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Abstract Objectives As of December, 1st, 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, resulted in more than 1 472 917 deaths worldwide and death toll is still increasing exponentially. Many COVID-19 infected people are asymptomatic or experience moderate symptoms and recover without medical intervention. However, older people and those with comorbid hypertension, diabetes, obesity, or heart disease are at higher risk of mortality. Because current therapeutic options for COVID-19 patients are limited specifically for this elderly population at risk, Biophytis is developing BIO101 (20-hydroxyecdysone, a Mas receptor activator) as a new treatment option for managing patients with SARS-CoV-2 infection at the severe stage. The angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2. Interaction between ACE2 and SARS-CoV2 spike protein seems to alter the function of ACE2, a key player in the renin-angiotensin system (RAS). The clinical picture of COVID-19 includes acute respiratory distress syndrome (ARDS), cardiomyopathy, multiorgan dysfunction and shock, all of which might result from an imbalance of the RAS. We propose that RAS balance could be restored in COVID-19 patients through MasR activation downstream of ACE2 activity, with 20-hydroxyecdysone (BIO101) a non-peptidic Mas receptor (MasR) activator. Indeed, MasR activation by 20-hydroxyecdysone harbours anti-inflammatory, anti-thrombotic, and anti-fibrotic properties. BIO101, a 97% pharmaceutical grade 20-hydroxyecdysone could then offer a new therapeutic option by improving the respiratory function and ultimately promoting survival in COVID-19 patients that develop severe forms of this devastating disease. Therefore, the objective of this COVA study is to evaluate the safety and efficacy of BIO101, whose active principle is 20-hydroxyecdysone, in COVID-19 patients with severe pneumonia. Trial design Randomized, double-blind, placebo-controlled, multi-centre, group sequential and adaptive which will be conducted in 2 parts. Part 1: Ascertain the safety and tolerability of BIO101 and obtain preliminary indication of the activity of BIO101, in preventing respiratory deterioration in the target population Part 2: Re-assessment of the sample size needed for the confirmatory part 2 and confirmation of the effect of BIO101 observed in part 1 in the target population. The study is designed as group sequential to allow an efficient run-through, from obtaining an early indication of activity to a final confirmation. And adaptive – to allow accumulation of early data and adapt sample size in part 2 in order to inform the final design of the confirmatory part of the trial. Participants Inclusion criteria 1. Age: 45 and above 2. A confirmed diagnosis of COVID-19 infection, within the last 14 days, prior to randomization, as determined by PCR or other approved commercial or public health assay, in a specimen as specified by the test used. 3. Hospitalized, in observation or planned to be hospitalized due to COVID-19 infection symptoms with anticipated hospitalization duration ≥3 days 4. With evidence of pneumonia based on all of the following: a. Clinical findings on a physical examination b. Respiratory symptoms developed within the past 7 days 5. With evidence of respiratory decompensation that started not more than 4 days before start of study medication and present at screening, meeting one of the following criteria, as assessed by healthcare staff: a. Tachypnea: ≥25 breaths per minute b. Arterial oxygen saturation ≤92% c. A special note should be made if there is suspicion of COVID-19-related myocarditis or pericarditis, as the presence of these is a stratification criterion 6. Without a significant deterioration in liver function tests: a. ALT and AST ≤ 5x upper limit of normal (ULN) b. Gamma-glutamyl transferase (GGT) ≤ 5x ULN c. Total bilirubin ≤ 5×ULN 7. Willing to participate and able to sign an informed consent form (ICF). Or, when relevant, a legally authorized representative (LAR) might sign the ICF on behalf of the study participant 8. Female participants should be: at least 5 years post-menopausal (i.e., persistent amenorrhea 5 years in the absence of an alternative medical cause) or surgically sterile; OR a. Have a negative urine pregnancy test at screening b. Be willing to use a contraceptive method as outlined in inclusion criterion 9 from screening to 30 days after last dose. 9. Male participants who are sexually active with a female partner must agree to the use of an effective method of birth control throughout the study and until 3 months after the last administration of the investigational product. (Note: medically acceptable methods of contraception that may be used by the participant and/or partner include combined oral contraceptive, contraceptive vaginal ring, contraceptive injection, intrauterine device, etonogestrel implant, each supplemented with a condom, as well as sterilization and vasectomy). 10. Female participants who are lactating must agree not to breastfeed during the study and up to 14 days after the intervention. 11. Male participants must agree not to donate sperm for the purpose of reproduction throughout the study and until 3 months after the last administration of the investigational product. 12. For France only: Being affiliated with a European Social Security. Exclusion criteria 1. Not needing or not willing to remain in a healthcare facility during the study 2. Moribund condition (death likely in days) or not expected to survive for >7 days – due to other and non-COVID-19 related conditions 3. Participant on invasive mechanical ventilation via an endotracheal tube, or extracorporeal membrane oxygenation (ECMO), or high-flow Oxygen (delivery of oxygen at a flow of ≥16 L/min.). 4. Participant is not able to take medications by mouth (as capsules or as a powder, mixed in water). 5. Disallowed concomitant medication: Consumption of any herbal products containing 20-hydroxyecdysone and derived from Leuzea carthamoides; Cyanotis vaga or Cyanotis arachnoidea is not allowed (e.g. performance enhancing agents). 6. Any known hypersensitivity to any of the ingredients, or excipients of the study medication, BIO101. 7. Renal disease requiring dialysis, or known renal insufficiency (eGFR≤30 mL/min/1.73 m2, based on Cockcroft & Gault formula). 8. In France only: a. Non-affiliation to compulsory French social security scheme (beneficiary or right-holder). b. Being under tutelage or legal guardianship. Participants will be recruited from approximately 30 clinical centres in Belgium, France, the UK, USA and Brazil. Maximum patients’ participation in the study will last 28 days. Follow-up of participants discharged from hospital will be performed through post-intervention phone calls at 14 (± 2) and 60 (± 4) days. Intervention and comparator Two treatment arms will be tested in this study: interventional arm 350 mg b.i.d. of BIO101 (AP 20-hydroxyecdysone) and placebo comparator arm 350 mg b.i.d of placebo. Administration of daily dose is the same throughout the whole treatment period. Participants will receive the study medication while hospitalized for up to 28 days or until a clinical endpoint is reached (i.e., ‘negative’ or ‘positive’ event). Participants who are officially discharged from hospital care will no longer receive study medication. Main outcomes Primary study endpoint: The proportion of participants with ‘negative’ events up to 28 days. ‘Negative’ events are defined as respiratory deterioration and all-cause mortality. For the purpose of this study, respiratory deterioration will be defined as any of the following: Requiring mechanical ventilation (including cases that will not be intubated due to resource restrictions and triage). Requiring extracorporeal membrane oxygenation (ECMO). Requiring high-flow oxygen defined as delivery of oxygen at a flow of ≥16 L/min. Only if the primary endpoint is significant at the primary final analysis the following Key secondary endpoints will be tested in that order: Proportion of participants with events of respiratory failure at Day 28 Proportion of participants with ‘positive’ events at Day 28. Proportion of participants with events of all-cause mortality at Day 28 A ‘positive’ event is defined as the official discharge from hospital care by the department due to improvement in participant condition. Secondary and exploratory endpoints: In addition, a variety of functional measures and biomarkers (including the SpO2 / FiO2 ratio, viral load and markers related to inflammation, muscles, tissue and the RAS / MAS pathways) will also be collected. Randomization Randomization is performed using an IBM clinical development IWRS system during the baseline visit. Block-permuted randomization will be used to assign eligible participants in a 1:1 ratio. In part 1, randomization will be stratified by RAS pathway modulator use (yes/no) and co-morbidities (none vs. 1 and above). In Part 2, randomization will be stratified by centre, gender, RAS pathway modulator use (yes/no), co-morbidities (none vs. 1 and above), receiving Continuous Positive Airway Pressure/Bi-level Positive Airway Pressure (CPAP/BiPAP) at study entry (Yes/No) and suspicion of COVID-19 related myocarditis or pericarditis (present or not). Blinding (masking) Participants, caregivers, and the study team assessing the outcomes are blinded to group assignment. All therapeutic units (TU), BIO101 b.i.d. or placebo b.i.d., cannot be distinguished in compliance with the double-blind process. An independent data-monitoring committee (DMC) will conduct 2 interim analyses. A first one based on the data from part 1 and a second from the data from parts 1 and 2. The first will inform about BIO101 safety, to allow the start of recruitment into part 2 followed by an analysis of the efficacy data, to obtain an indication of activity. The second interim analysis will inform about the sample size that will be required for part 2, in order to achieve adequate statistical power. Numbers to be randomised (sample size) Number of participants randomized: up to 465, in total Part 1: 50 (to obtain the proof of concept in COVID-19 patients). Part 2: 310, potentially increased by 50% (up to 465, based on interim analysis 2) (to confirm the effects of BIO101 observed in part 1). Trial Status The current protocol Version is V 10.0, dated on 24.09.2020. The recruitment that started on September 1st 2020 is ongoing and is anticipated to finish for the whole study by March2021. Trial registration The trial was registered before trial start in trial registries: EudraCT , No. 2020-001498-63, registered May 18, 2020; and Clinicaltrials.gov, identifier NCT04472728 , registered July 15, 2020. Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Since December 2019, coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has changed our lives. Elderly and those with comorbidities represent the vast majority of patients hospitalized with severe COVID-19 symptoms, including acute respiratory disease syndrome and cardiac dysfunction. Despite a huge effort of the scientific community, improved treatment modalities limiting the severity and mortality of hospitalized COVID-19 patients are still required. Here, we compare the effectiveness of virus- and patients-centred strategies to reduce COVID-19 mortality. We also discuss the therapeutic options that might further reduce death rates associated with the disease in the future. Unexpectedly, extensive review of the literature suggests that SARS-CoV-2 viral load seems to be associated neither with the severity of symptoms nor with mortality of hospitalized patients with COVID-19. This may explain why, so far, virus-centred strategies using antivirals aiming to inhibit the viral replicative machinery have failed to reduce COVID-19 mortality in patients with respiratory failure. By contrast, anti-inflammatory treatments without antiviral capacities but centred on patients, such as dexamethasone or Tocilizumab®, reduce COVID-19 mortality. Finally, since the spike protein of SARS-CoV-2 binds to angiotensin converting enzyme 2 and inhibits its function, we explore the different treatment options focussing on rebalancing the renin-angiotensin system. This new therapeutic strategy could hopefully further reduce the severity of respiratory failure and limit COVID-19 mortality in elderly patients.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debat ed, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse m yoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1-7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abol ished by angiotensin(1-7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consi stent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1-7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E. [ABSTRACT FROM AUTHOR]

Aging is associated with impaired vascular repair following ischemic insult, largely due to reparative dysfunctions of progenitor cells. Activation of Mas receptor (MasR) was shown to reverse aging-associated vasoreparative dysfunction. This study tested the impact of MasR-deficiency on mobilization and vasoreparative functions with aging. Wild type (WT) or MasR-deficient mice (MasR−/− or MasR+/−) at 12–14 weeks (young) or middle age (11–12 months) (MA) were used in the study. Mobilization of lineage-negative, Sca-1-positive cKit-positive (LSK) cells in response to G-CSF or plerixafor was determined. Hindlimb ischemia (HLI) was induced by femoral artery ligation. Mobilization and blood flow recovery were monitored post-HLI. Radiation chimeras were made by lethal irradiation of WT or MasR−/− mice followed by administration of bone marrow cells from MasR−/− or WT mice, respectively. Nitric oxide (NO) generation by stromal-derived factor-1α (SDF) and mitochondrial reactive oxygen species (mitoROS) levels were determined by flow cytometry. Effect of A779 treatment on mobilization, blood flow recovery, and NO and ROS levels were determined in young WT and MasR+/− mice. Circulating LSK cells in basal or in response to plerixafor or G-CSF or in response to ischemic injury were lower in MasR−/− mice compared to the WT. Responses in MasR+/− mice were similar to the WT at young age but at the middle age, impairments were observed. Impaired mobilization to ischemia or G-CSF was rescued in WT → MasR−/− chimeras. NO levels were lower and mitoROS were higher in MasR−/− LSK cells compared to WT cells. A779 precipitated dysfunctions in young-MasR+/− mice similar to that observed in MA-MasR+/−, and this accompanied decreased NO generation by SDF and enhanced mitoROS levels. This study shows that mice at MA do not exhibit vasoreparative dysfunction. Either partial or total loss of MasR precipitates advanced-aging phenotype likely due to lack of NO and oxidative stress. [ABSTRACT FROM AUTHOR]

GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis; the latter is an essential factor for iNOS activation that contributes neuronal loss in Huntington's disease (HD). The aim of the study was to investigate the neuroprotective effect of 2,4-diamino-6-hydroxypyrimidine (DAHP), GTPCH I enzyme inhibitor, against neuronal loss in 3-nitropropinic acid (3-NP)-induced HD in rats and to reveal the possible involved mechanisms mediated through PI3K/Akt axis and its correlation to Mas receptor (MasR). Rats received 3-NP (10 mg/kg/day; i.p.) with or without administration of DAHP (0.5 g/kg/day; i.p.) or wortmannin (WM), a PI3K inhibitor, (15 μg/kg/day; i.v.) for 14 days. DAHP improved cognitive, memory, and motor abnormalities induced by 3-NP, as confirmed by striatal histopathological specimens and immunohistochemical examination of GFAP. Moreover, DAHP treatment inhibited GTPCH I activity, resulting in decreased BH4 levels and iNOS activation. Also, DAHP upregulated the protein expression of survival protein; p85/p55 (pY458/199)-PI3K and pS473-Akt that, in turn, boosted the activation of striatal neurotrophic factors and receptor, pS133-CREB, BDNF and pY515-TrKB, which positively affect MasR protein expression and improve mitochondrial dysfunction, as indicated by enhancing both SDH and PGC-1α levels. Indeed, DAHP attenuates oxidative stress by increasing SOD activity and Nrf2 expression in addition to reducing neuro-inflammatory status by inhibiting NF-κB p65 and TNF-α expression. Interestingly, all the previous effects were blocked by co-administration of WM with DAHP. In conclusion, DAHP exerts neuroprotective effect against neuronal loss induced by 3-NP administration via inhibition of GTPCH I and iNOS activity and activation of MasR/PI3K/Akt/CREB/BDNF/TrKB axis besides its antioxidant and anti-inflammatory effect. [ABSTRACT FROM AUTHOR]

This study investigated the impact of intrarenal angiotensin 1–7 (Ang [1–7]) infusion on renal excretory function in a rat model of hypertension. Eleven‐week‐old spontaneously hypertensive rats (SHRs, n = 7) and Han Wistar controls (NCR, n = 7) were anaesthetised with sodium pentobarbital (60 mg/kg i.p.) and prepared for the measurement of mean arterial pressure (MAP) and left renal function during renal interstitial infusion of Ang (1–7) (50 ng/min). The kidneys were harvested, the renal cortex and medulla separated, prepared for measurement of Ang II and Ang (1–7) and Western blot determination of AT1 and Mas receptor protein expression. MAP, glomerular filtration rate (GFR), urine flow (UF) and absolute sodium excretion (UNaV) were 109 ± 16 mmHg, 4.4 ± 1.0 mL/min/kg, 102 ± 16 µL/min/kg and 16 ± 3 µmol/min/kg, respectively in the NCR and 172 ± 24 mmHg, 3.4 ± 0.7 mL/min/kg, 58 ± 30 μL/min/kg and 8.6 ± 4.8 μmol/min/kg respectively in the SHR. Ang (1–7) increased UF (31%), UNaV (50%) and fractional sodium excretion (FENa+) (22%) in the NCR group (all p < 0.05) but had no effect on GFR in either group. The magnitudes of the Ang (1–7)‐induced increases in UF and UNaV were significantly blunted in the SHR group (model × drug p < 0.05). The renal cortical AT1: Mas receptor expression ratio was significantly higher in the SHR group (p < 0.05) but renal Ang II and Ang (1–7) levels were not statistically different between groups. The Ang (1–7)‐induced increases in sodium and water excretion were impaired in the SHR group in the context of an unstimulated RAS. The decrease in responsiveness of the SHR kidney to Ang (1–7) appears to be associated with higher levels of AT1 receptor expression in the renal cortex. [ABSTRACT FROM AUTHOR]

Background and purpose: Chronic kidney disease is among the common diseases in the world. Studies show that women are more protected against renal diseases compared to men. On the other hand, vasodilatory axises of renin angiotensin system (angiotensin 1-7 (Ang 1-7)- Mas receptor (MasR)), kallikrein-kinin, and nitric oxide (NO) play key roles in kidney function and circulation in females. Therefore, the purpose of this study was to examine the effect of bradykinin in renal blood flow (RBF) response to Ang 1-7 when MasR was blocked in female rats. Materials and methods: Female Wistar rats were divided into three groups (control, Bradykinine, and Bradykinin + MasR antagonist (A779)). The animals were anesthetized and catheterized in the carotid and femoral arteries and jugular vein to measure mean arterial pressure (MAP), renal perfusion pressure (RPP), and drug administration, respectively. The left kidney was exposed (in situ) and placed in a kidney cup to measure RBF. After the equilibration period, A779 and bradykinin (50 μg/kg/h, 150 μg/kg/h, respectively) were injected and vascular responses to Ang1-7 (100, 300, and 1000 ng kg−1min−1) infusion were determined. At the end of the experiment, blood samples were collected for measuring the level of nitrite. Results: The MAP and RPP were not significantly different between the three groups. Bradykinin exhibited vasodilatory effect on RBF in response to Ang 1-7 in female rats, however, A779 administration increased RBF at low dose of Ang 1-7(Pdose=0.001). The serum level of nitrite significantly decreased when the MasR was blocked (P= 0.04). Conclusion: Bradykinin enhanced RBF response to Ang 1-7 infusion. The effect of bradykinin on RBF response to Ang 1-7 may be modulated via the NO pathway. Interaction of these factors might help to broaden our vision for treatment strategy in future.

GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis; the latter is an essential factor for iNOS activation that contributes neuronal loss in Huntington’s disease (HD). The aim of the study was to investigate the neuroprotective effect of 2,4-diamino-6-hydroxypyrimidine (DAHP), GTPCH I enzyme inhibitor, against neuronal loss in 3-nitropropinic acid (3-NP)-induced HD in rats and to reveal the possible involved mechanisms mediated through PI3K/Akt axis and its correlation to Mas receptor (MasR). Rats received 3-NP (10 mg/kg/day; i.p.) with or without administration of DAHP (0.5 g/kg/day; i.p.) or wortmannin (WM), a PI3K inhibitor, (15 μg/kg/day; i.v.) for 14 days. DAHP improved cognitive, memory, and motor abnormalities induced by 3-NP, as confirmed by striatal histopathological specimens and immunohistochemical examination of GFAP. Moreover, DAHP treatment inhibited GTPCH I activity, resulting in decreased BH4 levels and iNOS activation. Also, DAHP upregulated the protein expression of survival protein; p85/p55 (pY458/199)-PI3K and pS473-Akt that, in turn, boosted the activation of striatal neurotrophic factors and receptor, pS133-CREB, BDNF and pY515-TrKB, which positively affect MasR protein expression and improve mitochondrial dysfunction, as indicated by enhancing both SDH and PGC-1α levels. Indeed, DAHP attenuates oxidative stress by increasing SOD activity and Nrf2 expression in addition to reducing neuro-inflammatory status by inhibiting NF-κB p65 and TNF-α expression. Interestingly, all the previous effects were blocked by co-administration of WM with DAHP. In conclusion, DAHP exerts neuroprotective effect against neuronal loss induced by 3-NP administration via inhibition of GTPCH I and iNOS activity and activation of MasR/PI3K/Akt/CREB/BDNF/TrKB axis besides its antioxidant and anti-inflammatory effect.

Aim: The influence of the physiochemical properties of dendrimer nanoparticles on cardiac contractility and hemodynamics are not known. Herein, we investigated (a) the effect of polyamidoamine (PAMAM) dendrimer generation (G7, G6, G5, G4 and G3) and surface chemistry (-NH2, -COOH and -OH) on cardiac function in mammalian hearts following ischemia-reperfusion (I/R) injury, and (b) determined if any PAMAM-induced cardiotoxicity could be mitigated by Angiotensin-(1-7) (Ang-(1-7), a cardioprotective agent. Methods: Hearts isolated from male Wistar rats underwent regional I/R and/or treatment with different PAMAM dendrimers, Ang-(1-7) or its MAS receptors antagonists. Thirty minutes of regional ischemia through ligation of the left anterior descending coronary artery was followed by 30 min of reperfusion. All treatments were initiated 5 min prior to reperfusion and maintained during the first 10 min of reperfusion. Cardiac function parameters for left ventricular contractility, hemodynamics and vascular dynamics data were acquired digitally, whereas cardiac enzymes and infarct size were used as measures of cardiac injury. Results: Treatment of isolated hearts with increasing doses of G7 PAMAM dendrimer progressively exacerbated recovery of cardiac contractility and hemodynamic parameters post-I/R injury. Impairment of cardiac function was progressively less on decreasing dendrimer generation with G3 exhibiting little or no cardiotoxicity. Cationic PAMAMs (-NH2) were more toxic than anionic (-COOH), with neutral PAMAMs (-OH) exhibiting the least cardiotoxicity. Cationic G7 PAMAM-induced cardiac dysfunction was significantly reversed by Ang-(1-7) administration. These cardioprotective effects of Ang-(1-7) were significantly revoked by administration of the MAS receptor antagonists, A779 and D-Pro7-Ang-(1-7). Conclusions: PAMAM dendrimers can impair the recovery of hearts from I/R injury in a dose-, dendrimer-generation-(size) and surface-charge dependent manner. Importantly, PAMAM-induced cardiotoxicity could be mitigated by Ang-(1-7) acting through its MAS receptor. Thus, this study highlights the activation of Ang-(1-7)/Mas receptor axis as a novel strategy to overcome dendrimer-induced cardiotoxicity.

Calcitriol, an active form of vitamin D, has immunomodulatory and anti-inflammatory properties. Vitamin D levels have inverse correlation with sepsis outcomes and obesity may aggravate the severity of the diseases. This study administered calcitriol to investigate its impact on sepsis-induced acute lung injury (ALI) in obese mice. Mice were fed a high-fat diet to induce obesity and were randomly assigned to control or sepsis groups, which were intravenously administered either saline (SS) or calcitriol (SD). Sepsis was induced by cecal ligation and puncture (CLP). Saline or calcitriol was injected 1 h after CLP via tail vein. Mice were sacrificed at either 12 or 24 h post-CLP and survival rates were observed. The results demonstrated that sepsis caused upregulation of inflammatory mediators and downregulation of renin-angiotensin system (RAS)-associated gene expressions in the lungs of obese mice. Cluster of differentiation 68 (CD68) expression and myeloperoxidase (MPO) activities also increased. Calcitriol treatment lowered expressions of blood and lung inflammatory mediators at 12 and/or 24 h after CLP. The RAS-proinflammatory-associated angiotensin type 1 receptor (AT1R) was lower while anti-inflammatory Mas receptor and AT2R expressions were higher at 12 h after CLP than those in the SS group. In addition, the SD group exhibited lower CD68 expression and MPO activity. Lower lung injury scores and higher survival rates were also noted in the SD group. The findings suggest that calcitriol treatment after sepsis induction upregulated RAS-associated anti-inflammatory pathway and decreased immune cell infiltration, which may have alleviated the severity of ALI of obese mice.

Central micturition control and urine storage involve a multisynaptic neuronal circuit for the efferent control of the urinary bladder. Electrical stimulation of the lateral preoptic area (LPA) at the level of the decussation of the anterior commissure in cats evokes relaxation of the bladder, whereas ventral stimulation of LPA evokes vigorous contraction. Endogenous Angiotensin-(1–7) [(Ang-(1–7)] synthesis depends on ACE-2, and its actions on binding to Mas receptors, which were found in LPA neurons. We aimed to investigate the Ang-(1–7) actions into the LPA on intravesical pressure (IP) and cardiovascular parameters. The gene and protein expressions of Mas receptors and ACE-2 were also evaluated in the LPA. Angiotensin-(1–7) (5 nmol/μL) or A-779 (Mas receptor antagonist, 50 nmol/μL) was injected into the LPA in anesthetized female Wistar rats; and the IP, mean arterial pressure (MAP), heart rate (HR), and renal conductance (RC) were recorded for 30 min. Unilateral injection of Ang-(1–7) into the LPA increased IP (187.46 ± 37.23%) with peak response at ∼23–25-min post-injection and yielded no changes in MAP, HR, and RC. Unilateral or bilateral injections of A-779 into the LPA decreased IP (−15.88 ± 2.76 and −27.30 ± 3.40%, respectively) and elicited no changes in MAP, HR, and RC. The genes and the protein expression of Mas receptors and ACE-2 were found in the LPA. Therefore, the LPA is an important part of the circuit involved in the urinary bladder control, in which the Ang-(1–7) synthetized into the LPA activates Mas receptors for increasing the IP independent on changes in RC and cardiovascular parameters.

Background The activation of AT2 (angiotensin II type 2 receptor ) and Mas receptor by angiotensin II and angiotensin‐(1‐7), respectively, is the primary process that counteracts activation of the canonical renin‐angiotensin system (RAS). Although inhibition of canonical RAS could delay the progression of physiological aging, we recently reported that deletion of Mas had no impact on the aging process in mice. Here, we used male mice with a deletion of only AT2 or a double deletion of AT2 and Mas to clarify whether these receptors contribute to the aging process in a complementary manner, primarily by focusing on aging‐related muscle weakness. Methods and Results Serial changes in grip strength of these mice up to 24 months of age showed that AT2/Mas knockout mice, but not AT2 knockout mice, had significantly weaker grip strength than wild‐type mice from the age of 18 months. AT2/Mas knockout mice exhibited larger sizes, but smaller numbers and increased frequency of central nucleation (a marker of aged muscle) of single skeletal muscle fibers than AT2 knockout mice. Canonical RAS‐associated genes, inflammation‐associated genes, and senescence‐associated genes were highly expressed in skeletal muscles of AT2/Mas knockout mice. Muscle angiotensin II content increased in AT2/Mas knockout mice. Conclusions Double deletion of AT2 and Mas in mice exaggerated aging‐associated muscle weakness, accompanied by signatures of activated RAS, inflammation, and aging in skeletal muscles. Because aging‐associated phenotypes were absent in single deletions of the receptors, AT2 and Mas could complement each other in preventing local activation of RAS during aging.

Central micturition control and urine storage involve a multisynaptic neuronal circuit for the efferent control of the urinary bladder. Electrical stimulation of the lateral preoptic area (LPA) at the level of the decussation of the anterior commissure in cats evokes relaxation of the bladder, whereas ventral stimulation of LPA evokes vigorous contraction. Endogenous Angiotensin-(1–7) [(Ang-(1–7)] synthesis depends on ACE-2, and its actions on binding to Mas receptors, which were found in LPA neurons. We aimed to investigate the Ang-(1–7) actions into the LPA on intravesical pressure (IP) and cardiovascular parameters. The gene and protein expressions of Mas receptors and ACE-2 were also evaluated in the LPA. Angiotensin-(1–7) (5 nmol/μL) or A-779 (Mas receptor antagonist, 50 nmol/μL) was injected into the LPA in anesthetized female Wistar rats; and the IP, mean arterial pressure (MAP), heart rate (HR), and renal conductance (RC) were recorded for 30 min. Unilateral injection of Ang-(1–7) into the LPA increased IP (187.46 ± 37.23%) with peak response at ∼23–25-min post-injection and yielded no changes in MAP, HR, and RC. Unilateral or bilateral injections of A-779 into the LPA decreased IP (−15.88 ± 2.76 and −27.30 ± 3.40%, respectively) and elicited no changes in MAP, HR, and RC. The genes and the protein expression of Mas receptors and ACE-2 were found in the LPA. Therefore, the LPA is an important part of the circuit involved in the urinary bladder control, in which the Ang-(1–7) synthetized into the LPA activates Mas receptors for increasing the IP independent on changes in RC and cardiovascular parameters. [ABSTRACT FROM AUTHOR]

Tourniquet is a well-established model of hind limb ischemia–reperfusion (HLI/R) in rats. Nevertheless, measures should be taken to alleviate the expected injury from ischemia/ reperfusion (I/R). In the present study, 30 adult male Sprague-Dawley rats were randomly divided into 5 groups (n = 6): control, HLI/R, HLI/R given candesartan (1 mg/kg, P.O); HLI/R given Coenzyme Q10 (CoQ10) (10 mg/kg, P.O); HLI/R given candesartan (0.5 mg/kg) and CoQ10 (5 mg/kg). The drugs were administered for 7 days starting one hour after reperfusion. Candesartan and CoQ10 as well as their combination suppressed gastrocnemius content of angiotensin II while they raised angiotensin-converting enzyme 2 (ACE2) activity, angiotensin (1–7) expression, and Mas receptor mRNA level. Consequently, candesartan and/or CoQ10 reversed the oxidative stress and inflammatory changes that occurred following HLI/R as demonstrated by the rise of SOD activity and the decline of MDA, TNF-α, and IL-6 skeletal muscle content. Additionally, candesartan and/or CoQ10 diminished gastrocnemius active caspase-3 level and phospho-p38 MAPK protein expression. Our study proved that CoQ10 enhanced the beneficial effect of candesartan in a model of tourniquet-induced HLI/R by affecting classical and non-classical renin-angiotensin system (RAS) pathway. To our knowledge, this is the first study showing the impact of CoQ10 on skeletal muscle RAS in rats. [ABSTRACT FROM AUTHOR]

Aims: Recently, dipeptidyl peptidase 3 (DPP3) has been discovered as the peptidase responsible for cleavage of angiotensin (1–7) [Ang (1–7)]. Ang (1–7) is part of the angiotensin‐converting enzyme–Ang (1–7)–Mas pathway which is considered to antagonize the renin–angiotensin–aldosterone system (RAAS). Since DPP3 inhibits the counteracting pathway of the RAAS, we hypothesize that DPP3 might be deleterious in the setting of heart failure. However, no data are available on DPP3 in chronic heart failure. We therefore investigated the clinical characteristics and outcome related to elevated DPP3 concentrations in patients with worsening heart failure. Methods and results: Dipeptidyl peptidase 3 was measured in 2156 serum samples of patients with worsening heart failure using luminometric immunoassay (DPP3‐LIA) by 4TEEN4 Pharmaceuticals GmbH, Hennigsdorf, Germany. Predictors of DPP3 levels were selected using multiple linear regression with stepwise backward selection. Median DPP3 concentration was 11.45 ng/mL with a range from 2.8 to 84.9 ng/mL. Patients with higher DPP3 concentrations had higher renin [78.3 (interquartile range, IQR 26.3–227.7) vs. 120.7 IU/mL (IQR 34.74–338.9), P < 0.001, for Q1–3 vs. Q4] and aldosterone [88 (IQR 44–179) vs. 116 IU/mL (IQR 46–241), P < 0.001, for Q1–3 vs. Q4] concentrations. The strongest independent predictors for higher concentration of DPP3 were log‐alanine aminotransferase, log‐total bilirubin, the absence of diabetes, higher osteopontin, fibroblast growth factor‐23 and N‐terminal pro‐B‐type natriuretic peptide concentrations (all P < 0.001). In univariable survival analysis, DPP3 was associated with mortality and the combined endpoint of death or heart failure hospitalization (P < 0.001 for both). After adjustment for confounders, this association was no longer significant. Conclusions: In patients with worsening heart failure, DPP3 is a marker of more severe disease with higher RAAS activity. It may be deleterious in heart failure by counteracting the Mas receptor pathway. Procizumab, a specific antibody against DPP3, might be a potential future treatment option for patients with heart failure. [ABSTRACT FROM AUTHOR]

The aortic endothelium is crucial in preserving vascular tone through endothelium-derived vasodilators and vasoconstrictors. Dysfunction in the endothelium is an early indicator of cardiovascular diseases. Our study explores the therapeutic potential of a dual-acting peptide (DAP) to co-activate Mas and pGCA receptors and restore the balance between vasodilators and vasoconstrictors on endothelial dysfunction in DOCA-salt-induced hypertensive rats. DOCA-salt was administered to male wistar rats to induce hypertension, and various parameters, including blood pressure (BP), water intake and body weight were monitored. DAP, Ang1–7, BNP, and losartan were administered to hypertensive rats for three weeks. Histological analysis and isometric tension studies were carried out to assess endothelial function. In addition to this, we used primary aortic endothelial cells for detailed mechanistic investigations. DOCA-salt administration significantly elevated systolic, diastolic, mean arterial BP, and water intake whereas, downregulated the gene expression of Mas and pGCA receptors. However, DAP co-administration attenuated BP increase, upregulated the gene expression of Mas and pGCA receptors, normalized serum and urinary parameters, and effectively reduced fibrosis, inflammation, and vascular calcification. Notably, DAP outperformed the standard drug, Losartan. Our findings indicate that DAP restores aortic function by balancing the NO and ET1-induced pathways. Co-activating Mas and pGCA receptors with DAP mitigates vascular damage and enhances endothelial function, emphasizing its potential to maintain a delicate balance between vasodilatory NO and vasoconstrictor ET1 in endothelial dysfunction. [Display omitted] • Ang1-7 & BNP, endogenous ligands of Mas & pGCA receptors, show heart & kidney protection, but use is limited by short half-life. • Simultaneous activation of multiple receptors may enhance effects beyond individual receptor activation. • Ang1-7 & BNP induce vasodilation, crucial for endothelial-dependent smooth muscle contraction and relaxation • New therapeutic targeting nitric oxide/cGMP & ET-1 pathways is the standalone therapy for vascular diseases. • Further research needed to explore Mas and pGCA receptors' role in regulating NO and ET1 balance in cardiovascular conditions [ABSTRACT FROM AUTHOR]

Objective To investigate whether TO90 inhibits angiotensin II (Ang Ⅱ)-induced inflammatory response by regulating the expressions of ACE2/Ang-(1-7)/MAS receptor axis in human retinal pigment epithelial cells. Methods In vitro cultured ARPE-19 cells were treated with Ang Ⅱ for 48 h, and the changes in the expressions of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1 (MCP-1) were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA) to verify the establishment of the inflammatory cell model. The effect of TO90 on the inflammatory response of Ang Ⅱ-treated ARPE-19 cells was evaluated by detecting the expression of IL-6, IL-8, MCP-1, ACE2 and MAS receptor using real-time PCR and Western blotting. ARPE-19 cells treated with Ang Ⅱ, TO90+Ang Ⅱ, A779 (a MAS receptor inhibitor)+TO90+Ang Ⅱ, or A779+Ang Ⅱ were examined for expressions of IL-6, IL-8, MCP-1, ACE2 and MAS receptors to further explore the effect of TO90 on Ang Ⅱ-induced inflammation response. Results Ang Ⅱ treatment resulted in significantly increased expressions of IL-6, IL-8 and MCP-1 at both the mRNA and protein levels in ARPE-19 cells (P < 0.05), and these effects were obviously attenuated by pretreatment of the cells with TO90 (P < 0.05), which also caused significantly increased expressions of ACE2 and MAS receptor in the cells (P < 0.05). In ARPE-19 cells treated with TO90+Ang Ⅱ, inhibition of MAS receptor with A779 significantly enhanced the expression of IL-6, IL-8, MCP-1, while inhibited the expression of ACE2 and MAS receptor at both the mRNA and protein levels (P < 0.05). Conclusion TO90 inhibits Ang II-induced inflammatory response by up-regulating the expressions of ACE2/Ang-(1-7)/MAS receptor axis in ARPE-19 cells.

Objective. Epidemiological studies confirm that hypertensive patients respond differently to renin-angiotensin system (RAS) inhibition depending on their gender. The aim of present work is to focus on sex-dependent differences in RAS regulation under conditions of increased salt intake.

Moderate renal ischemia/reperfusion (I/R) injury is one of the major causes of kidney failure. We examined the role of Mas receptor (MasR) antagonist (A779) alone and combined with angiotensin II (Ang II) type 2 receptor (AT2R) antagonist (PD123319) on renal hemodynamic responses to Ang II after moderate I/R in male and female rats. Anaesthetized Wistar rats underwent 30 min partial ischemia by reduction of renal perfusion pressure (RPP) and subjected to block vasodepressor receptors followed by Ang II (100 and 300 ng/kg/min) infusion. Mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) responses were assessed during graded Ang II infusion at controlled RPP. Thirty min post reperfusion, the Ang II infusion reduced RBF and increased RVR in a dose-related fashion (P < 0.05). However, A779 alone or A779 plus PD123319 infusion increased the RBF and RVR responses to Ang II infusion significantly (P < 0.05) in female but not in the male rats. MasR antagonist altered the RBF and RVR responses to Ang II infusion in female, and these responses were not altered statistically in dual blockade of MasR and AT2R. These findings suggest the important role of Mas receptor in renal vascular response to Ang II in female rats after moderate I/R.

ObjectivesRheumatoid arthritis (RA) is a disabling disease with a high incidence that is regularly accompanied by cardiovascular complications. Several studies have suggested that renin–angiotensin–aldosterone system (RAAS) is closely associated with RA. The aim of this study was to investigate the mechanisms underlying Angiotensin-(1–7) [Ang-(1–7)] and its Mas receptor agonist (AVE0991) on joint inflammation and cardiac complications in a collagen-induced arthritis (CIA) model.MethodsCollagen type II was injected into DBA/1 mice to construct an arthritis model. CIA mice were treated with Ang-(1–7) (2.0 mg/kg intraperitoneally) and AVE0991 (3.0 mg/kg intraperitoneally). The serum levels of inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 β, IL-6, and C-reactive protein (CRP)] were determined by ELISA. The mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling pathways in joint tissues and the transforming growth factor (TGF)-β/Smad pathway and levels of α-Smooth muscle action (SMA) and β-myosin heavy chain (MHC) protein expression in cardiac tissues were assessed by western blots. The levels of TGF-β/Smad pathway, α-SMA, and β-MHC RNA in cardiac tissues were analyzed by real time-PCR. The levels of receptor activator of nuclear factor kappa ligand (RANKL) and promoting matrix metalloproteinase (MMP)-3 expression in the ankle joints were detected by immunohistochemistry and real time-PCR.ResultsAng-(1–7) and AVE0991 reduced the levels of inflammatory cytokines and inhibited the MAPKs and NF-κB signaling pathways in ankle joint tissues, reduced RANKL and MMP3 expression, and ameliorated local joint inflammation and bone destruction compared with the control group. In addition, Ang-(1–7) and AVE0991 attenuated the TGF-β/Smad signaling pathway, reduced the levels of α-SMA and β-MHC expression, and diminished inflammatory cell infiltration into the myocardial interstitium and myocardial interstitial fibrosis in the hearts of CIA mice.ConclusionsAng-(1–7) alleviated joint damage caused by inflammation likely through the attenuation of NF-κB and MAPK pathways and ameliorated inflammation-induced cardiac fibrosis and activation of the TGF-β/Smad pathway. Moreover, Ang-(1–7) was likely mediated through the Mas receptor. This study provides theoretical evidence for exploring novel clinical therapeutic approaches for RA and its cardiac complications.

Background: Defective absorption of acute allergic airway inflammation is involved in the initiation and development of chronic asthma. After allergen exposure, there is a rapid recruitment of macrophages around the airways, which promote acute inflammatory responses. The Ang-(1−7)/Mas receptor axis reportedly plays protective roles in various tissue inflammation and remodeling processes in vivo. However, the exact role of Mas receptor and their underlying mechanisms during the pathology of acute allergic airway inflammation remains unclear. Objective: We investigated the role of Mas receptor in acute allergic asthma and explored its underlying mechanisms in vitro, aiming to find critical molecules and signal pathways. Methods: Mas receptor expression was assessed in ovalbumin (OVA)-induced acute asthmatic murine model. Then we estimated the anti-inflammatory role of Mas receptor in vivo and explored expressions of several known inflammatory cytokines as well as phosphorylation levels of MAPK pathways. Mas receptor functions and underlying mechanisms were studied further in the human bronchial epithelial cell line (16HBE). Results: Mas receptor expression decreased in acute allergic airway inflammation. Multiplex immunofluorescence co-localized Mas receptor and EpCAM, indicated that Mas receptor may function in the bronchial epithelium. Activating Mas receptor through AVE0991 significantly alleviated macrophage infiltration in airway inflammation, accompanied with down-regulation of CCL2 and phosphorylation levels of MAPK pathways. Further studies in 16HBE showed that AVE0991 pre-treatment inhibited LPS-induced or anisomycin-induced CCL2 increase and THP-1 macrophages migration via JNK pathways. Conclusion: Our findings suggested that Mas receptor activation significantly attenuated CCL2 dependent macrophage recruitments in acute allergic airway inflammation through JNK pathways, which indicated that Mas receptor, CCL2 and phospho-JNK could be potential targets against allergic airway inflammation.

Objectives: Rheumatoid arthritis (RA) is a disabling disease with a high incidence that is regularly accompanied by cardiovascular complications. Several studies have suggested that renin–angiotensin–aldosterone system (RAAS) is closely associated with RA. The aim of this study was to investigate the mechanisms underlying Angiotensin-(1–7) [Ang-(1–7)] and its Mas receptor agonist (AVE0991) on joint inflammation and cardiac complications in a collagen-induced arthritis (CIA) model. Methods: Collagen type II was injected into DBA/1 mice to construct an arthritis model. CIA mice were treated with Ang-(1–7) (2.0 mg/kg intraperitoneally) and AVE0991 (3.0 mg/kg intraperitoneally). The serum levels of inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 β, IL-6, and C-reactive protein (CRP)] were determined by ELISA. The mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF- κ B) signaling pathways in joint tissues and the transforming growth factor (TGF)- β /Smad pathway and levels of α -Smooth muscle action (SMA) and β -myosin heavy chain (MHC) protein expression in cardiac tissues were assessed by western blots. The levels of TGF- β /Smad pathway, α -SMA, and β -MHC RNA in cardiac tissues were analyzed by real time-PCR. The levels of receptor activator of nuclear factor kappa ligand (RANKL) and promoting matrix metalloproteinase (MMP)-3 expression in the ankle joints were detected by immunohistochemistry and real time-PCR. Results: Ang-(1–7) and AVE0991 reduced the levels of inflammatory cytokines and inhibited the MAPKs and NF- κ B signaling pathways in ankle joint tissues, reduced RANKL and MMP3 expression, and ameliorated local joint inflammation and bone destruction compared with the control group. In addition, Ang-(1–7) and AVE0991 attenuated the TGF- β /Smad signaling pathway, reduced the levels of α -SMA and β -MHC expression, and diminished inflammatory cell infiltration into the myocardial interstitium and myocardial interstitial fibrosis in the hearts of CIA mice. Conclusions: Ang-(1–7) alleviated joint damage caused by inflammation likely through the attenuation of NF- κ B and MAPK pathways and ameliorated inflammation-induced cardiac fibrosis and activation of the TGF- β /Smad pathway. Moreover, Ang-(1–7) was likely mediated through the Mas receptor. This study provides theoretical evidence for exploring novel clinical therapeutic approaches for RA and its cardiac complications. [ABSTRACT FROM AUTHOR]

The renin–angiotensin system (RAS) not only plays an important role in controlling blood pressure but also participates in almost every process to maintain homeostasis in mammals. Interest has recently increased because SARS viruses use one RAS component (ACE2) as a target-cell receptor. The occurrence of RAS in the basal ganglia suggests that the system may be targeted to improve the therapy of neurodegenerative diseases. RAS-related data led to the hypothesis that RAS receptors may interact with each other. The aim of this paper was to find heteromers formed by Mas and angiotensin receptors and to address their functionality in neurons and microglia. Novel interactions were discovered by using resonance energy transfer techniques. The functionality of individual and interacting receptors was assayed by measuring levels of the second messengers cAMP and Ca2+ in transfected human embryonic kidney cells (HEK-293T) and primary cultures of striatal cells. Receptor complex expression was assayed by in situ proximity ligation assay. Functionality and expression were assayed in parallel in primary cultures of microglia treated or not with lipopolysaccharide and interferon-γ (IFN-γ). The proximity ligation assay was used to assess heteromer expression in parkinsonian and dyskinetic conditions. Complexes formed by Mas and the angiotensin AT1 or AT2 receptors were identified in both a heterologous expression system and in neural primary cultures. In the heterologous system, we showed that the three receptors—MasR, AT1R, and AT2R—can interact to form heterotrimers. The expression of receptor dimers (AT1R-MasR or AT2R-MasR) was higher in microglia than in neurons and was differentially affected upon microglial activation with lipopolysaccharide and IFN-γ. In all cases, agonist-induced signaling was reduced upon coactivation, and in some cases just by coexpression. Also, the blockade of signaling of two receptors in a complex by the action of a given (selective) receptor antagonist (cross-antagonism) was often observed. Differential expression of the complexes was observed in the striatum under parkinsonian conditions and especially in animals rendered dyskinetic by levodopa treatment. The negative modulation of calcium mobilization (mediated by AT1R activation), the multiplicity of possibilities on RAS affecting the MAPK pathway, and the disbalanced expression of heteromers in dyskinesia yield new insight into the operation of the RAS system, how it becomes unbalanced, and how a disbalanced RAS can be rebalanced. Furthermore, RAS components in activated microglia warrant attention in drug-development approaches to address neurodegeneration. [ABSTRACT FROM AUTHOR]

Background and Aims: Previous studies have demonstrated the anti-hypertensive effect of chronic intermittent hypobaric hypoxia (CIHH) in hypertensive rats. The present study investigated the anti-hypertensive effect of CIHH in spontaneously hypertensive rats (SHR) and the role of the renin-angiotensin system (RAS) in anti-hypertensive effect of CIHH. Methods: Fifteen-week-old male SHR and WKY rats were divided into four groups: the SHR without CIHH treatment (SHR-CON), the SHR with CIHH treatment (SHR-CIHH), the WKY without CIHH treatment (WKY-CON), and the WKY with CIHH treatment (WKY-CIHH) groups. The SHR-CIHH and WKY-CIHH rats underwent 35-days of hypobaric hypoxia simulating an altitude of 4,000 m, 5 h per day. Arterial blood pressure and heart rate were recorded by biotelemetry, and angiotensin (Ang) II, Ang1–7, interleukin (IL)-6, tumor necrosis factor-alpha (TNF)-α, and IL-10 in serum and the mesenteric arteries were measured by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. The microvessel tension recording technique was used to determine the contraction and relaxation of the mesenteric arteries. Hematoxylin and eosin and Masson's staining were used to observe vascular morphology and fibrosis. Western blot was employed to detect the expression of the angiotensin-converting enzyme (ACE), ACE2, AT1, and Mas proteins in the mesenteric artery. Results: The biotelemetry result showed that CIHH decreased arterial blood pressure in SHR for 3–4 weeks (P < 0.01). The ELISA and immunohistochemistry results showed that CIHH decreased Ang II, but increased Ang1–7 in serum and the mesenteric arteries of SHR. In the CIHH-treated SHR, IL-6 and TNF-α decreased in serum and the mesenteric arteries, and IL-10 increased in serum (P < 0.05–0.01). The microvessel tension results revealed that CIHH inhibited vascular contraction with decreased Ang1–7 in the mesenteric arteries of SHR (P < 0.05–0.01). The staining results revealed that CIHH significantly improved vascular remodeling and fibrosis in SHR. The western blot results demonstrated that CIHH upregulated expression of the ACE2 and Mas proteins, and downregulated expression of the ACE and AT1 proteins (P < 0.05–0.01). Conclusion: CIHH decreased high blood pressure in SHR, possibly by inhibiting RAS activity, downregulating the ACE-Ang II-AT1 axis and upregulating the ACE2-(Ang1-7)-Mas axis, which resulted in antagonized vascular remodeling and fibrosis, reduced inflammation, and enhanced vascular relaxation. [ABSTRACT FROM AUTHOR]

Introduction: Both age and gender can influence kidney function as well as renin-angiotensin system, and Mas receptor (MasR) as one of the components of depressor arm has more expression in female gender. The association between the receptor and age in renal vascular responses to angiotensin II (AngII) in male and female rats is completely unclear. Therefore, the aim of this study was to examine the effects of age and sex on systemic and renal vascular responses to AngII in Wistar rats with or without MasR antagonist (A779). Methods and Materials: Anesthetized Wistar male and female rats with two age ranges of adult (8-12 weeks) and old (24-28 weeks) underwent arterial and venous cannulation. Mean arterial pressure (MAP), renal perfusion pressure (RPP), renal vascular resistance (RVR) and renal blood flow (RBF) in response to the infusion of AngII with or without A779 were recorded and evaluated. Results: The baseline data (before A779 and AngII infusion) showed no significant changes in MAP, RPP, RBF, and RVR values between 8-12- week male and female groups. Although, RBF in the phase had a significant change between 24-28- week male and female groups (P < 0.05). The administration of A779 or vehicle could not influence the mentioned parameters in all experimental groups. On the other hand, the gender difference was observed in RBF and RVR responses to AngII administration in 8-12- week groups receiving vehicle or A779 (P < 0.05). However, the gender difference was not seen between 24-28- week groups. Conclusion: Advancing age could impress sex difference in RBF and RVR responses to AngII infusion, and it seems that the MasR could not participate in the responses. [ABSTRACT FROM AUTHOR]

Heart failure (HF) causes decreased brain perfusion in older adults, and increased brain and systemic inflammation increases the risk of cognitive impairment and Alzheimer’s disease (AD). Glycosylated Ang-(1-7) MasR agonists (PNA5) has shown improved bioavailability, stability, and brain penetration compared to Ang-(1-7) native peptide. Despite promising results and numerous potential applications, clinical applications of PNA5 glycopeptide are limited by its short half-life, and frequent injections are required to ensure adequate treatment for cognitive impairment. Therefore, sustained-release injectable formulations of PNA5 glycopeptide are needed to improve its bioavailability, protect the peptide from degradation, and provide sustained drug release over a prolonged time to reduce injection administration frequency. Two types of poly(D,L-lactic-co-glycolic acid) (PLGA) were used in the synthesis to produce nanoparticles (≈0.769–0.35 µm) and microparticles (≈3.7–2.4 µm) loaded with PNA5 (ester and acid-end capped). Comprehensive physicochemical characterization including scanning electron microscopy, thermal analysis, molecular fingerprinting spectroscopy, particle sizing, drug loading, encapsulation efficiency, and in vitro drug release were conducted. The data shows that despite the differences in the size of the particles, sustained release of PNA5 was successfully achieved using PLGA R503H polymer with high drug loading (% DL) and high encapsulation efficiency (% EE) of >8% and >40%, respectively. While using the ester-end PLGA, NPs showed poor sustained release as after 72 h, nearly 100% of the peptide was released. Also, lower % EE and % DL values were observed (10.8 and 3.4, respectively). This is the first systematic and comprehensive study to report on the successful design, particle synthesis, physicochemical characterization, and in vitro glycopeptide drug release of PNA5 in PLGA nanoparticles and microparticles.

This study investigated the role of AT1, AT2 and Mas angiotensinergic receptors within the MeA in autonomic, cardiovascular and baroreflex changes evoked by a 10‐day (1 hr daily) repeated restraint stress (RRS) protocol. Analysis of cardiovascular function after the end of the RRS protocol indicated increased values of arterial pressure, without heart rate changes. Arterial pressure increase was not affected by acute MeA treatment after the RRS with either the selective AT1 receptor antagonist losartan, the selective AT2 receptor antagonist PD123319 or the selective Mas receptor antagonist A‐779. Analysis of heart rate variability indicated that RRS increased the sympathetic tone to the heart, which was inhibited by MeA treatment with either losartan, PD123319 or A‐779. Baroreflex function assessed using the pharmacological approach via intravenous infusion of vasoactive agents revealed a facilitation of tachycardia evoked by blood pressure decrease in chronically stressed animals, which was inhibited by MeA treatment with losartan. Conversely, baroreflex responses during spontaneous fluctuations of blood pressure were impaired by RRS, and this effect was not affected by injection of the angiotensinergic receptor antagonists into the MeA. Altogether, the data reported in the present study suggest an involvement of both angiotensinergic receptors present in the MeA in autonomic imbalance evoked by RRS, as well as an involvement of MeA AT1 receptor in the enhanced baroreflex responses during full range of blood pressure changes. Results also indicate that RRS‐evoked increase in arterial pressure and impairment of baroreflex responses during spontaneous variations of arterial pressure are independent of MeA angiotensinergic receptors. [ABSTRACT FROM AUTHOR]

Objectives: As of December, 1st, 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, resulted in more than 1 472 917 deaths worldwide and death toll is still increasing exponentially. Many COVID-19 infected people are asymptomatic or experience moderate symptoms and recover without medical intervention. However, older people and those with comorbid hypertension, diabetes, obesity, or heart disease are at higher risk of mortality. Because current therapeutic options for COVID-19 patients are limited specifically for this elderly population at risk, Biophytis is developing BIO101 (20-hydroxyecdysone, a Mas receptor activator) as a new treatment option for managing patients with SARS-CoV-2 infection at the severe stage. The angiotensin converting enzyme 2 (ACE2) serves as a receptor for SARS-CoV-2. Interaction between ACE2 and SARS-CoV2 spike protein seems to alter the function of ACE2, a key player in the renin-angiotensin system (RAS). The clinical picture of COVID-19 includes acute respiratory distress syndrome (ARDS), cardiomyopathy, multiorgan dysfunction and shock, all of which might result from an imbalance of the RAS. We propose that RAS balance could be restored in COVID-19 patients through MasR activation downstream of ACE2 activity, with 20-hydroxyecdysone (BIO101) a non-peptidic Mas receptor (MasR) activator. Indeed, MasR activation by 20-hydroxyecdysone harbours anti-inflammatory, anti-thrombotic, and anti-fibrotic properties. BIO101, a 97% pharmaceutical grade 20-hydroxyecdysone could then offer a new therapeutic option by improving the respiratory function and ultimately promoting survival in COVID-19 patients that develop severe forms of this devastating disease. Therefore, the objective of this COVA study is to evaluate the safety and efficacy of BIO101, whose active principle is 20-hydroxyecdysone, in COVID-19 patients with severe pneumonia.Trial Design: Randomized, double-blind, placebo-controlled, multi-centre, group sequential and adaptive which will be conducted in 2 parts. Part 1: Ascertain the safety and tolerability of BIO101 and obtain preliminary indication of the activity of BIO101, in preventing respiratory deterioration in the target population Part 2: Re-assessment of the sample size needed for the confirmatory part 2 and confirmation of the effect of BIO101 observed in part 1 in the target population. The study is designed as group sequential to allow an efficient run-through, from obtaining an early indication of activity to a final confirmation. And adaptive - to allow accumulation of early data and adapt sample size in part 2 in order to inform the final design of the confirmatory part of the trial.Participants: Inclusion criteria 1. Age: 45 and above 2. A confirmed diagnosis of COVID-19 infection, within the last 14 days, prior to randomization, as determined by PCR or other approved commercial or public health assay, in a specimen as specified by the test used. 3. Hospitalized, in observation or planned to be hospitalized due to COVID-19 infection symptoms with anticipated hospitalization duration ≥3 days 4. With evidence of pneumonia based on all of the following: a. Clinical findings on a physical examination b. Respiratory symptoms developed within the past 7 days 5. With evidence of respiratory decompensation that started not more than 4 days before start of study medication and present at screening, meeting one of the following criteria, as assessed by healthcare staff: a. Tachypnea: ≥25 breaths per minute b. Arterial oxygen saturation ≤92% c. A special note should be made if there is suspicion of COVID-19-related myocarditis or pericarditis, as the presence of these is a stratification criterion 6. Without a significant deterioration in liver function tests: a. ALT and AST ≤ 5x upper limit of normal (ULN) b. Gamma-glutamyl transferase (GGT) ≤ 5x ULN c. Total bilirubin ≤ 5×ULN 7. Willing to participate and able to sign an informed consent form (ICF). Or, when relevant, a legally authorized representative (LAR) might sign the ICF on behalf of the study participant 8. Female participants should be: at least 5 years post-menopausal (i.e., persistent amenorrhea 5 years in the absence of an alternative medical cause) or surgically sterile; OR a. Have a negative urine pregnancy test at screening b. Be willing to use a contraceptive method as outlined in inclusion criterion 9 from screening to 30 days after last dose. 9. Male participants who are sexually active with a female partner must agree to the use of an effective method of birth control throughout the study and until 3 months after the last administration of the investigational product. (Note: medically acceptable methods of contraception that may be used by the participant and/or partner include combined oral contraceptive, contraceptive vaginal ring, contraceptive injection, intrauterine device, etonogestrel implant, each supplemented with a condom, as well as sterilization and vasectomy). 10. Female participants who are lactating must agree not to breastfeed during the study and up to 14 days after the intervention. 11. Male participants must agree not to donate sperm for the purpose of reproduction throughout the study and until 3 months after the last administration of the investigational product. 12. For France only: Being affiliated with a European Social Security. Exclusion criteria 1. Not needing or not willing to remain in a healthcare facility during the study 2. Moribund condition (death likely in days) or not expected to survive for >7 days - due to other and non-COVID-19 related conditions 3. Participant on invasive mechanical ventilation via an endotracheal tube, or extracorporeal membrane oxygenation (ECMO), or high-flow Oxygen (delivery of oxygen at a flow of ≥16 L/min.). 4. Participant is not able to take medications by mouth (as capsules or as a powder, mixed in water). 5. Disallowed concomitant medication: Consumption of any herbal products containing 20-hydroxyecdysone and derived from Leuzea carthamoides; Cyanotis vaga or Cyanotis arachnoidea is not allowed (e.g. performance enhancing agents). 6. Any known hypersensitivity to any of the ingredients, or excipients of the study medication, BIO101. 7. Renal disease requiring dialysis, or known renal insufficiency (eGFR≤30 mL/min/1.73 m2, based on Cockcroft & Gault formula). 8. In France only: a. Non-affiliation to compulsory French social security scheme (beneficiary or right-holder). b. Being under tutelage or legal guardianship. Participants will be recruited from approximately 30 clinical centres in Belgium, France, the UK, USA and Brazil. Maximum patients' participation in the study will last 28 days. Follow-up of participants discharged from hospital will be performed through post-intervention phone calls at 14 (± 2) and 60 (± 4) days.Intervention and Comparator: Two treatment arms will be tested in this study: interventional arm 350 mg b.i.d. of BIO101 (AP 20-hydroxyecdysone) and placebo comparator arm 350 mg b.i.d of placebo. Administration of daily dose is the same throughout the whole treatment period. Participants will receive the study medication while hospitalized for up to 28 days or until a clinical endpoint is reached (i.e., 'negative' or 'positive' event). Participants who are officially discharged from hospital care will no longer receive study medication.Main Outcomes: Primary study endpoint: The proportion of participants with 'negative' events up to 28 days. 'Negative' events are defined as respiratory deterioration and all-cause mortality. For the purpose of this study, respiratory deterioration will be defined as any of the following: Requiring mechanical ventilation (including cases that will not be intubated due to resource restrictions and triage). Requiring extracorporeal membrane oxygenation (ECMO). Requiring high-flow oxygen defined as delivery of oxygen at a flow of ≥16 L/min. Only if the primary endpoint is significant at the primary final analysis the following Key secondary endpoints will be tested in that order: Proportion of participants with events of respiratory failure at Day 28 Proportion of participants with 'positive' events at Day 28. Proportion of participants with events of all-cause mortality at Day 28 A 'positive' event is defined as the official discharge from hospital care by the department due to improvement in participant condition. Secondary and exploratory endpoints: In addition, a variety of functional measures and biomarkers (including the SpO2 / FiO2 ratio, viral load and markers related to inflammation, muscles, tissue and the RAS / MAS pathways) will also be collected.Randomization: Randomization is performed using an IBM clinical development IWRS system during the baseline visit. Block-permuted randomization will be used to assign eligible participants in a 1:1 ratio. In part 1, randomization will be stratified by RAS pathway modulator use (yes/no) and co-morbidities (none vs. 1 and above). In Part 2, randomization will be stratified by centre, gender, RAS pathway modulator use (yes/no), co-morbidities (none vs. 1 and above), receiving Continuous Positive Airway Pressure/Bi-level Positive Airway Pressure (CPAP/BiPAP) at study entry (Yes/No) and suspicion of COVID-19 related myocarditis or pericarditis (present or not).Blinding (masking): Participants, caregivers, and the study team assessing the outcomes are blinded to group assignment. All therapeutic units (TU), BIO101 b.i.d. or placebo b.i.d., cannot be distinguished in compliance with the double-blind process. An independent data-monitoring committee (DMC) will conduct 2 interim analyses. A first one based on the data from part 1 and a second from the data from parts 1 and 2. The first will inform about BIO101 safety, to allow the start of recruitment into part 2 followed by an analysis of the efficacy data, to obtain an indication of activity. The second interim analysis will inform about the sample size that will be required for part 2, in order to achieve adequate statistical power. Numbers to be randomised (sample size) Number of participants randomized: up to 465, in total Part 1: 50 (to obtain the proof of concept in COVID-19 patients). Part 2: 310, potentially increased by 50% (up to 465, based on interim analysis 2) (to confirm the effects of BIO101 observed in part 1).Trial Status: The current protocol Version is V 10.0, dated on 24.09.2020. The recruitment that started on September 1st 2020 is ongoing and is anticipated to finish for the whole study by March2021.Trial Registration: The trial was registered before trial start in trial registries: EudraCT , No. 2020-001498-63, registered May 18, 2020; and Clinicaltrials.gov, identifier NCT04472728 , registered July 15, 2020.Full Protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. [ABSTRACT FROM AUTHOR]

The renin-angiotensin system (RAS) is best known for playing a major role in maintaining the physiology of the cardiovascular system. Dysregulation of the RAS pathway has been proposed as a link to some malignancies and contributes to cancer metastasis. Breast cancer is considered as one of the leading causes of cancer death in women and its prevention remains yet a challenge. Elements of RAS are expressed in both normal breast tissue and cancerous cells, signifying the essential role of RAS in breast cancer pathology. Sertraline, a widely used antidepressant, has shown anti-proliferative properties on a variety of malignancies. This study aimed to investigate the effect of sertraline and its combination with agonists and antagonists of RAS (A779, Ang 1-7 and losartan) on viability of MCF-7 cells along with their effect on apoptosis and distribution of cell cycle. Our results indicated that sertraline, losartan and Ang 1-7 significantly decreased cell viability, induced apoptosis and cell cycle arrest. A779 blunted the effect of sertraline on cell viability, ROS generation and cell cycle arrest. Combination treatment of sertraline with losartan as well as Ang 1-7 caused a remarkable decline in cell viability. In conclusion, results of the present study support the anti-cancer properties of sertraline, losartan and Ang 1-7 via induction of apoptosis and cell cycle arrest., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Negar Firouzabadi reports financial support was provided by 10.13039/501100004320Shiraz University of Medical Sciences. Dr. Negar Firouzabadi reports a relationship with 10.13039/501100004320Shiraz University of Medical Sciences that includes: funding grants. None declared. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: The pathogenesis of diabetes and its microvascular complications are intimately associated with renin angiotensin system dysregulation. Evidence suggests the angiotensin converting enzyme 2 (ACE2)/angiotensin 1-7 (Ang 1-7)/Mas receptor (MasR) axis regulates metabolic imbalances, inflammatory responses, reduces oxidative stress, and sustains microvascular integrity, thereby strengthening defences against diabetic conditions. This study aims to conduct a comprehensive analysis of the ACE2/Ang 1-7/MasR axis in diabetes and its microvascular complications over the past two decades, focusing on key contributors, research hotspots, and thematic trends., Methods: This cross-sectional bibliometric analysis of 349 English-language publications was performed using HistCite, VOSviewer, CiteSpace, and Bibliometrix R for visualization and metric analysis. Primary analytical metrics included publication count and keyword trend dynamics., Results: The United States, contributing 105 articles, emerged as the most productive country, with the University of Florida leading institutions with 18 publications. Benter IF was the most prolific author with 14 publications, and Clinical Science was the leading journal with 13 articles. A total of 151 of the 527 author's keywords with two or more occurrences clustered into four major clusters: diabetic microvascular pathogenesis, metabolic systems, type 2 diabetes, and coronavirus infections. Keywords such as "SARS", "ACE2", "coronavirus", "receptor" and "infection" displayed the strongest citation bursts. The thematic evolution in this field expanded from focusing on the renin angiotensin system (2002-2009) to incorporating ACE2 and diabetes metabolism (2010-2016). The latter period (2017-2023) witnessed a significant surge in diabetes research, reflecting the impact of COVID-19 and associated conditions such as diabetic retinopathy and cardiomyopathy., Conclusions: This scientometric study offers a detailed analysis of the ACE2/Ang 1-7/MasR axis in diabetes and its microvascular complications, providing valuable insights for future research directions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

We have previously demonstrated the presence of Angiotensin (Ang)-(1-7) in rat ovary homogenates and its stimulatory effect on estradiol and progesterone production. The present study was undertaken to identify the cellular localization of Ang-(1-7) and its receptor Mas in the rat ovary in the different phases of the estrous cycle. Ang-(1-7) and Mas were localized by immunohistochemistry and Mas mRNA expression was assessed by RT-PCR. Immunostaining for both Ang-(1-7) and Mas was found in all phases of the estrous cycle, particularly in the thecal and interstitial cells, as well as in regressing corpora lutea. However, granulosa cells were positive only in antral and preovulatory follicles at proestrus and estrus phases. This pattern contrasted with the distribution of the octapeptide Ang II, which was abundant in granulosa but not in theca cells. In addition, the expression of Mas mRNA was demonstrated in all estrous cycle phases. Angiotensin-converting enzyme activity did not vary between estrous cycle phases, whereas prolyl endopeptidase activity was significantly higher in diestrus and neutral endopeptidase activity was significantly higher in metestrus. These data provide the first evidence that new RAS components are dynamically expressed in the ovary across the rat estrous cycle. Further functional studies should clarify the role of Ang-(1-7) signaling through Mas receptor in the regulation of ovarian physiology. [ABSTRACT FROM AUTHOR]

G-protein–coupled receptors (GPCRs) are targets for around one third of currently approved and clinical prescribed drugs and represent the largest and most structurally diverse family of transmembrane signaling proteins, with almost 1000 members identified in the human genome. Upon agonist stimulation, GPCRs are internalized and trafficked inside the cell: they may be targeted to different organelles, recycled back to the plasma membrane or be degraded. Once inside the cell, the receptors may initiate other signaling pathways leading to different biological responses. GPCRs’ biological function may also be influenced by interaction with other receptors. Thus, the ultimate cellular response may depend not only on the activation of the receptor from the cell membrane, but also from receptor trafficking and/or the interaction with other receptors. This review is focused on angiotensin receptors and how their biological function is influenced by trafficking and interaction with others receptors.

Recently a cross talk between angiotensin 1-7 (Ang1-7) receptor (MasR) and angiotensin II receptors types 1 and 2 (AT1R and AT2R) has been highlighted. The effects of MasR antagonist (A779) compared to the vehicle on the renal blood flow (RBF) and renal vascular resistance (RVR) responses to Ang1-7 (300 ng/kg/min) infusion in the absence of Ang II receptors in male and female rats were determined at controlled renal perfusion pressure. Ang1-7 infusion did not alter mean arterial pressure in male and female rats. However, A779 compared to vehicle increased RBF (18% vs 3%) and decreased RVR (13% vs 4%) responses to Ang1-7 infusion significantly (P < 0.05) in male when AngII receptors were blocked. Such observation was not occurred in female animals. Finally it was concluded that renal vascular responses to Ang1-7 administration may not be exerted by MasR in male rats, and these responses are not mediated with AngII receptors.

To circumvent the major threats of low blood volume and low blood pressure, animals need powerful mechanisms for salt and water conservation, which is renin–angiotensin system (RAS). Activation of the RAS is, therefore, a useful response in many demanding situations. However, increased activity of the RAS, especially in combination with other cardiovascular risk factors, may lead to a cascade of deleterious effects such as hypertension, atherosclerosis, myocardial remodeling, heart failure, ischemic stroke, and diabetes mellitus. Although many pathophysiological actions of angiotensin (Ang) II may still be viewed as being homeostatic in principle, its over-activation can be detrimental.. Numerous experimental studies have indicated that angiotensin-converting enzyme (ACE) 2 efficiently hydrolyzes the potent vasoconstrictor Ang II to Ang 1–7. Thus, the axis formed by ACE 2/Ang 1–7/Mas appears to represent an endogenous counterregulatory pathway within the RAS, the actions of which are in opposition to the vasoconstrictor/proliferative arm of the RAS consisting of ACE, Ang II, and AT1 receptor. Although most of the well-known cardiovascular and renal effects of RAS are attributed to ACE, an important enzyme in the generation of Ang II, much less is known about the function of ACE 2. This review summarizes the recently published data on basic properties of ACE 2 and Ang 1–7 and the evidence from experimental and clinical studies of various pathological conditions related to the biological roles of ACE 2/Ang 1–7/Mas in the heart.

The renin-angiotensin system (RAS) plays an important role in erectile function. The RAS contains 2 major axes: one deleterious, composed of ACE-Ang II-AT1 receptor, and another protective, composed of ACE2-Ang-(1-7)-Mas receptor. While aging is a well-known cause for development of male sexual disorders, little is known about local regulation of the RAS in age-related erectile dysfunction (ED). The present study aimed to assess regulation of the RAS in aging-associated ED rat model and evaluate possible options for disease management through pharmacological modulation of the RAS. Penile tissues were harvested from 3-, 12-, and 24-month-old Wistar rats. Local expression of major RAS components and ED markers was measured by RT-PCR. Protein expression of RAS components was assessed by western blot. Collagen deposition was measured by Sirius Red and immunohistochemical staining. Evaluation of collagen content was also performed in penile sections of Mas-knockout mice by Sirius Red and Masson's trichrome stainings. Finally, the effect of Ang-(1-7) pretreatment on TGF-β–induced myofibroblast activation was studied in primary cavernosal and immortalized fibroblasts. Experimental results highlighted the essential role of the RAS in modulation of cavernosal fibrosis. The present study demonstrates local expression of angiotensinogen mRNA alongside with major RAS components, which suggests local autonomous functioning of the RAS within penile tissue. Gene expression analysis revealed strong positive correlation between ACE-Ang II-AT1 axis with markers for inflammation and fibrosis. While corpus cavernosum from 24-month-old rats was characterized by increased collagen deposition, protein expression of ACE, AT1, and Mas was shown to be upregulated in the penile tissue of this group. At the same time, penile sections from Mas-knockout mice (FVB/N background) were also shown to have increased collagen deposition. Finally, it was demonstrated that Ang-(1-7) treatment of primary cavernosal and immortalized fibroblasts was able to alleviate TGF-β–induced fibroblast-to-myofibroblast transition. The present study suggests Ang-(1-7) treatment as a possible strategy for pharmacological management of fibrosis-associated ED in aging. The link between the RAS and penile fibrosis, indicated by a holistic screening of different ED markers, was confirmed by in vivo and in vitro data. However, results, presented in the manuscript, need to be further reinforced by human data. Important to note, the main goal of the study was to characterize RAS regulation in aging condition rather than state any causal relationships. Present study characterizes RAS regulation in aging-associated ED and indicates its important role in cavernosal fibrosis. Bragina ME, Costa-Fraga F, Sturny M, et al. Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis. J Sex Med 2020;17:2129–2140. [ABSTRACT FROM AUTHOR]

Aims  Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking. Methods and results  Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it. Conclusion  MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7). [ABSTRACT FROM AUTHOR]

Diminazene aceturate (DIZE) has been described as an angiotensin‐converting enzyme 2 (ACE2) activator. We aimed to investigate DIZE effects on blood pressure (BP) of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats. BP was recorded in awake and unrestrained rats 24 hours after femoral artery catheterization. DIZE (15 mg/kg, s.c.) produced a fast BP decrease only in SHR (P <.01). Pre‐treatment with L‐NAME (10 mg/kg, iv) did not change the hypotensive effect on systolic BP whereas mitigated the DIZE effect on diastolic BP (∆ Emax: −31 ± 5 DIZE vs −15 ± 1 mm Hg DIZE + L‐NAME, P <.05). BP changes after DIZE remained unchanged after the treatment of rats with A‐779 (50 ug/kg, iv), a Mas receptor blocker. Vasodilatation curves to DIZE (10‐9 to 10‐4 mol/L) in mesenteric arteries confirmed the NO‐mediation on DIZE effects in SHR, as L‐NAME (300 μmol/L) reduced the vascular sensitivity (∆EC50: −5.12 ± 0.09 CONTROL vs −4.66 ± 0.08 L‐NAME, P <.05) and the magnitude of DIZE effect (area under the curve (AUC), 357.5 ± 8.2 DIZE vs 424.7 ± 11.6 L‐NAME; P <.001), whereas A‐779 (1 μmol/L) enhanced DIZE response (AUC, 357.5 ± 8.2 DIZE vs 309.8 ± 14.7 A‐779, P <.05). Our findings indicate that DIZE acutely reduces the BP in SHR possibly by a mechanism other than Mas receptor activation. This effect seems to be mediated, at least partially, by NO. [ABSTRACT FROM AUTHOR]

G-protein–coupled receptors (GPCRs) are targets for around one third of currently approved and clinical prescribed drugs and represent the largest and most structurally diverse family of transmembrane signaling proteins, with almost 1000 members identified in the human genome. Upon agonist stimulation, GPCRs are internalized and trafficked inside the cell: they may be targeted to different organelles, recycled back to the plasma membrane or be degraded. Once inside the cell, the receptors may initiate other signaling pathways leading to different biological responses. GPCRs' biological function may also be influenced by interaction with other receptors. Thus, the ultimate cellular response may depend not only on the activation of the receptor from the cell membrane, but also from receptor trafficking and/or the interaction with other receptors. This review is focused on angiotensin receptors and how their biological function is influenced by trafficking and interaction with others receptors. [ABSTRACT FROM AUTHOR]

The renin–angiotensin system (RAS) is one of the oldest and most extensively studied human peptide cascades, well-known for its role in regulating blood pressure. When aldosterone is included, RAAS is involved also in fluid and electrolyte homeostasis. There are two main axes of RAAS: (1) Angiotensin (1–7), angiotensin converting enzyme 2 and Mas receptor (ACE2–Ang(1–7)–MasR), (2) Angiotensin II, angiotensin converting enzyme 1 and angiotensin II type 1 receptor (ACE1–AngII–AT1R). In its entirety, RAAS comprises dozens of angiotensin peptides, peptidases and seven receptors. The first mentioned axis is known to counterbalance the deleterious effects of the latter axis. In addition to the systemic RAAS, tissue-specific regulatory systems have been described in various organs, evidence that RAAS is both an endocrine and an autocrine system. These local regulatory systems, such as the one present in the vascular endothelium, are responsible for long-term regional changes. A local RAAS and its components have been detected in many structures of the human eye. This review focuses on the local ocular RAAS in the anterior part of the eye, its possible role in aqueous humour dynamics and intraocular pressure as well as RAAS as a potential target for anti-glaucomatous drugs. Components of renin–angiotensin–aldosterone system have been detected in different structures of the human eye, introducing the concept of a local intraocular renin–angiotensin–aldosterone system (RAAS). Evidence is accumulating that the local ocular RAAS is involved in aqueous humour dynamics, regulation of intraocular pressure, neuroprotection and ocular pathology making components of RAAS attractive candidates when developing new effective ways to treat glaucoma. [ABSTRACT FROM AUTHOR]

Background and purpose: Chronic kidney disease is among the common diseases in the world. Studies show that women are more protected against renal diseases compared to men. On the other hand, vasodilatory axises of renin angiotensin system (angiotensin 1-7 (Ang 1-7)- Mas receptor (MasR)), kallikrein-kinin, and nitric oxide (NO) play key roles in kidney function and circulation in females. Therefore, the purpose of this study was to examine the effect of bradykinin in renal blood flow (RBF) response to Ang 1-7 when MasR was blocked in female rats. Materials and methods: Female Wistar rats were divided into three groups (control, Bradykinine, and Bradykinin + MasR antagonist (A779)). The animals were anesthetized and catheterized in the carotid and femoral arteries and jugular vein to measure mean arterial pressure (MAP), renal perfusion pressure (RPP), and drug administration, respectively. The left kidney was exposed (in situ) and placed in a kidney cup to measure RBF. After the equilibration period, A779 and bradykinin (50 μg/kg/h, 150 μg/kg/h, respectively) were injected and vascular responses to Ang1-7 (100, 300, and 1000 ng kg−1min−1) infusion were determined. At the end of the experiment, blood samples were collected for measuring the level of nitrite. Results: The MAP and RPP were not significantly different between the three groups. Bradykinin exhibited vasodilatory effect on RBF in response to Ang 1-7 in female rats, however, A779 administration increased RBF at low dose of Ang 1-7(Pdose=0.001). The serum level of nitrite significantly decreased when the MasR was blocked (P= 0.04). Conclusion: Bradykinin enhanced RBF response to Ang 1-7 infusion. The effect of bradykinin on RBF response to Ang 1-7 may be modulated via the NO pathway. Interaction of these factors might help to broaden our vision for treatment strategy in future. [ABSTRACT FROM AUTHOR]

The renin angiotensin system (RAS), which is classically known for blood pressure regulation, has functions beyond this. There are two axes of RAS that work to counterbalance each other and are active throughout the body, including the CNS. The pathological axis, consisting of angiotensin II (A1-8), angiotensin converting enzyme (ACE) and the angiotensin II type 1 receptor (AT1R), is upregulated in many CNS diseases, including multiple sclerosis (MS). MS is an autoimmune and neurodegenerative disease of the CNS characterized by inflammation, demyelination and axonal degeneration. Published research has described increased expression of AT1R and ACE in tissues from MS patients and in animal models of MS such as experimental autoimmune encephalomyelitis (EAE). In contrast to the pathological axis, little is known about the protective axes of RAS in MS and EAE. In other neurological conditions the protective axis, which includes A1–7, ACE2, angiotensin II type 2 receptor and Mas receptor, has been shown to have anti-inflammatory, regenerative and neuroprotective effects. Here we show, for the first time, changes in the protective arm of RAS in both EAE and MS CNS tissue. We observed a significant increase in expression of the protective arm during stages of disease stabilization in EAE, and in MS tissue showing evidence of remyelination. These data provide evidence that the protective arm of RAS, through both ligand and receptor expression, is associated with reductions in the pathological processes that occur in the earlier stages of MS and EAE, possibly slowing the neurodegenerative process and enhancing neural repair. [ABSTRACT FROM AUTHOR]

AVE0991, a nonpeptide angiotensin-(1–7) mimic, has similar protective effects for cardiovascular system to Ang-(1–7). In this article, we aimed to explore the effects of AVE0991 and Ang-(1–7) on abdominal aortic aneurysm (AAA) induced by Ang II in apolipoprotein E knockout mice. The mice AAA model was established by Ang II infusion, and then mice received different treatment with saline, Ang II (1.44 mg/kg/day), different dose AVE0991 (0.58 or 1.16 μmol/kg/day), or Ang-(1–7) (400 ng/kg/min). The incidence of AAA was 76%, 48%, 28%, and 24% in the vehicle, the low-dose AVE0991, high-dose AVE0991, and the Ang-(1–7) group, respectively. In comparison with control group, AVE0991 and Ang-(1–7) treatment significantly increased smooth muscle cells and decreased macrophage accumulation, the expression levels of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α), and the expression and activity of metalloproteinases 2 and 9 in mice AAA model or in human smooth muscle cells (hVSMCs). The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation, whereas the positive impacts were reversed by co-administration with the Mas antagonist A779 (400 ng/kg/min) and AVE0991 in Ang II–infused mice or in hVSMCs. Therefore, AVE0991 and Ang-(1–7) might be novel and promising interventions in the prevention and treatment of AAA. Key messages: • AVE0991 dose-dependently inhibited Ang II–induced AAA formation in Apoe−/− mice. • Ang-(1–7) played the same protective role as high-dose AVE0991. • Inhibition of Mas receptor with A779 could reverse the protective effect of AVE0991. • The therapeutic effects may be contributed to reduction of oxidative stress and downregulation of P38 and ERK1/2 signal pathways via Mas receptor activation. [ABSTRACT FROM AUTHOR]

Neprilysin (NEP) is an endogenously induced peptidase for modulating production and degradation of various peptides in humans. It is most abundantly present in kidney and regulates the intrinsic renal homeostatic mechanism. Recently, drugs inhibiting NEP have been approved for the use in heart failure. In the context of increased prevalence of ischemia associated renal failure, NEP could be an attractive target for treating kidney failure. In the kidney, targeting NEP may possess potential benefits as well as adverse consequences. The unfavorable outcomes of NEP are mainly attributed to the degradation of the natriuretic peptides (NPs). NPs are involved in the inhibition of the renin–angiotensin–aldosterone system (RAAS) and activation of the sympathetic system contributing to the tubular and glomerular injury. In contrary, NEP exerts the beneficial effect by converting angiotensin-1 (Ang I) to angiotensin-(1–7) (Ang-(1–7)), thus activating MAS-related G-protein coupled receptor. MAS receptor antagonizes angiotensin type I receptor (AT-1R), reduces reactive oxygen species (ROS) and inflammation, thus ameliorating renal injury. However, the association of NEP with complex cascades of renal ischemia remains vague. Therefore, there is a need to evaluate the putative mechanism of NEP and its overlap with other signaling cascades in conditions of renal ischemia. [ABSTRACT FROM AUTHOR]

BACKGROUND Essential hypertension is associated with increased plasma concentrations of extracellular vesicles (EVs). We aimed to determine the role of monocyte miR-27a in EVs on arterial Mas receptor expression, and its involvement in the pathogenesis of hypertension. METHODS THP-1 cells were transfected with miR-27a mimic and miR-27a inhibitor, and EVs were collected. Mas receptor expression and endothelial nitric oxide synthase (eNOS) phosphorylation were determined by immunoblotting. Sprague–Dawley (SD) rats received EVs via tail-vein injection. Blood pressure (BP) was measured with the tail-cuff method. The vasodilatory response of mesenteric arteries was measured using a small vessel myograph. RESULTS EVs from THP-1 cells increased rat BP by impairing Ang-(1–7)-mediated vasodilation in mesenteric arteries, which was further exaggerated by EVs from lipopolysaccharides-treated THP-1 cells. As the receptor and key signaling of Ang-(1–7), next experiments found that Mas receptor expression and eNOS phosphorylation were decreased in mesenteric arteries from EVs-treated SD rats. Screening studies found miR-27a in EVs may be involved in this process. Through transfection with miR-27a inhibitor or miR-27a mimic, we found that miR-27a downregulates Mas receptor expression in endothelial cells. Injection of EVs from miR-27a-transfected HEK-293 cells decreased Mas receptor and eNOS phosphorylation in mesenteric arteries, impaired Ang-(1–7)-mediated vasodilation and increased BP. Earlier effects were reversed using cells with downregulation of miR-27 in EVs. CONCLUSIONS Monocyte miR-27a in EVs decreases Mas receptor expression and eNOS phosphorylation in endothelium, impairs Ang-(1–7)-mediated vasodilation, and causes hypertension. Understanding the contributions of EVs in the pathogenesis of hypertension may facilitate their use as a diagnostic biomarker. [ABSTRACT FROM AUTHOR]

Portal hypertension (PHT) resulting from splanchnic vasodilatation is a major cause of morbidity and mortality in patients with cirrhosis. The renin-angiotensin system (RAS) plays an important role in splanchnic vasodilatation in cirrhosis. This study investigated whether acute blockade of the vasodilatory receptors of the alternate RAS, Mas (MasR), Mas-related G-protein coupled receptor type D (MrgD), and angiotensin II type-2 receptor (AT2R) improves PHT in cirrhotic and non-cirrhotic portal hypertensive rats and counteracts systemic hypotension associated with angiotensin II type 1 receptor (AT1R) blockade. Cirrhotic bile duct ligated (BDL) or carbon tetrachloride (CCl4) injected and non-cirrhotic partial portal vein ligated (PPVL) rats were used for measurement of portal pressure (PP) and mean arterial pressure before and after an intravenous bolus injection of the MasR, MrgD, and AT2R blockers, A779, D-Pro7-Ang-(1-7) (D-Pro) and PD123319, respectively. Separate groups of rats received a combined treatment with A779 or D-Pro given 20 min after AT1R blocker losartan. Mesenteric expression of MasR, MrgD, and AT2R and circulating levels of peptide blockers were also measured. Treatment with A779 and D-Pro significantly reduced PP in cirrhotic rat models. Despite rapid degradation of A779 and D-Pro in the rat circulation, the PP lowering effect of the blockers lasted for up to 25 min. We also found that PD123319 reduced PP in CCl4 rats, possibly by blocking the MasR and/or MrgD since AT2R expression in cirrhotic mesenteric vessels was undetectable, whereas the expression of MasR and MrgD was markedly elevated. While losartan resulted in a marked reduction in PP, its profound systemic hypotensive effect was not counteracted by the combination therapy with A779 or D-Pro. In marked contrast, none of the receptor blockers had any effect on PP in non-cirrhotic PPVL rats whose mesenteric expression of MasR and MrgD was unchanged. We conclude that in addition to MasR, MrgD, a newly discovered receptor for Angiotensin-(1-7), plays a key role in splanchnic vasodilatation in cirrhosis. This implies that both MasR and MrgD are potential therapeutic targets to treat PHT in cirrhotic patients. We also conclude that the alternate RAS may not contribute to the development of splanchnic vasodilatation in non-cirrhotic PHT.

The MasR receptor (MasR) is an orphan G protein-coupled receptor proposed as a candidate for mediating the angiotensin (Ang)-converting enzyme 2-Ang-(1–7) protective axis of renin-angiotensin system. This receptor has been suggested to participate in several physiological processes including cardio- and reno-protection and regulation of the central nervous system function. Although the knowledge of the signaling mechanisms associated with MasR is essential for therapeutic purposes, these are still poorly understood. Accordingly, in the current study we aimed to characterize the signaling pathways triggered by the MasR. To do that, we measured cAMP and Ca2+ levels in both naïve and MasR transfected cells in basal conditions and upon incubation with putative MasR ligands. Besides, we evaluated activation of ERK1/2 by Ang-(1–7) in MasR transfected cells. Results indicated the existence of a high degree of MasR constitutive activity toward cAMP modulation. This effect was not mediated by the PDZ-binding motif of the MasR but by receptor coupling to Gαi-adenylyl cyclase signaling pathway. Incubation of MasR transfected cells with Ang-(1–7) or the synthetic ligand AVE 0991 amplified MasR negative modulation of cAMP levels. On the other hand, we provided evidence for lack of MasR-associated modulation of Ca2+ levels by Ang-(1–7). Finally, it was determined that the MasR attenuated Ang-(1–7)-induced ERK1/2 phosphorylation mediated by AT1R. We provided further characterization of MasR signaling mechanisms regarding its constitutive activity and response to putative ligands. This information could prove useful to better describe MasR physiological role and development of therapeutic agents that could modulate its action.

Background: Muscle aging is associated with a consistent decrease in the ability of muscle tissue to regenerate following intrinsic muscle degradation, injury or overuse. Age-related imbalance of protein synthesis and degradation, mainly regulated by AKT/mTOR pathway, leads to progressive loss of muscle mass. Maintenance of anabolic and regenerative capacities of skeletal muscles may be regarded as a therapeutic option for sarcopenia and other muscle wasting diseases. Our previous studies have demonstrated that BIO101, a pharmaceutical grade 20-hydroxyecdysone, increases protein synthesis through the activation of MAS receptor involved in the protective arm of renin-angiotensin-aldosterone system. The purpose of the present study was to assess the anabolic and pro-differentiating properties of BIO101 on C2C12 muscle cells in vitro and to investigate its effects on adult and old mice models in vivo., Methods: The effects of BIO101 on C2C12 differentiation were assessed using myogenic transcription factors and protein expression of major kinases of AKT/mTOR pathway by Western blot. The in vivo effects of BIO101 have been investigated in BIO101 orally-treated (50 mg/kg/day) adult mice (3 months) for 28 days. To demonstrate potential beneficial effect of BIO101 treatment in a sarcopenic mouse model, we use orally treated 22-month-old C57Bl6/J mice, for 14 weeks with vehicle or BIO101. Mice body and muscle weight were recorded. Physical performances were assessed using running capacity and muscle contractility tests., Results: Anabolic properties of BIO101 were confirmed by the rapid activation of AKT/mTOR, leading to an increase of C2C12 myotubes diameters (+26%, P < 0.001). Pro-differentiating effects of BIO101 on C2C12 myoblasts were revealed by increased expression of muscle-specific differentiation transcription factors (MyoD, myogenin), resulting in increased fusion index and number of nuclei per myotube (+39% and +53%, respectively, at day 6). These effects of BIO101 were like those of angiotensin (1-7) and were abolished with the use of A779, a MAS receptor specific antagonist. Chronic BIO101 oral treatment induced AKT/mTOR activation and anabolic effects accompanied with improved physical performances in adult and old animals (maximal running distance and maximal running velocity)., Conclusions: Our data suggest beneficial anabolic and pro-differentiating effects of BIO101 rendering BIO101 a potent drug candidate for treating sarcopenia and possibly other muscle wasting disorders., (© 2023 Biophytis. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)