutes. Functional bioinformatic analysis revealed a cluster of proteins involved in cell cycle and cell division regulation. Conclusion: This study provides evidence that Ang-(1-5) is an endogenous AT 2 R agonist with high efficacy towards the AT 2 R. The early signaling phosphorylation pattern resembles those of other protective RAS agonists, such as C21 and Ang-(1-7).

Angiotensin II type 2 receptor pharmacological agonist, C21, reduces the inflammation and pain hypersensitivity in mice with joint inflammatory pain

Primary and secondary hyperalgesia develop in response to chronic joint inflammation due to peripheral and central mechanisms. Synovial macrophage and spinal microglia are involved in pain sensitization in arthritis. The level of angiotensin II type 2 receptor (AT2R) is related to the severity of arthritis. This study aimed to determine the role of AT2R in primary and secondary hyperalgesia in joint inflammatory pain in mice. After intra-articular CFA injection, primary hyperalgesia in the ipsilateral knee joint was measured by pressure application meter and gait analysis, secondary hypersensitivity in ipsilateral hind-paw was measured by von-Frey and Hargreaves tests following a combination of global AT2R-deficient (Agtr2-/-) mice and AT2R pharmacological agonist C21. Synovial macrophage and spinal microglia were collected for flow cytometry. Morphological reconstruction of microglia was detected by immunostaining. AT2R expression was investigated by quantitative polymerase chain reaction and western blot. Neuronal hyperactivity was evaluated by c-Fos and CGRP immunostaining. We found that pain hypersensitivity and synovial inflammation in Agtr2-/- mice were significantly exacerbated compared with wild-type mice; conversely, systemically administrated C21 attenuated both of the symptoms. Additionally, spinal microglia were activated, and an abundant increase of spinal AT2R was expressed on activated microglia in response to peripheral joint inflammation. Intrathecally-administrated C21 reversed the secondary hypersensitivity, accompanied by alleviation of spinal microglial activation, spinal neuronal hyperactivity, and calcitonin gene-related peptide content. These findings revealed a beneficial role of AT2R activating stimulation against pain hypersensitivity in joint inflammatory pain via direct modulation of synovial macrophage and spinal microglial activity.

COMPOUND 21 STIMULATES THE ACTIVATION OF AKT AND ERK1/2 IN MOUSE ADIPOSE TISSUE IN VIVO

Objective: To analyze signaling events triggered by in vivo stimulation of the AT2 receptor. Design and method: Male B57Bl/6 mice were used as model at the age of 3 month. Mice were subjected to a bolus in vivo intravenous injection (cava vein) of saline buffer containing either the AT2 agonist compound 21 (0.25 mg/Kg), a mixture of C21 (0.25 mg/Kg) and the AT2 antagonist PD123319 (2.5 mg/Kg) or saline for baseline assesment under anesthesia. A group of animals that received insulin (0.25 mg/Kg) were used as positive control. The heart and the inguinal adipose tissue were removed after 1 and 3 min respectively, flash-frozen and kept at -70 C until analysis. Proteins from heart and adipose tissue were solubilized in a buffer containing 1% Triton together withprotease and phosphatase inhibitors. Protein content was measured by the BCA method and homogenates were subjected to Western Blotting using specific anti-phospho Akt or anti-phospho ERK1/2 antibodies. The content of Akt and ERK content was detected through incubation with either anti-Akt or anti-ERK1/2 antibodies. Specific bands were detected by chemiluminescence. Protein loading control was checked by coomassie blue staining of every PVDF membrane employed. Results: Injection of C21 resulted in the activation of both Akt and ERK1/2 in mouse adipose tissue. The extent of activation of Akt and ERK1/2 attained was equivalent to approximately 30–40% of the levels of activation of these enzymes obtained after an injection of a dose of insulin known to generate maximum activation of these signaling components. Co-infusion of C21 and PD123319 abolished the in vivo activation of Akt and ERK1/2, indicating that these events were mediated by the AT2 receptor. Conclusions: In vivo administration of the AT2 receptor agonist C21 resulted in the stimulation of the phosphorylation of both Akt and ERK1/2 at stimulatory sites indicative of activation of these enzymes. Co-infusion of C21 with the specific AT2 receptor antangonist blunted this effect. It is concluded that the AT2 receptor is capable of recruiting both Akt as ERK1/2 as signaling molecules in mouse adipose tissue in vivo.

2-Alkyl substituted benzimidazoles as a new class of selective AT2 receptor ligands

Ligands comprising a benzimidazole rather than the imidazole ring that is common in AT2R ligands e.g. in the AT2R agonist C21, can provide both high affinity and receptor selectivity. In particular, compounds encompassing benzimidazoles, substituted in the 2-position with small bulky groups such as an isopropyl (Ki = 4.0 nM) or a tert-butyl (Ki = 5.3 nM) or alternatively a thiazole heterocycle (Ki = 5.1 nM) demonstrate high affinity and AT2R selectivity. An n-butyl chain, as found in the AT1R selective sartans, makes the ligand less receptor selective. The isobutyl group on the biaryl scaffold present in most AT2R selective ligands reported so far was originally derived from the nonselective potent AT1R/AT2R ligand L-162,313. Notably, in all ligands discussed herein, the isobutyl group was substituted by an n-propyl group and ligands with high affinity to AT2R were provided and in addition the majority of them demonstrate a favorable AT2R/AT1R selectivity. The introduction of fluoro atoms in various positions had no pronounced effect on the affinity data. Ligands with a thiazole or a tert-butyl group attached to the 2-position and with a terminal trifluoromethyl butoxycarbonyl sidechain exhibited a similar stability as C21 in human liver microsomes, while other ligands examined were less stable in the microsome assay.

Angiotensin II AT2 receptor ligands with phenylthiazole scaffolds

The syntheses and the AT1R and AT2R binding data of a series of new small molecule ligands are reported. These ligands comprise a phenylthiazole scaffold rather than the biphenyl or phenylthiophene scaffolds found in essentially all of the previously described ligands originating from the nonselective AT1R/AT2R ligand L-162,313 and the AT2R selective agonist C21, the latter now in Phase II/III clinical trials. A phenylthiazole rather than the phenylthiophene scaffold that is present in the AT2R selective agonist C21 and in the AT2R selective antagonist C38 had a deleterious effect on the affinity to AT2R. Nevertheless, a significant improvement could be accomplished by introduction of a small bulky alkyl group in the 2-position of the imidazole ring attached through a methylene group bridge to the phenylthiazole scaffold. Hence, a combination of a 2-tert-butyl or a 2-isopropyl group and a butoxycarbonyl furnished potent AT2R selective ligands. Furthermore, a high affinity ligand derived from L-162,313 and exhibiting a > 35 fold selectivity for AT1R was identified (10). The ligand 21 with the 2-tert-butyl group and ∼ 35 fold selectivity for AT2R, demonstrated high stability in human, rat and mouse liver microsomes and a very attractive profile with regard to the inhibition of common drug-metabolizing CYP enzymes. Thus, very low levels of inhibition of CYP 3A (5%), 2D6 (12%), 2C8 (26%), 2C9 (23%) and 2B6 (24%) were observed with the 2-tert-butyl derivative comprising the methoxycarbonyl sulfonamide function, levels that are significantly lower than those obtained with C21 under the same experimental conditions.

AT1R blockade together with AT2R stimulation prevents aortic aneurysm in mice with progressively severe Marfan syndrome

Angiotensin II receptor signaling regulate vascular tone by stimulating either vasoconstriction (AT1R) or vasodilation (AT2R). AT1R antagonism (via losartan) has been shown to mitigate thoracic aortic aneurysm (TAA) progression in mouse models of Marfan syndrome (MFS). Here we monitored multiple pathological readouts of TAA in MFS mice chronically treated for 90 days with either losartan, the AT2R agonist C21, or both. While C21 had no apparent effects on arterial disease by itself, it significantly enhanced losartan’s efficacy by preventing death of MFS mice from ruptured TAA during the treatment period. Specifically, the combination therapy normalized the diameters of the aortic root and ascending aorta, the architecture of the vessel wall, endothelial‐dependent relaxation, and circumferential wall strength (a strong biomechanical indicator of aneurysmal vulnerability). Computational analyses of bulk RNASeq data predicted that the combination therapy largely decreased expression of abnormally upregulated genes, principally those associated with inflammatory pathways. By contrast, these in silico analyses predicted no modifications of signaling pathways associated with dysregulated mitochondrial energy metabolism. Ongoing investigations are using single cell RNASeq to identify the different cell types targeted by the two drugs used in combination therapy, as well as potential cell‐cell interactions that underlie the efficacy of the combination therapy.

Angiotensin AT2 Receptor Activation during Ischemic Injury Protects against High Salt‐Induced Hypertension and Proteinuria in the Long‐term: Role of IL‐17A Cells

Short time renal ischemia/reperfusion (I/R) causes reversible acute kidney injury (AKI) that often recovers within 5‐7 days. However, a single event of AKI predisposes and makes kidneys susceptible to develop chronic kidney disease (CKD) in conditions such as obesity and hypertension. Recently we have shown that angiotensin type 2 receptor (AT2R) activation by the agonist compound C21 (C21) protects against I/R‐induced AKI in the early injury phase in terms of suppressing pro‐inflammatory cytokines and improving functional injury markers such as reducing proteinuria and blood urea nitrogen (BUN). The late recovery phase (day 5) in the I/R and I/R treated with C21 remained same i.e., the C21 treated and un‐treated animals fully recovered functionally to the same extent but with higher anti‐inflammatory IL‐10 producing and protective Treg cell (FoxP3+) in I/R+C21 compared I/R groups. However, the long‐term protective effect of the AT2R agonist C21 on these recovered kidneys against chronic injury is unknown. To address this question, male Sprague‐Dawley (SD) rats were administered C21 (0.3 mg/kg, i.p.) prior to 30‐minute bilateral renal ischemia and allowed to recover for 5 days, with daily C21 treatment (I/R+ C21), without treatment (I/R) and sham control. On 7th day, all the three groups were placed on high salt (1% NaCl) in drinking water for two weeks but without C21 treatment. On day 6 (prior to initiating high salt treatment), week 1 and 2 of the high salt treatment, renal function parameters (proteinuria), and T cells were quantitated in the blood and kidney (only at 2 weeks). On 6th day, all the three groups showed similar level of proteinuria, suggesting same renal function. However, at week 1 and 2, I/R group had significantly greater proteinuria, suggesting a function injury. Similarly, I/R group showed higher mean arterial blood pressure at two weeks compared with the I/R+C21 and sham control. Consistent to the higher proteinuria and blood pressure in I/R group, there were higher infiltrating proinflammatory (IL‐17A) cells in the kidney in I/R group compared with sham control and I/R+C21 groups. Also, kidney infiltrating CD45 cells, T cells and CD4 T cells were found to be similar among all the groups. Overall, the data indicate that I/R pre‐disposes the kidneys to high salt‐induced chronic injury which is completely prevented by AT2R agonist treatment during AKI phase. Moreover, AT2R’s protective effect could be related to preventing the T cell modulation into proinflammatory IL‐17A cells.