Renin-Angiotensin System Inhibitors in Patients With COVID-19: A Meta-Analysis of Randomized Controlled Trials Led by the International Society of Hypertension.

Background Published randomized controlled trials are underpowered for binary clinical end points to assess the safety and efficacy of renin-angiotensin system inhibitors (RASi) in adults with COVID-19. We therefore performed a meta-analysis to assess the safety and efficacy of RASi in adults with COVID-19. Methods and Results MEDLINE, EMBASE, ClinicalTrials.gov, and the Cochrane Controlled Trial Register were searched for randomized controlled trials that randomly assigned patients with COVID-19 to RASi continuation/commencement versus no RASi therapy. The primary outcome was all-cause mortality at ≤30 days. A total of 14 randomized controlled trials met the inclusion criteria and enrolled 1838 participants (aged 59 years, 58% men, mean follow-up 26 days). Of the trials, 11 contributed data. We found no effect of RASi versus control on all-cause mortality (7.2% versus 7.5%; relative risk [RR], 0.95; [95% CI, 0.69-1.30]) either overall or in subgroups defined by COVID-19 severity or trial type. Network meta-analysis identified no difference between angiotensin-converting enzyme inhibitors versus angiotensin II receptor blockers. RASi users had a nonsignificant reduction in acute myocardial infarction (2.1% versus 3.6%; RR, 0.59; [95% CI, 0.33-1.06]), but increased risk of acute kidney injury (7.0% versus 3.6%; RR, 1.82; [95% CI, 1.05-3.16]), in trials that initiated and continued RASi. There was no increase in need for dialysis or differences in congestive cardiac failure, cerebrovascular events, venous thromboembolism, hospitalization, intensive care admission, inotropes, or mechanical ventilation. Conclusions This meta-analysis of randomized controlled trials evaluating angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers versus control in patients with COVID-19 found no difference in all-cause mortality, a borderline decrease in myocardial infarction, and an increased risk of acute kidney injury with RASi. Our findings provide strong evidence that RASi can be used safely in patients with COVID-19.

AT2 activation does not influence brain damage in the early phase after experimental traumatic brain injury in male mice.

Antagonism of the angiotensin II type 1 receptor (AT1) improves neurological function and reduces brain damage after experimental traumatic brain injury (TBI), which may be partly a result of enhanced indirect angiotensin II type 2 receptor (AT2) stimulation. AT2 stimulation was demonstrated to be neuroprotective via anti-inflammatory, vasodilatory, and neuroregenerative mechanisms in experimental cerebral pathology models. We recently demonstrated an upregulation of AT2 after TBI suggesting a protective mechanism. The present study investigated the effect of post-traumatic (5 days after TBI) AT2 activation via high and low doses of a selective AT2 agonist, compound 21 (C21), compared to vehicle-treated controls. No differences in the extent of the TBI-induced lesions were found between both doses of C21 and the controls. We then tested AT2-knockdown animals for secondary brain damage after experimental TBI. Lesion volume and neurological outcomes in AT2-deficient mice were similar to those in wild-type control mice at both 24 h and 5 days post-trauma. Thus, in contrast to AT1 antagonism, AT2 modulation does not influence the initial pathophysiological mechanisms of TBI in the first 5 days after the insult, indicating that AT2 plays only a minor role in the early phase following trauma-induced brain damage.

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

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

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.

The protective effect of Angiotensin AT2-receptor stimulation in Neuromyelitis optica spectrum disorder is independent of astrocyte-derived BDNF.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an antibody-mediated autoimmune inflammatory disease of the central nervous system (CNS), resulting in primary astrocytopathy. We have previously shown that Angiotensin AT2-receptor (AT2R) stimulation with the specific agonist Compound 21 (C21) attenuated NMOSD-like pathology. Recent studies have proposed that the mechanism behind protective effects of AT2R includes induction of brain derived neurotrophic factor (BDNF). Astrocytes are a major cellular source of BDNF. In this study we used mice with conditional BDNF deficiency in astrocytes (GfapF) to examine the involvement of astrocyte-derived BDNF in NMOSD-like pathology and in mediating the protective effect of AT2R stimulation. METHODS: Anti-aquaporin-4 IgG (AQP4-IgG) from an NMOSD patient and human complement (C) were co-injected intrastriatally to GfapF and wildtype littermate BDNFfl/fl mice (WT), together with either C21 or vehicle at day 0, followed by intrathecal injection of C21 or vehicle at day 2 and tissue collection at day 4. RESULTS: Intracerebral/intrathecal injection of C21, alone or in combination with AQP4-IgG + C, induced BDNF expression in WT mice. Injection of AQP4-IgG + C induced NMOSD-like pathology, including loss of AQP4 and GFAP. There was no difference in the severity of pathological changes between GfapF and WT mice. C21 treatment significantly and equally ameliorated NMOSD-like pathology in both WT and GfapF mice. CONCLUSION: Our findings indicate that astrocyte-derived BDNF neither reduces the severity of NMOSD-like pathology nor is it necessary for the protective effect of AT2R stimulation in NMOSD-like pathology.

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.

Prospective meta-analysis protocol on randomised trials of renin-angiotensin system inhibitors in patients with COVID-19: an initiative of the International Society of Hypertension.

INTRODUCTION: Whether ACE inhibitors (ACEi) or angiotensin II receptor blocker (ARB) therapy should be continued, initiated or ceased in patients with COVID-19 is uncertain. Given the widespread use of ACEi/ARBs worldwide, guidance on the use of these drugs is urgently needed. This prospective meta-analysis aims to pool data from randomised controlled trials (RCTs) to assess the safety and efficacy of ACEi/ARB therapy in adults infected with SARS-CoV-2. METHODS AND ANALYSIS: RCTs will be eligible if they compare patients with COVID-19 randomised to ACEi/ARB continuation or commencement versuss no ACEi/ARB therapy; study duration ≥14 days; recruitment completed between March 2020 and May 2021. The primary outcome will be all-cause mortality at ≤30 days. Secondary outcomes will include mechanical ventilation, admission to intensive care or cardiovascular events at short-term follow-up (≤30 days) and all-cause mortality at longer-term follow-up (>1 month). Prespecified subgroup analyses will assess the effect of sex; age; comorbidities; smoking status; ethnicity; country of origin on all-cause mortality. A search of ClinicalTrials.gov has been performed, which will be followed by a formal search of trial registers, preprint servers, MEDLINE, EMBASE and Cochrane Central Register of Controlled Trials to identify RCTs that meet inclusion criteria. To date, a search of ClinicalTrials.gov identified 21 potentially eligible trials for this meta-analysis. We will request trial investigators/sponsors to contribute standardised grouped tabular outcome data. ETHICS AND DISSEMINATION: Ethics approval and informed consent will be the responsibility of the individual RCTs. Dissemination of results will occur by peer-reviewed publication. The results of our analysis can inform public health policy and clinical decision making regarding ACEi/ARB use in patients with COVID-19 on a global scale.

Mas Receptor Activation Contributes to the Improvement of Nitric Oxide Bioavailability and Vascular Remodeling During Chronic AT1R (Angiotensin Type-1 Receptor) Blockade in Experimental Hypertension.

Angiotensin (1-7) production increases during AT1R (angiotensin type-1 receptor) blockade. The contribution of Ang (1-7) (angiotensin [1-7]) and its receptor (MasR) to the favorable effect of angiotensin receptor blockers on remodeling and function of resistance arteries remains unclear. We sought to determine whether MasR contributes to the improvement of vascular structure and function during chronic AT1R blockade. Spontaneously hypertensive rats were treated with Ang (1-7) or olmesartan ± MasR antagonist A-779, or vehicle, for 14 days. Blood pressure was measured by tail cuff methodology. Mesenteric arteries were dissected and mounted on a pressurized micromyograph to evaluate media-to-lumen ratio (M/L) and endothelial function. Expression of MasR and eNOS (endothelial nitric oxide synthase) was evaluated by immunoblotting, plasma nitrate by colorimetric assay, and reactive oxygen species production by dihydroethidium staining. Independently of blood pressure, olmesartan significantly reduced M/L and improved NO bioavailability, A-779 prevented these effects. Likewise, Ang (1-7) significantly reduced M/L and NO bioavailability. MasR expression was significantly increased by Ang (1-7) as well as by olmesartan, and it was blunted in the presence of A-779. Both Ang (1-7) and olmesartan increased eNOS expression and plasma nitrite which were reduced by A-779. Superoxide generation was attenuated by olmesartan and Ang (1-7) and was blunted in the presence of A-779. These MasR-mediated actions were independent of AT2R activation since olmesartan and Ang (1-7) increased MasR expression and reduced M/L in Ang II (angiotensin II)-infused AT2R knockout mice, independently of blood pressure control. A-779 prevented these effects. Hence, MasR activation may contribute to the favorable effects of AT1R antagonism on NO bioavailability and microvascular remodeling, independently of AT2R activation and blood pressure control.

Activation of AT2 receptors prevents diabetic complications in female db/db mice by NO-mediated mechanisms.

BACKGROUND AND PURPOSE: The AT2 receptor plays a role in metabolism by opposing the actions triggered by the AT1 receptors. Activation of AT2 receptors has been shown to enhance insulin sensitivity in both normal and insulin resistance animal models. In this study, we investigated the mechanism by which AT2 receptors activation improves metabolism in diabetic mice. EXPERIMENTAL APPROACH: Female diabetic (db/db) and non-diabetic (db/+) mice were treated for 1 month with the selective AT2 agonist, compound 21 (C21, 0.3 mg·kg-1 ·day-1 , s.c.). To evaluate whether the effects of C21 depend on NO production, a subgroup of mice was treated with C21 plus a sub-pressor dose of the NOS inhibitor l-NAME (0.1 mg·ml-1 , drinking water). KEY RESULTS: C21-treated db/db mice displayed improved glucose and pyruvate tolerance compared with saline-treated db/db mice. Also, C21-treated db/db mice showed reduced liver weight and decreased hepatic lipid accumulation compared with saline-treated db/db mice. Insulin signalling analysis showed increased phosphorylation of the insulin receptor, Akt and FOXO1 in the livers of C21-treated db/db mice compared with saline-treated counterparts. These findings were associated with increased adiponectin levels in plasma and adipose tissue and reduced adipocyte size in inguinal fat. The beneficial effects of AT2 receptors activation were associated with increased eNOS phosphorylation and higher levels of NO metabolites and were abolished by l-NAME. CONCLUSION AND IMPLICATIONS: Chronic C21 infusion exerts beneficial metabolic effects in female diabetic db/db mice, alleviating type 2 diabetes complications, through a mechanism that involves NO production.

The role of the renin-angiotensin system in skin physiology and pathophysiology.

Since its first description around the year 2000, the local renin-angiotensin system (RAS) in skin has been subject of an increasing number of studies with many additions over the last two to three years. A focus of research has been investigations on the role of cutaneous angiotensin receptors and locally synthesised angiotensin II in wound healing, in dermatoses associated with skin fibrosis and in melanoma. This review will provide an introduction into the RAS with emphasis on information relevant for the cutaneous RAS. It will discuss the role of the RAS in skin physiology, followed by a detailed review of the existing literature addressing the role of local angiotensin II and angiotensin AT1 and AT2 receptors in wound healing and in various skin diseases such as hypertrophic scars/keloids, scleroderma, dystrophic epidermolysis bullosa, Dupuytren's disease, squamous cell carcinoma, melanoma and psoriasis. In a final section, the potential relevance of drugs, which interfere with the RAS, for future therapy of dermatological disorders is discussed. Collectively, research about the RAS in skin can currently be described as an area, which has gained increasing attention by basic researchers, thus resulting in a multitude of preclinical studies pointing to the potential relevance of components of the RAS as drug targets in dermatological diseases. With a few small clinical studies already performed successfully for indications such as hypertrophic scars and keloids, it can be said that the skin RAS is now in the critical phase of translation from preclinical evidence to clinical use.

The renin-angiotensin system in cutaneous hypertrophic scar and keloid formation.

Hypertrophic scar and keloid are two types of fibroproliferative conditions that result from excessive extracellular matrix production. The underlying pathological mechanism is not entirely clear. Activation of the renin-angiotensin system (RAS) is associated with fibrosis in various organs. RAS components including angiotensin II (Ang II), angiotensin AT1 and AT2 receptors, and angiotensin-converting enzyme (ACE) are expressed in the skin and act independently from the plasma RAS. AT1 receptors, which are usually the dominating receptor subtype, promote fibrosis and scar formation, while AT2 receptors inhibit the aforementioned AT1 receptor-coupled effects. Elevated angiotensin II (Ang II) levels acting on the AT1 receptor contribute to skin scar formation through increased expression of inflammatory factors such as interleukin-6 (IL-6), angiogenic factors such as vascular endothelial growth factor (VEGF) and fibrinogenic factors such as transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF), while at the same time suppressing the anti-fibrotic tissue inhibitors of matrix metalloproteinase (TIMPs). First, small clinical trials have provided evidence that inhibition of the ACE/Ang II/ AT1 receptor axis may be effective in the treatment of hypertrophic scars/keloids. This review provides a detailed overview of the current literature on the RAS in skin, wound healing and scar formation and discusses the translational potential of targeting this hormonal system for treatment and prevention of hypertrophic scars and keloids.

May Measurement Month 2019: The Global Blood Pressure Screening Campaign of the International Society of Hypertension.

Elevated blood pressure remains the single biggest risk factor contributing to the global burden of disease and mortality. May Measurement Month is an annual global screening campaign aiming to improve awareness of blood pressure at the individual and population level. Adults (≥18 years) recruited through opportunistic sampling were screened at sites in 92 countries during May 2019. Ideally, 3 blood pressure readings were measured for each participant, and data on lifestyle factors and comorbidities were collected. Hypertension was defined as a systolic blood pressure ≥140 mm Hg, or a diastolic blood pressure ≥90 mm Hg (mean of the second and third readings) or taking antihypertensive medication. When necessary, multiple imputation was used to estimate participants' mean blood pressure. Mixed-effects models were used to evaluate associations between blood pressure and participant characteristics. Of 1 508 130 screenees 482 273 (32.0%) had never had a blood pressure measurement before and 513 337 (34.0%) had hypertension, of whom 58.7% were aware, and 54.7% were on antihypertensive medication. Of those on medication, 57.8% were controlled to <140/90 mm Hg, and 28.9% to <130/80 mm Hg. Of all those with hypertension, 31.7% were controlled to <140/90 mm Hg, and 350 825 (23.3%) participants had untreated or inadequately treated hypertension. Of those taking antihypertensive medication, half were taking only a single drug, and 25% reported using aspirin inappropriately. This survey is the largest ever synchronized and standardized contemporary compilation of global blood pressure data. This campaign is needed as a temporary substitute for systematic blood pressure screening in many countries worldwide.

Highlights from the International Society of Hypertension's New Investigators Network during 2019.

: The New Investigators Committee (NIC) of the International Society of Hypertension (ISH) is a dynamic group of junior doctors and scientists, actively involved in various society activities. This report highlights the events (scientific meetings and summer schools) and activities (social media, mentorship and networking) during 2019 including May Measurement Month and collaborative efforts with the ISH Women in Hypertension Research Committee (WiHRC). The ISH NIC is proud to sponsor awards for outstanding work by junior and emerging researchers at hypertension conferences and also provides opportunities to showcase their work on our social media features such as 'Our Fellows Work' and the New Investigator Spotlight of the month. In 2020, the ISH NIC aims to promote women in leadership roles and to foster strong collaborations with and between society committees and other scientific organizations.

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

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

Anti-fibrotic mechanisms of angiotensin AT2 -receptor stimulation.

The angiotensin AT2 -receptor is a main receptor of the protective arm of the renin-angiotensin system. Understanding of this unconventional G-protein coupled receptor has significantly advanced during the past decade, largely because of the availability of a selective non-peptide AT2 -receptor agonist, which allowed the conduct of a multitude of studies in animal disease models. This article reviews such preclinical studies that in their entirety provide strong evidence for an anti-fibrotic effect mediated by activation of the AT2 -receptor. Prevention of the development of fibrosis by AT2 -receptor stimulation has been demonstrated in lungs, heart, blood vessels, kidney, pancreas and skin. In lungs, AT2 -receptor stimulation was even able to reverse existing fibrosis. The article further discusses intracellular signalling mechanisms mediating the AT2 -receptor-coupled anti-fibrotic effect, including activation of phosphatases and subsequent interference with pro-fibrotic signalling pathways, induction of matrix-metalloproteinases and hetero-dimerization with the AT1 -receptor, the TGF-ßRII-receptor or the RXFP1-receptor for relaxin. Knowledge of the anti-fibrotic effects of the AT2 -receptor is of particular relevance because drugs targeting this receptor have entered clinical development for indications involving fibrotic diseases.

The renin-angiotensin system: going beyond the classical paradigms.

Thirty years ago, a novel axis of the renin-angiotensin system (RAS) was unveiled by the discovery of angiotensin-(1-7) [ANG-(1-7)] generation in vivo. Later, angiotensin-converting enzyme 2 (ACE2) was shown to be the main mediator of this reaction, and Mas was found to be the receptor for the heptapeptide. The functional analysis of this novel axis of the RAS that followed its discovery revealed numerous protective actions in particular for cardiovascular diseases. In parallel, similar protective actions were also described for one of the two receptors of ANG II, the ANG II type 2 receptor (AT2R), in contrast to the other, the ANG II type 1 receptor (AT1R), which mediates deleterious actions of this peptide, e.g., in the setting of cardiovascular disease. Very recently, another branch of the RAS was discovered, based on angiotensin peptides in which the amino-terminal aspartate was replaced by alanine, the alatensins. Ala-ANG-(1-7) or alamandine was shown to interact with Mas-related G protein-coupled receptor D, and the first functional data indicated that this peptide also exerts protective effects in the cardiovascular system. This review summarizes the presentations given at the International Union of Physiological Sciences Congress in Rio de Janeiro, Brazil, in 2017, during the symposium entitled "The Renin-Angiotensin System: Going Beyond the Classical Paradigms," in which the signaling and physiological actions of ANG-(1-7), ACE2, AT2R, and alatensins were reported (with a focus on noncentral nervous system-related tissues) and the therapeutic opportunities based on these findings were discussed.

Chronic administration of the angiotensin type 2 receptor agonist C21 improves insulin sensitivity in C57BL/6 mice.

The renin-angiotensin system modulates insulin action. Angiotensin type 1 receptor exerts a deleterious effect, whereas the angiotensin type 2 receptor (AT2R) appears to have beneficial effects providing protection against insulin resistance and type 2 diabetes. To further explore the role of the AT2R on insulin action and glucose homeostasis, in this study we administered C57Bl/6 mice with the synthetic agonist of the AT2R C21 for 12 weeks (1 mg/kg per day; ip). Vehicle-treated animals were used as control. Metabolic parameters, glucose, and insulin tolerance, in vivo insulin signaling in main insulin-target tissues as well as adipose tissue levels of adiponectin, and TNF-α were assessed. C21-treated animals displayed decreased glycemia together with unaltered insulinemia, increased insulin sensitivity, and increased glucose tolerance compared to nontreated controls. This was accompanied by a significant decrease in adipocytes size in epididymal adipose tissue and significant increases in both adiponectin and UCP-1 expression in this tissue. C21-treated mice showed an increase in both basal Akt and ERK1/2 phosphorylation levels in the liver, and increased insulin-stimulated Akt activation in adipose tissue. This positive modulation of insulin action induced by C21 appeared not to involve the insulin receptor. In C21-treated mice, adipose tissue and skeletal muscle became unresponsive to insulin in terms of ERK1/2 phosphorylation levels. Present data show that chronic pharmacological activation of AT2R with C21 increases insulin sensitivity in mice and indicate that the AT2R has a physiological role in the conservation of insulin action.

The Selective Angiotensin II Type 2 Receptor Agonist, Compound 21, Attenuates the Progression of Lung Fibrosis and Pulmonary Hypertension in an Experimental Model of Bleomycin-Induced Lung Injury.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic lung disease characterized by scar formation and respiratory insufficiency, which progressively leads to death. Pulmonary hypertension (PH) is a common complication of IPF that negatively impacts clinical outcomes, and has been classified as Group III PH. Despite scientific advances, the dismal prognosis of IPF and associated PH remains unchanged, necessitating the search for novel therapeutic strategies. Accumulating evidence suggests that stimulation of the angiotensin II type 2 (AT2) receptor confers protection against a host of diseases. In this study, we investigated the therapeutic potential of Compound 21 (C21), a selective AT2 receptor agonist in the bleomycin model of lung injury. A single intra-tracheal administration of bleomycin (2.5 mg/kg) to 8-week old male Sprague Dawley rats resulted in lung fibrosis and PH. Two experimental protocols were followed: C21 was administered (0.03 mg/kg/day, ip) either immediately (prevention protocol, BCP) or after 3 days (treatment protocol, BCT) of bleomycin-instillation. Echocardiography, hemodynamic, and Fulton's index assessments were performed after 2 weeks of bleomycin-instillation. Lung tissue was processed for gene expression, hydroxyproline content (a marker of collagen deposition), and histological analysis. C21 treatment prevented as well as attenuated the progression of lung fibrosis, and accompanying PH. The beneficial effects of C21 were associated with decreased infiltration of macrophages in the lungs, reduced lung inflammation and diminished pulmonary collagen accumulation. Further, C21 treatment also improved pulmonary pressure, reduced muscularization of the pulmonary vessels and normalized cardiac function in both the experimental protocols. However, there were no major differences in any of the outcomes measured from the two experimental protocols. Collectively, our findings indicate that stimulation of the AT2 receptor by C21 attenuates bleomycin-induced lung injury and associated cardiopulmonary pathology, which needs to be further explored as a promising approach for the clinical treatment of IPF and Group III PH.

Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase.

The Angiotensin II type 2 receptor (AT2R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT2R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT2R-mediated phosphorylation of eNOS at Ser1177, activation of phosphatases and dephosphorylation of eNOS at Tyr657 and Thr495 are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT2R-agonist Compound 21 (C21) (1 µM) in the presence or absence of either PD123319 (10 µM; AT2R antagonist), l-NG-Nitroarginine methyl ester (l-NAME) (10 µM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na3VO4) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser1177) or inhibitory eNOS residues (eNOS-Tyr657, eNOS-Thr495) was determined by Western blotting. Phosphorylation of Akt at Ser473 was measured to estimate Akt activity. AT2R stimulation significantly increased NO release from HAEC, which was blocked by PD123319, l-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT2R stimulation resulted in phosphorylation of eNOS-Ser1177 and dephosphorylation of eNOS-Tyr657 and eNOS-Thr495 Phosphorylation at eNOS-Ser1177 was prevented by inhibition of Akt with MK-2206. From these data, we conclude that AT2R stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr657, eNOS-Thr495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.

Protective effects of the angiotensin II AT2 receptor agonist compound 21 in ischemic stroke: a nose-to-brain delivery approach.

Significant neuroprotective effects of angiotensin II type 2 (AT2) receptor (AT2 receptor) agonists in ischemic stroke have been previously demonstrated in multiple studies. However, the routes of agonist application used in these pre-clinical studies, direct intracerebroventricular (ICV) and systemic administration, are unsuitable for translation into humans; in the latter case because AT2 receptor agonists are blood-brain barrier (BBB) impermeable. To circumvent this problem, in the current study we utilized the nose-to-brain (N2B) route of administration to bypass the BBB and deliver the selective AT2 receptor agonist Compound 21 (C21) to naïve rats or rats that had undergone endothelin 1 (ET-1)-induced ischemic stroke. The results obtained from the present study indicated that C21 applied N2B entered the cerebral cortex and striatum within 30 min in amounts that are therapeutically relevant (8.4-9 nM), regardless of whether BBB was intact or disintegrated. C21 was first applied N2B at 1.5 h after stroke indeed provided neuroprotection, as evidenced by a highly significant, 57% reduction in cerebral infarct size and significant improvements in Bederson and Garcia neurological scores. N2B-administered C21 did not affect blood pressure or heart rate. Thus, these data provide proof-of-principle for the idea that N2B application of an AT2 receptor agonist can exert neuroprotective actions when administered following ischemic stroke. Since N2B delivery of other agents has been shown to be effective in certain human central nervous system diseases, the N2B application of AT2 receptor agonists may become a viable mode of delivering these neuroprotective agents for human ischemic stroke patients.