Unlocking the protective potential of the angiotensin type 2 receptor (AT2R) in acute lung injury and age-related pulmonary dysfunction.

Despite its known importance in the cardiovascular system, the specific role and impact of the angiotensin type 2 receptor (AT2R) in lung physiology and pathophysiology remain largely elusive. In this study, we highlight the distinct and specialized lung-specific roles of AT2R, primarily localized to an alveolar fibroblast subpopulation, in contrast to the angiotensin type 1 receptor (AT1R), which is almost exclusively expressed in lung pericytes. Evidence from our research demonstrates that the disruption of AT2R (AT2R-/y), is associated with a surge in oxidative stress and impaired lung permeability, which were further intensified by Hyperoxic Acute Lung Injury (HALI). With aging, AT2R-/y mice show an increase in oxidative stress, premature enlargement of airspaces, as well as increased mortality when exposed to hyperoxia as compared to age-matched WT mice. Our investigation into Losartan, an AT1R blocker, suggests that its primary HALI lung-protective effects are channeled through AT2R, as its protective benefits are absent in AT2R-/y mice. Importantly, a non-peptide AT2R agonist, Compound 21 (C21), successfully reverses lung oxidative stress and TGFß activation in wild-type (WT) mice exposed to HALI. These findings suggest a possible paradigm shift in the therapeutic approach for lung injury and age-associated pulmonary dysfunction, from targeting AT1R with angiotensin receptor blockers (ARBs) towards boosting the protective function of AT2R.

Potential nephroprotective effects of angiotensin II type 2 receptor agonist Compound 21 in renal ischemia-reperfusion injury.

This study examined the reno-protective potential of Compound 21 during renal ischemia-reperfusion injury by regulating the PI3K expression. 20 adult male Swiss-albino mice, aged 8-12 weeks and weighing 20-30g, were randomly assigned to four equal groups: sham, control, vehicle, and Compound 21. Serum urea, creatinine, inflammatory mediators, tissue 8-isoprostane, and myeloperoxidase were quantified using ELISA. Compared to the sham group, blood levels of urea, creatinine, TNF-α, IL-6, and IL-10 were significantly higher in the ischemia-reperfusion group than in the sham group (p<0.05). However, these indicators were significantly lower in the Compound 21 group (p<0.05). Histological analysis revealed significant renal tissue damage in the ischemia-reperfusion group (p<0.05), which was significantly reduced in the Compound 21 group (p<0.05). PCR results showed that PI3K expression was significantly lower (p<0.05) in the control group compared to the sham group but significantly higher in the Compound 21 group (p<0.05). Furthermore, P-AKT expression levels in the control group were considerably lower than in the sham group (p<0.05). On the other hand, the level of P-AKT expression in the Compound 21 group was significantly upregulated compared to the control group (p<0.05). The findings revealed that Compound 21 could mitigate renal dysfunction induced by ischemia-reperfusion injury in male mice through modulation of the PI3K/AKT signaling pathway, resulting in decreased levels of pro-inflammatory cytokines and renal oxidative stress markers.

Angiotensin II type 2 receptor as a novel activator of brown adipose tissue in obesity.

The angiotensin II type 2 receptor (AT2R) exerts vasorelaxant, anti-inflammatory, and antioxidant properties. In obesity, its activation counterbalances the adverse cardiovascular effects of angiotensin II mediated by the AT1R. Preliminary results indicate that it also promotes brown adipocyte differentiation in vitro. Our hypothesis is that AT2R activation could increase BAT mass and activity in obesity. Five-week-old male C57BL/6J mice were fed a standard or a high-fat (HF) diet for 6 weeks. Half of the animals were treated with compound 21 (C21), a selective AT2R agonist, (1 mg/kg/day) in the drinking water. Electron transport chain (ETC), oxidative phosphorylation, and UCP1 proteins were measured in the interscapular BAT (iBAT) and thoracic perivascular adipose tissue (tPVAT) as well as inflammatory and oxidative parameters. Differentiation and oxygen consumption rate (OCR) in the presence of C21 was tested in brown preadipocytes. In vitro, C21-differentiated brown adipocytes showed an AT2R-dependent increase of differentiation markers (Ucp1, Cidea, Pparg) and increased basal and H+ leak-linked OCR. In vivo, HF-C21 mice showed increased iBAT mass compared to HF animals. Both their iBAT and tPVAT showed higher protein levels of the ETC protein complexes and UCP1, together with a reduction of inflammatory and oxidative markers. The activation of the AT2R increases BAT mass, mitochondrial activity, and reduces markers of tissue inflammation and oxidative stress in obesity. Therefore, insulin reduction and better vascular responses are achieved. Thus, the activation of the protective arm of the renin-angiotensin system arises as a promising tool in the treatment of obesity.

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 agonist treatment of doxorubicin induced heart failure.

Doxorubicin (DOX) is an anthracycline derivative used for treatment of malignancies; however, its clinical use is limited by its cardiotoxicity. We investigated the effects of angiotensin II type 2 receptor agonist compound 21 (C21) on DOX induced heart failure in rat heart. We compared C21 with losartan (LOS), an AT 1 receptor antagonist used for treating heart failure. We allocated 40 rats into five groups of eight: saline treated control group, DOX group administered a single 20 mg/kg dose of DOX, DOX + C21 group administered 0.3 mg/kg C21 for 21 days following the 20 mg/kg dose of DOX, DOX + losartan (LOS) group administered a 21 day regimen of 20 mg/kg LOS following the single dose of DOX, and a DOX + LOS + C21 group administered 0.3 mg/kg C21 and 20 mg/kg LOS for 21 days following the single dose of DOX. We assessed histopathology and conducted echocardiograpic and hemodynamic measurements. Left ventricular ejection fraction (EF) was reduced only in the DOX treated group. C21, LOS and C21 + LOS therapy prevented decreased EF due to DOX. Less histopathology was observed in the DOX + LOS + C21 group than for the other treatment groups. Application of C21 decreased DOX induced cardiac injury similar to LOS. Combined use of C21 and LOS was most beneficial for DOX induced heart failure.

CGP42112: the full AT2 receptor agonist and its role in the renin-angiotensin-aldosterone system: no longer misunderstood.

For years, the AT2R-selective ligand CGP42112 has been erroneously characterized as a partial agonist, partly due to its ability to also interact with the AT1R at high concentrations. As late as 2009, it was still being characterized as an antagonist as well. In this perspective/opinion piece, we try to resolve the ambiguity that surrounds the efficacy of this compound by extensively reviewing the literature, tracing its beginnings to 1989, showing that CGP42112 has never been convincingly shown to be a partial agonist or an antagonist at the AT2R. While CGP42112 is now routinely characterized as an AT2R agonist, regrettably, there is a paucity of studies that can validate its efficacy as a full agonist at the AT2R, leaving the door open for continuing speculation regarding the extent of its efficacy. Hopefully, the information presented in this perspective/opinion piece will firmly establish CGP42112 as a full agonist at the AT2R such that it can once again be used as a tool to study the AT2R.

Angiotensin-(1-9) in hypertension.

Angiotensin-(1-9) [Ang-(1-9)] is a peptide of the non-canonical renin-angiotensin system (RAS) synthesized from angiotensin I by the monopeptidase angiotensin-converting enzyme type 2 (ACE2). Using osmotic minipumps, infusion of Ang-(1-9) consistently reduces blood pressure in several rat hypertension models. In these animals, hypertension-induced end-organ damage is also decreased. Several pieces of evidence suggest that Ang-(1-9) is the endogenous ligand that binds and activates the type-2 angiotensin II receptor (AT2R). Activation of AT2R triggers different tissue-specific signaling pathways. This phenomenon could be explained by the ability of AT2R to form different heterodimers with other G protein-coupled receptors. Because of the antihypertensive and protective effects of AT2R activation by Ang-(1-9), associated with a short half-life of RAS peptides, several synthetic AT2R agonists have been synthesized and assayed. Some of them, particularly CGP42112, C21 and novokinin, have demonstrated antihypertensive properties. Only two synthetic AT2R agonists, C21 and LP2-3, have been tested in clinical trials, but none of them like an antihypertensive. Therefore, Ang-(1-9) is a promising antihypertensive drug that reduces hypertension-induced end-organ damage. However, further research is required to translate this finding successfully to the clinic.

Contralesional angiotensin type 2 receptor activation contributes to recovery in experimental stroke.

We and others have previously shown that angiotensin II receptor type 2 receptor (AT2R) is upregulated in the contralesional hemisphere after stroke in normoglycemic Wistar rats. In this study, we examined the expression of AT2R in type 2 diabetic Goto-Kakizaki (GK) rats and control Wistars after stroke. We also tested the contribution of the contralesional AT2R in recovery after stroke through a specific knockdown of the AT2R in this hemisphere only. Two experiments were conducted. In the first experiment, GK rats were subjected to middle cerebral artery occlusion (MCAO) and treated with the angiotensin II receptor type 1 receptor (AT1R) blocker candesartan or saline at reperfusion. Stroke outcomes, as well as AT2R expression, were examined and compared to control Wistars at 24 h. In the second experiment, localized AT2R knockdown was achieved through intrastriatal injection of short hairpin RNA (shRNA) lentiviral particles or non-targeting control into the left-brain hemisphere of Wistar rats. After 14 days, rats were subjected to right MCAO and treated with the AT2R agonist, Compound 21 (C21), or saline for 7 days. Behavioral outcomes were assessed for up to 10 days. In the first experiment, stroke reduced the expression of AT2R in GK rats. Candesartan treatment failed to improve the neurobehavioral outcomes, preserve vascular integrity or reduce oxidative/nitrative stress or apoptotic markers at 24 h post stroke in these animals. In the second experiment, contralesional AT2R knockdown reduced the C21-mediated functional recovery after stroke. In conclusion, contralesional AT2R upregulation after stroke is blunted in diabetic rats which show reduced sensitivity to post-stroke candesartan treatment. Contralesional AT2R could be involved in C21-mediated functional recovery after stroke.

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.

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 âˆ¼ 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.

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.

AT2R activation increases in vitro angiogenesis in pregnant human uterine artery endothelial cells.

Angiogenesis is vital during pregnancy for remodeling and enhancing vasodilation of maternal uterine arteries, and increasing uterine blood flow. Abnormal angiogenesis is associated with decreased uteroplacental blood flow and development of pregnancy disorders such as gestational hypertension, preeclampsia, fetal growth restriction, preterm delivery, stillbirth, and miscarriage. The mechanisms that contribute to normal angiogenesis remain obscure. Our previous studies demonstrated that expression of the angiotensin type 2 receptor (AT2R) is increased while the angiotensin type 1 receptor (AT1R) is unchanged in the endothelium of uterine arteries, and that AT2R-mediated pregnancy adaptation facilitates enhanced vasodilation and uterine arterial blood flow. However, the role of AT2R in regulating angiogenesis during pregnancy has never been studied. This study examines whether or not AT2R activation induces angiogenesis and, if so, what mechanisms are involved. To this end, we used primary human uterine artery endothelial cells (hUAECs) isolated from pregnant and nonpregnant women undergoing hysterectomy. The present study shows that Compound 21, a selective AT2R agonist, induced proliferation of pregnant-hUAECs, but not nonpregnant-hUAECs, in a concentration-dependent manner, and that this C21-induced mitogenic effect was blocked by PD123319, a selective AT2R antagonist. The mitogenic effects induced by C21 were inhibited by blocking JNK-but not ERK, PI3K, and p38-signaling pathways. In addition, C21 concentration dependently increased cell migration and capillary-like tube formation in pregnant-hUAECs. The membrane-based antibody array showed that C21 increased expression of multiple angiogenic proteins, including EGF, bFGF, leptin, PLGF, IGF-1, and angiopoietins. Our qPCR analysis demonstrates that C21-induced increase in expression of these angiogenic proteins correlates with a proportional increase in mRNA expression, indicating that AT2R activates angiogenic proteins at the transcriptional level. In summary, the present study shows that AT2R activation induces angiogenesis of hUAECs in a pregnancy-specific manner through JNK-mediated pathways with associated transcriptional upregulation of multiple proangiogenic proteins.

Compound 21, a Direct AT2R Agonist, Induces IL-10 and Inhibits Inflammation in Mice Following Traumatic Brain Injury.

Recent studies demonstrated that the angiotensin type 2 receptor (AT2R) agonist, compound 21 (C21), provides neuroprotection and enhances recovery in experimental stroke. However, C21 has never been tested in traumatic brain injury (TBI). Here, we aim to examine whether C21 confers protection after TBI. Unilateral cortical impact injury was induced in young adult C57BL/6 mice. C21 (0.03 mg/kg, i.p.) was administered at 1 h and 3 h post-TBI. After neurological severity score (NSS) assessments, all animals were sacrificed for immunoblotting analysis at 24 h post-TBI. C21 treatment significantly ameliorated NSS and reduced TBI's biomarkers [high mobility group box 1 (HMGB1), aquaporin-4 (AQ4)] and inflammatory markers [interlukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α)] in the pericontusional areas compared to saline TBI. Further, C21 treatment induced interleukin-10 (IL-10) and phosphorylation of endothelial nitric oxide synthase (eNOS) after TBI. C21 also attenuated pro-apoptotic activation of poly (ADP-ribose) polymerase (PARP) and caspase-3. These findings support the therapeutic potential of C21 against TBI.

AT2R stimulation with C21 prevents arterial stiffening and endothelial dysfunction in the abdominal aorta from mice fed a high-fat diet.

The aim of the present study was to evaluate the effect of Compound 21 (C21), a selective AT2R agonist, on the prevention of endothelial dysfunction, extracellular matrix (ECM) remodeling and arterial stiffness associated with diet-induced obesity (DIO). Five-week-old male C57BL/6J mice were fed a standard (Chow) or high-fat diet (HF) for 6 weeks. Half of the animals of each group were simultaneously treated with C21 (1 mg/kg/day, in the drinking water), generating four groups: Chow C, Chow C21, HF C, and HF C21. Vascular function and mechanical properties were determined in the abdominal aorta. To evaluate ECM remodeling, collagen deposition and TGF-ß1 concentrations were determined in the abdominal aorta and the activity of metalloproteinases (MMP) 2 and 9 was analyzed in the plasma. Abdominal aortas from HF C mice showed endothelial dysfunction as well as enhanced contractile but reduced relaxant responses to Ang II. This effect was abrogated with C21 treatment by preserving NO availability. A left-shift in the tension-stretch relationship, paralleled by an augmented ß-index (marker of intrinsic arterial stiffness), and enhanced collagen deposition and MMP-2/-9 activities were also detected in HF mice. However, when treated with C21, HF mice exhibited lower TGF-ß1 levels in abdominal aortas together with reduced MMP activities and collagen deposition compared with HF C mice. In conclusion, these data demonstrate that AT2R stimulation by C21 in obesity preserves NO availability and prevents unhealthy vascular remodeling, thus protecting the abdominal aorta in HF mice against the development of endothelial dysfunction, ECM remodeling and arterial stiffness.

Angiotensin II type 2 receptor agonist, compound 21, prevents tubular epithelial cell damage caused by renal ischemia.

During ischemic acute kidney injury (AKI), loss of cytoskeletal integrity and disruption of intercellular junctions are rapid events in response to ATP depletion. Angiotensin II type 2 receptor (AT2R) is overexpressed in injury situations and its stimulation by angiotensin II (AngII) is related to beneficial renal effects. Its role on ischemic AKI has not been deeply studied. The aim of the present study was to investigate whether pretreatment with the AT2R agonist, C21, prevents ischemic renal epithelial cell injury. Studies in a model of 40 min of renal ischemia followed by 24 h of reperfusion (IR) in rats demonstrated that C21 pretreatment attenuated renal dysfunction and induced better preservation of tubular architecture. In addition, we studied the expression of Rho GTPases, RhoA and Cdc42, since they are key proteins in the regulation of the actin cytoskeleton and the stability of epithelial intercellular junctions. IR downregulated RhoA and Cdc42 abundance in rat kidneys. C21 pretreatment prevented RhoA reduction and increased Cdc42 abundance compared to controls. We also used an in vitro model of ATP depletion in MDCK cells grown on filter support. Using immunofluorescence we observed that in MDCK cells, C21 pretreatment prevented the ATP depletion-induced reduction of actin in brush border microvilli and in stress fibers. Moreover, C21 prevented membrane E-cadherin reduction, and RhoA and Cdc42 downregulation. The present study describes for the first time a renoprotective effect of the AT2R agonist, C21, against AKI, and provides evidence supporting that stimulation of AT2R triggers cytoprotective mechanisms against an ischemic event.

Angiotensin II biphasically regulates cell differentiation in human iPSC-derived kidney organoids.

Human kidney organoid technology holds promise for novel kidney disease treatment strategies and utility in pharmacological and basic science. Given the crucial roles of the intrarenal renin-angiotensin system (RAS) and angiotensin II (ANG II) in the progression of kidney development and injury, we investigated the expression of RAS components and effects of ANG II on cell differentiation in human kidney organoids. Human induced pluripotent stem cell-derived kidney organoids were induced using a modified 18-day Takasato protocol. Gene expression analysis by digital PCR and immunostaining demonstrated the formation of renal compartments and expression of RAS components. The ANG II type 1 receptor (AT1R) was strongly expressed in the early phase of organoid development (around day 0), whereas ANG II type 2 receptor (AT2R) expression levels peaked on day 5. Thus, the organoids were treated with 100 nM ANG II in the early phase on days 0-5 (ANG II-E) or during the middle phase on days 5-10 (ANG II-M). ANG II-E was observed to decrease levels of marker genes for renal tubules and proximal tubules, and the downregulation of renal tubules was inhibited by an AT1R antagonist. In contrast, ANG II-M increased levels of markers for podocytes, the ureteric tip, and the nephrogenic mesenchyme, and an AT2R blocker attenuated the ANG II-M-induced augmentation of podocyte formation. These findings demonstrate RAS expression and ANG II exertion of biphasic effects on cell differentiation through distinct mediatory roles of AT1R and AT2R, providing a novel strategy to establish and further characterize the developmental potential of human induced pluripotent stem cell-derived kidney organoids.NEW & NOTEWORTHY This study demonstrates angiotensin II exertion of biphasic effects on cell differentiation through distinct mediatory roles of angiotensin II type 1 receptor and type 2 receptor in human induced pluripotent stem cell-derived kidney organoids, providing a novel strategy to establish and further characterize the developmental potential of the human kidney organoids.

Update on Angiotensin II Subtype 2 Receptor: Focus on Peptide and Nonpeptide Agonists.

Angiotensin II (Ang II) is the most dominant effector component of the renin-angiotensin system (RAS) that generally acts through binding to two main classes of G protein-coupled receptors, namely Ang II subtype 1 receptor (AT1R) and angiotensin II subtype 2 receptor (AT2R). Despite some controversial reports, the activation of AT2R generally antagonizes the effects of Ang II binding on AT1R. Studying AT2R signaling, function, and its specific ligands in cell culture or animal studies has confirmed its beneficial effects throughout the body. These characteristics classify AT2R as part of the protective arm of the RAS that, along with functions of Ang (1-7) through Mas receptor signaling, modulates the harmful effects of Ang II on AT1R in the activated classic arm of the RAS. Although Ang II is the primary ligand for AT2R, we have summarized other natural or synthetic peptide and nonpeptide agonists with critical evaluation of their structure, mechanism of action, and biologic activity. SIGNIFICANCE STATEMENT: AT2R is one of the main components of the RAS and has a significant prospective for mediating the beneficial action of the RAS through its protective arm on the body's homeostasis. Targeting AT2R offers substantial clinical application possibilities for modulating various pathological conditions. This review provided concise information regarding the AT2R peptide and nonpeptide agonists and their potential clinical applications for various diseases.

C21 preserves endothelial function in the thoracic aorta from DIO mice: role for AT2, Mas and B2 receptors.

Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

Role of the central renin­angiotensin system in hypertension (Review).

Present in more than one billion adults, hypertension is the most significant modifiable risk factor for mortality resulting from cardiovascular disease. Although its pathogenesis is not yet fully understood, the disruption of the renin­angiotensin system (RAS), consisting of the systemic and brain RAS, has been recognized as one of the primary reasons for several types of hypertension. Therefore, acquiring sound knowledge of the basic science of RAS and the underlying mechanisms of the signaling pathways associated with RAS may facilitate the discovery of novel therapeutic targets with which to promote the management of patients with cardiovascular and kidney disease. In total, 4 types of angiotensin II receptors have been identified (AT1R­AT4R), of which AT1R plays the most important role in vasoconstriction and has been most extensively studied. It has been found in several regions of the brain, and its distribution is highly associated with that of angiotensin­like immunoreactivity in nerve terminals. The effect of AT1R involves the activation of multiple media and signaling pathways, among which the most important signaling pathways are considered to be AT1R/JAK/STAT and Ras/Raf/MAPK pathways. In addition, the regulation of the nuclear factor κ­light­chain­enhancer of activated B cells (NF­κB) and cyclic AMP response element­binding (CREB) pathways is also closely related to the effect of ATR1. Their mechanisms of action are related to pro­inflammatory and sympathetic excitatory effects. Central AT1R is involved in almost all types of hypertension, including spontaneous hypertension, salt­sensitive hypertension, obesity­induced hypertension, renovascular hypertension, diabetic hypertension, L­NAME­induced hypertension, stress­induced hypertension, angiotensin II­induced hypertension and aldosterone­induced hypertension. There are 2 types of central AT1R blockade, acute blockade and chronic blockade. The latter can be achieved by chemical blockade or genetic engineering. The present review article aimed to highlight the prevalence, functions, interactions and modulation means of central AT­1R in an effort to assist in the treatment of several pathological conditions. The identification of angiotensin­derived peptides and the development of AT­2R agonists may provide a wider perspective on RAS, as well as novel therapeutic strategies.

Complex interactions between the angiotensin II type 1 receptor, the epidermal growth factor receptor and TRIO-dependent signaling partners.

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.