Utilizing venous occlusion plethysmography to assess vascular effects: A study with buloxibutid, an angiotensin II type 2 receptor agonist.

Buloxibutid (also known as C21) is a potent and selective angiotensin II type 2 receptor (AT2R) agonist, in development for oral treatment of fibrotic lung disease. This phase I, open-label, pharmacodynamic study investigated vascular effects of buloxibutid in five healthy male volunteers. Subjects were administered intra-arterial infusions of buloxibutid for 5 min in ascending doses of 3, 10, 30, 100, and 200 µg/min, infused sequentially in the forearm. Infusions of sodium nitroprusside (SNP) solution in doses of 0.8-3.2 µg/min were administered as a positive control. Forearm blood flow (FBF) was measured by venous occlusion plethysmography. Safety and tolerability of intra-arterial administrations of buloxibutid were evaluated. Following infusion of buloxibutid in doses of 3-200 µg/min, the range of increase in FBF was 27.8%, 17.2%, 37.0%, 28.5%, and 60.5%, compared to the respective baseline. The largest increase was observed in the highest dose group. Infusions of SNP as a positive control, increased FBF 230-320% compared to baseline. Three adverse events (AEs) of mild intensity, not related to buloxibutid or SNP, were reported for two subjects. Two of these AEs were related to study procedures. There were no clinically relevant changes in arterial blood pressure during the study period. Intra-arterial infusion of buloxibutid in low, ascending doses increased FBF, indicating that buloxibutid may be effective in conditions associated with endothelial dysfunction. Venous occlusion plethysmography was found to be a useful method to explore pharmacodynamic vascular effects of novel AT2R agonists, while avoiding systemic adverse effects.

Angiotensin AT2 receptors reduce inflammation and fibrosis in cardiovascular remodeling.

The angiotensin AT2 receptor (AT2R), an important member of the "protective arm" of the renin-angiotensin system (RAS), has been recently defined as a therapeutic target in different pathological conditions. The AT2R activates complex signalling pathways linked to cellular proliferation, differentiation, anti-inflammation, antifibrosis, and induction or inhibition of apoptosis. The anti-inflammatory effect of AT2R activation is commonly associated with reduced fibrosis in different models. Current discoveries demonstrated a direct impact of AT2Rs on the regulation of cytokines, transforming growth factor beta1 (TGF-beta1), matrix metalloproteases (MMPs), and synthesis of the extracellular matrix components. This review article summarizes current knowledge on the AT2R in regard to immunity, inflammation and fibrosis in the heart and blood vessels. In particular, the differential influence of the AT2R on cardiovascular remodeling in preclinical models of myocardial infarction, heart failure and aneurysm formation are discussed. Overall, these studies demonstrate that AT2R stimulation represents a promising therapeutic approach to counteract myocardial and aortic damage in cardiovascular diseases.

The antihypertensive action of C-phycocyanin is related to the prevention of angiotensin II-caused vascular dysfunction in chronic kidney disease.

C-phycocyanin (CPC) is a photosynthetic protein found in Arthrospira maxima with a nephroprotective and antihypertensive activity that can prevent the development of hemodynamic alterations caused by chronic kidney disease (CKD). However, the complete nutraceutical activities are still unknown. This study aims to determine if the antihypertensive effect of CPC is associated with preventing the impairment of hemodynamic variables through delaying vascular dysfunction. Twenty-four normotensive male Wistar rats were divided into four groups: (1) sham + 4 mL/kg/d vehicle (100 mM of phosphate buffer, PBS) administered by oral gavage (og), (2) sham + 100 mg/kg/d og of CPC, (3) CKD induced by 5/6 nephrectomy (CKD) + vehicle, (4) CKD + CPC. One week after surgery, the CPC treatment began and was administrated daily for four weeks. At the end treatment, animals were euthanized, and their thoracic aorta was used to determine the vascular function and expression of AT1, AT2, and Mas receptors. CKD-induced systemic arterial hypertension (SAH) and vascular dysfunction by reducing the vasorelaxant response of angiotensin 1-7 and increasing the contractile response to angiotensin II. Also, CKD increased the expression of the AT1 and AT2 receptors and reduced the Mas receptor expression. Remarkably, the treatment with CPC prevented SAH, renal function impairment, and vascular dysfunction in the angiotensin system. In conclusion, the antihypertensive activity of CPC is associated with avoiding changes in the expression of AT1, AT2, and Mas receptors, preventing vascular dysfunction development and SAH in rats with CKD.

Delayed Administration of an Angiotensin II Type 2 Receptor Agonist Promotes Functional Recovery of the Brain and Heart After Traumatic Brain Injury.

Cardiac injury is a common complication following traumatic brain injury (TBI) that can lead to poor clinical outcomes. Angiotensin II type 2 receptor (AT2R) activation exerts protective roles in the brain and heart, yet its potential impact on TBI or TBI-induced cardiac deficits remains elusive. The goal of this study was to investigate the influence of AT2R activation on recovery after TBI-induced cognitive and cardiac injury using the selective nonpeptide AT2R agonist compound 21 (C21). TBI was induced by cortical impact injury in male adult C57BL/6J mice, and the mice received C21 (0.03 mg/kg, intraperitoneally) starting from 24 h after TBI and continuing once daily. C21 facilitated cognitive function recovery until 1 month after TBI. C21 alleviated blood-brain barrier leakage and brain edema and inhibited the expression of proinflammatory cytokines in the brain after 3 consecutive days of treatment. C21 improved cerebral blood flow after 1 month, although the lesion volume was not affected. C21 also reduced the expression of proinflammatory cytokines in the heart after a 3-day consecutive treatment. Meanwhile, C21 benefited cardiac function, as identified by increased left ventricular ejection fraction 1 month after TBI. In addition, C21 alleviated TBI-induced cardiac hypertrophy and fibrosis; however, blood pressure was not affected. Our results demonstrate that AT2R activation ameliorates TBI-induced neurological and cardiac deficits.

Intergenerational inheritance induced by a high-fat diet causes hyperphagia and reduced hypothalamic sensitivity to insulin and leptin in the second-generation of rats.

OBJECTIVE: The aim was to investigate the intergenerational inheritance induced by a high-fat diet on sensitivity to insulin and leptin in the hypothalamic control of satiety in second-generation offspring, which were fed a control diet. METHODS: Progenitor rats were fed a high-fat or a control diet for 59 d until weaning. The first-generation and second-generation offspring were fed the control diet until 90 d of age. Body mass and adiposity index of the progenitors fed the high-fat diet and the second-generation offspring from progenitors fed the high-fat diet were evaluated as were the gene expression of DNA methyltransferase 3a, angiotensin-converting enzyme type 2, angiotensin II type 2 receptor, insulin and leptin signaling pathway (insulin receptor, leptin receptor, insulin receptor substrate 2, protein kinase B, signal transducer and transcriptional activator 3, pro-opiomelanocortin, and neuropeptide Agouti-related protein), superoxide dismutase activity, and the concentration of carbonyl protein and satiety-regulating neuropeptides, pro-opiomelanocortin and neuropeptide Agouti-related protein, in the hypothalamus. RESULTS: The progenitor group fed a high-fat diet showed increased insulin resistance and reduced insulin-secreting beta-cell function and reduced food intake, without changes in caloric intake. The second-generation offspring from progenitors fed a high-fat diet, compared with second-generation offspring from progenitors fed a control diet group, had decreased insulin-secreting beta-cell function and increased food and caloric intake, insulin resistance, body mass, and adiposity index. Furthermore, second-generation offspring from progenitors fed a high-fat diet had increased DNA methyltransferase 3a, neuropeptide Agouti-related protein, angiotensin II type 1 receptor, and nicotinamide adenine dinucleotide phosphate oxidase p47phox gene expression, superoxide dismutase activity, and neuropeptide Agouti-related protein concentration in the hypothalamus. In addition, there were reduced in gene expression of the insulin receptor, leptin receptor, insulin receptor substrate 2, pro-opiomelanocortin, angiotensin II type 2 receptor, angiotensin-converting enzyme type 2, and angiotensin-(1-7) receptor and pro-opiomelanocortin concentration in the second-generation offspring from progenitors fed the high-fat diet. CONCLUSIONS: Overall, progenitors fed a high-fat diet induced changes in the hypothalamic control of satiety of the second-generation offspring from progenitors fed the high-fat diet through intergenerational inheritance. These changes led to hyperphagia, alterations in the hypothalamic pathways of insulin, and leptin and adiposity index increase, favoring the occurrence of different cardiometabolic disorders in the second-generation offspring from progenitors fed the high-fat diet fed only with the control diet.

N-(Heteroaryl)thiophene sulfonamides as angiotensin AT2 receptor ligands.

Two series of N-(heteroaryl)thiophene sulfonamides, encompassing either a methylene imidazole group or a tert-butylimidazolylacetyl group in the meta position of the benzene ring, have been synthesized. An AT2R selective ligand with a Ki of 42 nM was identified in the first series and in the second series, six AT2R selective ligands with significantly improved binding affinities and Ki values of <5 nM were discovered. The binding modes to AT2R were explored by docking calculations combined with molecular dynamics simulations. Although some of the high affinity ligands exhibited fair stability in human liver microsomes, comparable to that observed with C21 undergoing clinical trials, most ligands displayed a very low metabolic stability with t½ of less than 10 min in human liver microsomes. The most promising ligand, with an AT2R Ki value of 4.9 nM and with intermediate stability in human hepatocytes (t½ = 77 min) caused a concentration-dependent vasorelaxation of pre-contracted mouse aorta.

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

Adriamycin (ADR) causes irreversible damage to the heart, leading to ADR-induced cardiomyopathy (ACM). Angiotensin-(1-9) [Ang-(1-9)] is a peptide from the counter-regulatory renin-angiotensin system, but the effects on ACM is unclear. Our study was aimed to explore the effects and underlying molecular mechanisms of Ang-(1-9) against ACM in Wistar rats. Rats were injected intraperitoneally with ADR via six equal doses (each containing 2.5 mg/kg) within a period of 2 weeks to induce ACM. After 2 weeks of ADR treatment, the rats were treated with Ang-(1-9) (200 ng/kg/min) or angiotensin type 2 receptor (AT2R) antagonist PD123319 (100 ng/kg/min) for 4 weeks. Although Ang-(1-9) treatment did not influence blood pressure, it significantly improved left ventricular function and remodeling in ADR-treated rats, by inhibiting collagen deposition, the expression of TGF-ß1, inflammatory response, cardiomyocyte apoptosis and oxidative stress. Moreover, Ang-(1-9) reduced ERK1/2 and P38 MAPK phosphorylation. The therapeutic effects of Ang-(1-9) were blocked by the AT2R antagonist PD123319, which also offset the down-regulation protein expression of pERK1/2 and pP38 MAPK induced by Ang-(1-9). These data suggest that Ang-(1-9) improved left ventricular function and remodeling in ADR-treated rats by an AT2R/ ERK1/2 and P38 MAPK-dependent mechanism. Thus, the Ang-(1-9)/AT2R axis may provide a novel and promising target to the prevention and treatment of ACM.

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 that C21 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.

Regulation of vascular angiotensin II type 1 and type 2 receptor and angiotensin-(1-7)/MasR signaling in normal and hypertensive pregnancy.

Normal pregnancy (Norm-Preg) is associated with a slight reduction in blood pressure (BP) and decreased BP response to vasoconstrictor stimuli such as angiotensin II (Ang II), although the renin-angiotensin-aldosterone system (RAAS) is upregulated. Preeclampsia (PE) is a complication of pregnancy manifested as hypertension-in-pregnancy (HTN-Preg), and dysregulation of angiotensin biosynthesis and signaling have been implicated. Ang II activates vascular Ang II type-1 receptor (AT1R) and Ang II type-2 receptor (AT2R), while angiotensin-(1-7) promotes Ang-(1-7)/MasR signaling. The role of AT1R in vasoconstriction and the activated cellular mechanisms are well-characterized. The sensitivity of vascular AT1R to Ang II and consequent activation of vasoconstrictor mechanisms decrease during Norm-Preg, but dramatically increase in HTN-Preg. Placental ischemia in late pregnancy could also initiate the release of AT1R agonistic autoantibodies (AT1AA) with significant impact on endothelial dysfunction and activation of contraction pathways in vascular smooth muscle including [Ca2+]c and protein kinase C. On the other hand, the role of AT2R and Ang-(1-7)/MasR in vascular relaxation, particularly during Norm-Preg and PE, is less clear. During Norm-Preg, increases in the expression/activity of vascular AT2R and Ang-(1-7)/MasR promote the production of endothelium-derived relaxing factors such as nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factor leading to generalized vasodilation. Aortic segments of Preg rats show prominent endothelial AT2R staining and increased relaxation and NO production in response to AT2R agonist CGP42112A, and treatment with AT2R antagonist PD123319 enhances phenylephrine-induced contraction. Decreased vascular AT2R and Ang-(1-7)/MasR expression and receptor-mediated mechanisms of vascular relaxation have been suggested in HTN-Preg animal models, but their role in human PE needs further testing. Changes in angiotensin-converting enzyme-2 (ACE2) have been observed in COVID-19 patients, and whether ACE2 influences the course of COVID-19 viral infection/immunity in Norm-Preg and PE is an intriguing area for research.

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.

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.

A chromatographic method for determining the interaction between a drug and two target proteins by fabricating a dual-heterogeneous surface.

Characterization of the drug-target interactions is pivotal throughout the whole procedure of drug development. Most of the current assays, particularly, chromatographic methods lack the capacity to reveal drug adsorption on the muti-target surface. To this end, we derived a reliable and workable mathematical equation for revealing drug bindings to dual targets on the heterogeneous surface starting from the mass balance equation. The derivatization relied on the correlation of drug injection amounts with their retention factors. Experimental validation was performed by determining the binding parameters of three canonical drugs on a heterogeneous surface, which was fabricated by fusing angiotensin receptor type I and type II receptors (AT1R and AT2R) at the terminuses of circularly permuted HaloTag (cpHaloTag) and immobilizing the whole fusion protein onto 6-bromohexanoic acid modified silica gel. We proved that immobilized AT1R-cpHalo-AT2R maintained the original ligand- and antibody-binding activities of the two receptors in three weeks. The association constants of valsartan, candesartan, and telmisartan to AT1R were (6.26±0.14) × 105, (9.66±0.71) × 105, and (3.17±0.03) × 105 L/mol. In the same column, their association constants to AT2R were (1.25±0.04) × 104, (2.30±0.08) × 104, and (8.51±0.06) × 103 L/mol. The patterns of the association constants to AT1R/AT2R (candesartan>valsartan>telmisartan) were in good line with the data by performing nonlinear chromatography on control columns containing immobilized AT1R or AT2R alone. This provided proof of the fact that the derivatization allowed the determination of drug bindings on the heterogeneous surface with the utilization of a single series of injections and linear regression. We reasoned that is simple enough to model the bindings of drug adsorption on commercially available adsorbents in fundamental or industrial fields, thus having the potential to become a universal method for analyzing the bindings of a drug to the heterogeneous surface containing multiple targets.

Angiotensin II type 2 receptor signalling as a pain target: Bench, bedside and back-translation.

Translating promising preclinical pain relief data for novel molecules from drug discovery to positive clinical trial outcomes is challenging. The angiotensin II type 2 (AT2) receptor is a clinically-validated target based upon positive proof-of-concept clinical trial data in patients with post-herpetic neuralgia. This trial was conducted because AT2 receptor antagonists evoked pain relief in rodent models of neuropathic pain. EMA401 was selected as the drug candidate based upon its suitable preclinical toxicity and safety profile and good pharmacokinetics. Herein, we provide an overview of the discovery, preclinical and clinical development of EMA401, for the alleviation of peripheral neuropathic pain.

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.

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.

Evolving views on the first two ligands of the angiotensin II type 2 receptor. From putative antagonists to potential agonists?

The renin-angiotensin system is one of the most complex regulatory systems that controls multiple organ functions. One of its key components, angiotensin II (Ang II), stimulates two G-protein coupled class A receptors: the Ang II type 1 (AT1) receptor and the Ang II type 2 (AT2) receptor. While stimulation of the AT1 receptor causes G-protein-dependent signaling and arrestin recruitment, the AT2 receptor seems to have a constitutively active-like conformation and appears to act via G-protein-dependent and -independent pathways. Overstimulation of the AT1 receptor may lead to unwanted effects like inflammation and fibrosis. In contrast, stimulation of the AT2 receptor leads to opposite effects thus restoring the balance. However, the role of the AT2 receptor has become controversial due to beneficial effects of putative AT2 receptor antagonists. The two first synthetic AT2 receptor-selective ligands, peptide CGP42112 and small molecule PD123319, were initially both considered antagonists. CGP42112 was subsequently considered a partial agonist and it was recently demonstrated to be a full agonist. Based on the search-term PD123319 in Pubmed, 1652 studies have investigated putative AT2 receptor antagonist PD123319. Here, we put forward literature that shows beneficial effects of PD123319 alone, even at doses too low for antagonist efficacy. These beneficial effects appear compatible with agonist-like activity via the AT2 receptor. Taken together, a more consistent image of a therapeutic role of stimulated AT2 receptor emerges which may clarify current controversies.

Angiotensin II type 2 receptor-mediated dilation is greater in the cutaneous microvasculature of premenopausal women compared with men.

Differential activation of the renin-angiotensin system (RAS) likely contributes to sex differences in cardiovascular outcomes in premenopausal women compared with age-matched men. Women demonstrate reduced activation of the vasoconstrictor angiotensin II type 1 receptors (AT1R) compared with men, and evidence suggests that women also likely have increased sensitivity of the vasodilatory angiotensin II type 2 receptors (AT2R). However, few in vivo studies have directly examined sex differences in AT2R-mediated dilation, or the balance between AT1R- and AT2R-mediated vascular responses in humans. Using the cutaneous microcirculation as a model, we hypothesized that AT2R-mediated dilation would be greater in premenopausal women compared with men, and that AT1R-blockade would augment AT2R-mediated dilation to a greater extent in men than in women. Twelve healthy women (22 ± 3 yr) and 12 men (23 ± 5 yr) had two intradermal microdialysis fibers placed in the ventral forearm for graded infusions of compound 21 (AT2R agonist; 10-12 to 10-8 M) in a control fiber site and a site treated with 43 µM losartan (AT1R antagonist). Red blood cell flux was measured continuously by laser-Doppler flowmetry, and cutaneous vascular conductance [CVC = flux/mean arterial pressure (MAP)] was normalized to maximum [%max; 28 mM sodium nitroprusside (SNP) + 43 °C]. Women had greater AT2R-mediated dilation compared with men (women: 25 ± 4 vs. men: 15 ± 2%max, P = 0.03). Local AT1R inhibition increased AT2R-mediated dilation in men (losartan: 26 ± 4 vs. control: 15 ± 2%max, P < 0.001) but had no effect in women (losartan: 27 ± 6 vs. control: 25 ± 4%max, P > 0.05). These data suggest that premenopausal women have a greater AT2R-mediated vasodilation response than men, and that AT1R activation inhibits AT2R-mediated dilation in men, but not in women.NEW & NOTEWORTHY Premenopausal women have greater protection against cardiovascular disease than age-matched men. However, the role of vasoconstrictor angiotensin II type 1 receptors (AT1R) and vasodilatory angiotensin II type 2 receptors (AT2R) in mediating these sex differences is unclear. Here, we demonstrate that women have greater AT2R-mediated vasodilation than men and that AT1R negates AT2R-mediated dilation in men, but not in women.

The contribution of the AT1 receptor to erythropoiesis.

The renin-angiotensin system (RAS) comprises a broad set of functional peptides and receptors that play a role in cardiovascular homeostasis and contribute to cardiovascular pathologies. Angiotensin II (Ang II) is the most potent peptide hormone produced by the RAS due to its high abundance and its strong and pleiotropic impact on the cardiovascular system. Formation of Ang II takes place in the bloodstream and additionally in tissues in the so-called local RAS. Of the two Ang II receptors (AT1 and AT2) that Ang II binds to, AT1 is the most expressed throughout the mammalian body. AT1 expression is not restricted to cells of the cardiovascular system but in fact AT1 protein is found in nearly all organs, hence, Ang II takes part in several modulatory physiological processes one of which is erythropoiesis. In this review, we present multiple evidence supporting that Ang II modulates physiological and pathological erythropoiesis processes trough the AT1 receptor. Cumulative evidence indicates that Ang II by three distinct mechanisms influences erythropoiesis: 1) stimulation of renal erythropoietin synthesis; 2) direct action on bone marrow precursor cells; and 3) modulation of sympathetic nerve activity to the bone marrow. The text highlights clinical and preclinical evidence focusing on mechanistic studies using rodent models.

Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties.

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.

Restoring Angiotensin Type 2 Receptor Function Reverses PFOS-Induced Vascular Hyper-Reactivity and Hypertension in Pregnancy.

Perfluorooctane sulfonic acid (PFOS) exposure during pregnancy induces hypertension with decreased vasodilatory angiotensin type-2 receptor (AT2R) expression and impaired vascular reactivity and fetal weights. We hypothesized that AT2R activation restores the AT1R/AT2R balance and reverses gestational hypertension by improving vascular mechanisms. Pregnant Sprague-Dawley rats were exposed to PFOS through drinking water (50 µg/mL) from gestation day (GD) 4-20. Controls received drinking water with no detectable PFOS. Control and PFOS-exposed rats were treated with AT2R agonist Compound 21 (C21; 0.3 mg/kg/day, SC) from GD 15-20. In PFOS dams, blood pressure was higher, blood flow in the uterine artery was reduced, and C21 reversed these to control levels. C21 mitigated the heightened contraction response to Ang II and enhanced endothelium-dependent vasorelaxation in uterine arteries of PFOS dams. The observed vascular effects of C21 were correlated with reduced AT1R levels and increased AT2R and eNOS protein levels. C21 also increased plasma bradykinin production in PFOS dams and attenuated the fetoplacental growth restriction. These data suggest that C21 improves the PFOS-induced maternal vascular dysfunction and blood flow to the fetoplacental unit, providing preclinical evidence to support that AT2R activation may be an important target for preventing or treating PFOS-induced adverse maternal and fetal outcomes.