Biomechanical Effects of Angiotensin 1-7 in Diabetes Rats Femur / Efectos Biomecánicos de la Angiotensina 1-7 en el Fémur de Ratas Diabéticas

SUMMARY: It is known that diabetes mellitus has late complications, including microvascular and macrovascular diseases. Diabetes can affect bones through biochemical markers of bone structure, density, and turnover. This study aimed to biomechanically investigate the bone-protective effects of angiotensin 1-7 (Ang 1-7), one of the active peptides in the renin-angiotensin system, in rats with diabetes. Thirty male Wistar albino rats, three months old and weighing 250-300 g, were divided into four groups: diabetes, Ang 1- 7, diabetes plus Ang 1-7, and control. One month later, diabetes developed in rats; the rats were sacrificed, and their right femur was removed. Three-point bending biomechanical tests were performed on the femurs. The diabetic group had significantly higher bone fragility than the other groups (Pr >.05). Bone fragility was lower, and bone flexibility was higher in the Ang 1-7 groups (Pr>F value 0.05). As a result of our study, the effect of Ang 1-7 on the bones of rats with diabetes was investigated biomechanically. Ang 1-7 has a protective impact on the bones of rats with diabetes.

Se sabe que la diabetes mellitus tiene complicaciones tardías, incluyendo enfermedades microvasculares y macrovasculares. La diabetes puede afectar los huesos a través de los marcadores bioquímicos de la estructura, la densidad y el recambio óseo. Este estudio tuvo como objetivo investigar biomecánicamente los efectos protectores en los huesos de la angiotensina 1-7 (Ang 1-7), uno de los péptidos activos en el sistema renina-angiotensina, en ratas con diabetes. Treinta ratas albinas Wistar macho, de tres meses de edad y con un peso de 250-300 g, se dividieron en cuatro grupos: diabetes, Ang 1-7, diabetes más Ang 1-7 y control. Un mes después, se desarrolló diabetes en ratas; se sacrificaron los animales y se extrajo su fémur derecho. Se realizaron pruebas biomecánicas de flexión de tres puntos en los fémures. El grupo diabéticos tenía una fragilidad ósea significativamente mayor que los otros grupos (Pr > 0,05). La fragilidad ósea fue menor y la flexibilidad ósea fue mayor en los grupos Ang 1-7 (valor Pr>F 0,05). Como resultado de nuestro estudio, se determinó biomecánicamente el efecto de Ang 1-7 en los huesos de ratas con diabetes. Se concluye que Ang 1-7 tiene un impacto protector en los huesos de ratas diabéticas.

Angiotensin Type 2 and Mas Receptor Activation Prevents Myocardial Fibrosis and Hypertrophy through the Reduction of Inflammatory Cell Infiltration and Local Sympathetic Activity in Angiotensin II-Dependent Hypertension.

Compound 21 (C21), an AT2 receptor agonist, and Angiotensin 1-7 (Ang 1-7), through Mas receptor, play an important role in the modulation of the protective arm of the renin-angiotensin system. The aim of this study was to investigate in an experimental model of angiotensin II-dependent hypertension whether the activation of the potentially protective arm of the renin-angiotensin system, through AT2 or Mas receptor stimulation, counteracts the onset of myocardial fibrosis and hypertrophy, and whether these effects are mediated by inflammatory mechanism and/or sympathetic activation. Sprague Dawley rats (n = 67) were treated for 1 (n = 25) and 4 (n = 42) weeks and divided in the following groups: (a) Angiotensin II (Ang II, 200 ng/kg/min, osmotic minipumps, sub cutis); (b) Ang II+Compound 21 (C21, 0.3 mg/kg/day, intraperitoneal); (c) Ang II+Ang 1-7 (576 µg/kg/day, intraperitoneal); (d) Ang II+Losartan (50 mg/kg/day, per os); (e) control group (physiological saline, sub cutis). Systolic blood pressure was measured by tail cuff method and, at the end of the experimental period, the rats were euthanized and the heart was excised to evaluate myocardial fibrosis, hypertrophy, inflammatory cell infiltration and tyrosine hydroxylase expression, used as marker of sympathetic activity. Ang II caused a significant increase of blood pressure, myocardial interstitial and perivascular fibrosis and myocardial hypertrophy, as compared to control groups. C21 or Ang 1-7 administration did not modify the increase in blood pressure in Ang II treated rats, but both prevented the development of myocardial fibrosis and hypertrophy. Treatment with losartan blocked the onset of hypertension and myocardial fibrosis and hypertrophy in Ang II treated rats. Activation of AT2 receptors or Mas receptors prevents the onset of myocardial fibrosis and hypertrophy in Ang II-dependent hypertension through the reduction of myocardial inflammatory cell infiltration and tyrosine hydroxylase expression. Unlike what happens in case of treatment with losartan, the antifibrotic and antihypertrophic effects that follow the activation of the AT2 or Mas receptors are independent on the modulation of blood pressure.

The Effects of Angiotensin II or Angiotensin 1-7 on Rat Pial Microcirculation during Hypoperfusion and Reperfusion Injury: Role of Redox Stress.

Renin-angiotensin systems produce angiotensin II (Ang II) and angiotensin 1-7 (Ang 1-7), which are able to induce opposite effects on circulation. This study in vivo assessed the effects induced by Ang II or Ang 1-7 on rat pial microcirculation during hypoperfusion-reperfusion, clarifying the mechanisms causing the imbalance between Ang II and Ang 1-7. The fluorescence microscopy was used to quantify the microvascular parameters. Hypoperfusion and reperfusion caused vasoconstriction, disruption of blood-brain barrier, reduction of capillary perfusion and an increase in reactive oxygen species production. Rats treated with Ang II showed exacerbated microvascular damage with stronger vasoconstriction compared to hypoperfused rats, a further increase in leakage, higher decrease in capillary perfusion and marker oxidative stress. Candesartan cilexetil (specific Ang II type 1 receptor (AT1R) antagonist) administration prior to Ang II prevented the effects induced by Ang II, blunting the hypoperfusion-reperfusion injury. Ang 1-7 or ACE2 activator administration, preserved the pial microcirculation from hypoperfusion-reperfusion damage. These effects of Ang 1-7 were blunted by a Mas (Mas oncogene-encoded protein) receptor antagonist, while Ang II type 2 receptor antagonists did not affect Ang 1-7-induced changes. In conclusion, Ang II and Ang 1-7 triggered different mechanisms through AT1R or MAS receptors able to affect cerebral microvascular injury.

Ang(1-7) exerts Nrf2-mediated neuroprotection against amyloid beta-induced cognitive deficits in rodents.

Alzheimer's disease (AD) is a neurodegenerative disorder with cognitive deficits in an individual. Ang(1-7) exhibits neuroprotection against amyloid beta (Aß)-induced mitochondrial dysfunction and neurotoxicity in experimental conditions. Further, Ang(1-7) also exhibits nrf2-mediated antioxidant activity in experimental conditions. However, its therapeutic role on nrf2-mediated mitochondrial function is yet to be established in the Aß-induced neurotoxicity. The experimental dementia was induced in the male rats by intracerebroventricular administration of Aß(1-42) on day-1 (D-1) of the experimental schedule of 14 days. Ang(1-7) was administered once daily from D-1 toD-14 to the Aß-challenged rodents. Ang(1-7) attenuated Aß-induced increase in escape latency and decrease in the time spent in the target quadrant during Morris water maze and percentage of spontaneous alteration behavior during Y-maze tests in the rats. Further, Ang(1-7) attenuated Aß-induced cholinergic dysfunction in terms of decrease in the level of acetylcholine and activity of choline acetyltransferase, and increase in the activity of acetylcholinesterase, and increase in the level of Aß in rat hippocampus, pre-frontal cortex and amygdala. Furthermore, Ang(1-7) reversed Aß-induced decrease in the mitochondrial function, integrity and bioenergetics in all brain regions. Additionally, Ang(1-7) attenuated Aß-induced increase in the extent of apoptosis and decrease in the level of heme oxygenase-1 in all selected brain regions. Trigonelline significantly abolished the therapeutic effectiveness of Ang(1-7) on Aß-induced alterations in the behavioral, neurochemicals and molecular observations in the animals. Ang(1-7) may exhibit nrf2-mediated neuroprotection in these rodents. Hence, Ang(1-7) could be a potential therapeutic option in the pharmacotherapy of AD.

Angiotensin (1-7) Attenuates the Nociceptive Behavior Induced by Substance P and NMDA via Spinal MAS1.

The intrathecal (i.t.) injection of substance P (SP) and N-methyl-D-aspartate (NMDA) induce transient nociceptive response by activating neurokinin (NK) 1 and NMDA receptors, respectively. We have recently reported that angiotensin (Ang) (1-7), an N-terminal fragment of Ang II, could alleviate several types of pain including neuropathic and inflammatory pain by activating spinal MAS1. Here, we investigated whether Ang (1-7) can inhibit the SP- and NMDA-induced nociceptive response. The nociceptive response induced by an i.t. injection of SP or NMDA was assessed by measuring the duration of hindlimb scratching directed toward the flank, biting and/or licking of the hindpaw or the tail for 5 min. Localization of MAS1 and either NK1 or NMDA receptors in the lumbar superficial dorsal horn was determined by immunohistochemical observation. The nociceptive response induced by SP and NMDA was attenuated by the i.t. co-administration of Ang (1-7) (0.03-3 pmol) in a dose-dependent manner. The inhibitory effects of Ang (1-7) (3 pmol) were attenuated by A779 (100 pmol), a MAS1 antagonist. Moreover, immunohistochemical analysis showed that spinal MAS1 co-localized with NK1 receptors and NMDA receptors on cells in the dorsal horn. Taken together, the i.t. injection of Ang (1-7) attenuated the nociceptive response induced by SP and NMDA via spinal MAS1, which co-localized with NK1 and NMDA receptors. Thus, the spinal Ang (1-7)/MAS1 pathway could represent a therapeutic target to effectively attenuate spinal pain transmission caused by the activation of NK1 or NMDA receptors.

Chronic administration of pharmacological doses of angiotensin 1-7 and iodoangiotensin 1-7 has minimal effects on blood pressure, heart rate, and cognitive function of spontaneously hypertensive rats.

Cardiovascular diseases are the principal cause of death worldwide, with hypertension being the most common cardiovascular disease risk factor. High blood pressure (BP) is also associated with an increased risk of poor cognitive performance and dementia including Alzheimer's disease. Angiotensin 1-7 (Ang 1-7), a product of the renin-angiotensin system (RAS), exhibits central and peripheral actions to reduce BP. Recent data from our lab reveals that the addition of a non-radioactive iodine molecule to the tyrosine in position 4 of Ang 1-7 (iodoAng 1-7) makes it ~1000-fold more potent than Ang 1-7 in competing for the 125 I-Ang 1-7 binding site (Stoyell-Conti et al., 2020). Moreover, the addition of the non-radioactive iodine molecule increases (~4-fold) iodoAng 1-7's ability to bind to the AT1 receptor (AT1R), the primary receptor for Ang II. Preliminary data indicates that iodoAng 1-7 can also compete for the 125 I-Ang IV binding site with a low micromolar IC50. Thus, our aims were to compare the effects of chronic treatment of the Spontaneously Hypertensive Rat (SHR) with iodoAng 1-7 (non-radioactive iodine isotope) and Ang 1-7 on arterial pressure, heart rate, and cognitive function. For this study, male SHRs were divided into three groups and treated with Saline, Ang 1-7, or iodoAng 1-7 administrated subcutaneously using a 28-day osmotic mini pump. Systolic BP was measured non-invasively by the tail-cuff technique. Cognitive function was assessed by Y-Maze test and novel object recognition (NOR) test. We have demonstrated in SHRs that subcutaneous administration of high doses of iodoAng 1-7 prevented the increase in heart rate with age, while Ang 1-7 showed a trend toward preventing the increase in heart rate, possibly by improving baroreflex control of the heart. Conversely, neither Ang 1-7 nor iodoAng 1-7 administered subcutaneously affected BP nor cognitive function.

Angiotensin 1-7 protects against ventilator-induced diaphragm dysfunction.

Mechanical ventilation (MV) is a life-saving instrument used to provide ventilatory support for critically ill patients and patients undergoing surgery. Unfortunately, an unintended consequence of prolonged MV is the development of inspiratory weakness due to both diaphragmatic atrophy and contractile dysfunction; this syndrome is labeled ventilator-induced diaphragm dysfunction (VIDD). VIDD is clinically important because diaphragmatic weakness is an important contributor to problems in weaning patients from MV. Investigations into the pathogenesis of VIDD reveal that oxidative stress is essential for the rapid development of VIDD as redox disturbances in diaphragm fibers promote accelerated proteolysis. Currently, no standard treatment exists to prevent VIDD and, therefore, developing a strategy to avert VIDD is vital. Guided by evidence indicating that activation of the classical axis of the renin-angiotensin system (RAS) in diaphragm fibers promotes oxidative stress and VIDD, we hypothesized that activation of the nonclassical RAS signaling pathway via angiotensin 1-7 (Ang1-7) will protect against VIDD. Using an established animal model of prolonged MV, our results disclose that infusion of Ang1-7 protects the diaphragm against MV-induced contractile dysfunction and fiber atrophy in both fast and slow muscle fibers. Further, Ang1-7 shielded diaphragm fibers against MV-induced mitochondrial damage, oxidative stress, and protease activation. Collectively, these results reveal that treatment with Ang1-7 protects against VIDD, in part, due to diminishing oxidative stress and protease activation. These important findings provide robust evidence that Ang1-7 has the therapeutic potential to protect against VIDD by preventing MV-induced contractile dysfunction and atrophy of both slow and fast muscle fibers.

Central Administration of Angiotensin-(1-7) Improves Vasopressin Impairment and Hypotensive Response in Experimental Endotoxemia.

Angiotensin-(1-7) [Ang-(1-7)]/Mas receptor is a counter-regulatory axis that counteracts detrimental renin-angiotensin system (RAS) effects, especially regarding systemic inflammation, vasopressin (AVP) release, and hypothalamic-pituitary-adrenal (HPA) activation. However, it is not completely understood whether this system may control centrally or systemically the late phase of systemic inflammation. Thus, the aim of this study was to determine whether intracerebroventricular (i.c.v.) administration of Ang-(1-7) can modulate systemic inflammation through the activation of humoral pathways in late phase of endotoxemia. Endotoxemia was induced by systemic injection of lipopolysaccharide (LPS) (1.5 mg/kg, i.v.) in Wistar rats. Ang-(1-7) (0.3 nmol in 2 µL) promoted the release of AVP and attenuated interleukin-6 (IL-6) and nitric oxide (NO) levels but increased interleukin-10 (IL-10) in the serum of the endotoxemic rats. The central administration of Mas receptor antagonist A779 (3 nmol in 2 µL, i.c.v.) abolished these anti-inflammatory effects in endotoxemic rats. Furthermore, Ang-(1-7) applied centrally restored mean arterial blood pressure (MABP) without affecting heart rate (HR) and prevented vascular hyporesponsiveness to norepinephrine (NE) and AVP in animals that received LPS. Together, our results indicate that Ang-(1-7) applied centrally promotes a systemic anti-inflammatory effect through the central Mas receptor and activation of the humoral pathway mediated by AVP.

Targeting the Protective Arm of the Renin-Angiotensin System: Focused on Angiotensin-(1-7).

The in vivo application and efficacy of many therapeutic peptides is limited because of their instability and proteolytic degradation. Novel strategies for developing therapeutic peptides with higher stability toward proteolytic degradation would be extremely valuable. Such approaches could improve systemic bioavailability and enhance therapeutic effects. The renin-angiotensin system (RAS) is a hormonal system within the body essential for the regulation of blood pressure and fluid balance. The RAS is composed of two opposing classic and protective arms. The balance between these two arms is critical for the homeostasis of the body's physiologic function. Activation of the RAS results in the suppression of its protective arm, which has been reported in inflammatory and pathologic conditions such as arthritis, cardiovascular diseases, diabetes, and cancer. Clinical application of angiotensin-(1-7) [Ang-(1-7)], a RAS critical regulatory peptide, augments the protective arm and restores balance hampered by its enzymatic and chemical instability. Several attempts to increase the half-life and efficacy of this heptapeptide using more stable analogs and different drug delivery approaches have been made. This review article provides an overview of efforts targeting the RAS protective arm. It provides a critical analysis of Ang-(1-7) or its homologs' novel drug delivery systems using different administration routes, their pharmacological characterization, and therapeutic potential in various clinical settings. SIGNIFICANCE STATEMENT: Ang-(1-7) is a unique peptide component of the renin-angiotensin system with vast potential for clinical applications that modulate various inflammatory diseases. Novel Ang-(1-7) peptide drug delivery could compensate its lack of stability for effective clinical application.

Angiotensin-(1-7) attenuates collagen-induced arthritis via inhibiting oxidative stress in rats.

The present study was designed to investigate the anti-rheumatic effects and the mechanism of angiotensin (Ang)-(1-7) in rat models with collagen-induced arthritis (CIA). The CIA model was established using male Wistar rats by intradermal injection of bovine collagen-II in complete Freund's adjuvant at the base of the tail. The levels of angiotensin converting enzyme 2 (ACE2)/Ang-(1-7)/Mas receptor (MasR) were reduced in CIA rats. The attenuation of paw swelling and arthritis scores and improvement of indexes of spleen and thymus were done by Ang-(1-7) injection in CIA rats. The increased levels of inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the serum and hind paw were blocked by Ang-(1-7) administration. In addition, enhanced NADPH oxidase (Nox) activity, increased levels of superoxide anions and malondialdehyde (MDA), and weakened superoxide dismutase (SOD) activity, were all reversed by treatment with Ang-(1-7). Nox1 overexpression reversed the suppressing effects of Ang-(1-7) on paw swelling and arthritis scores in CIA rats. The Ang-(1-7)-induced improvement in spleen and thymus indexes in CIA rats was abolished by Nox1 overexpression. Nox1 overexpression reversed the inhibitory effects of Ang-(1-7) by increasing IL-1ß, IL-6, TNF-α, and IFN-γ levels in the serum and hind paw of CIA rats. These results demonstrated that Nox1 increased the oxidative stress in arthritis, and Ang-(1-7) improved rheumatism in arthritis via inhibiting oxidative stress.

Modeling the Molecular Impact of SARS-CoV-2 Infection on the Renin-Angiotensin System.

SARS-CoV-2 infection is mediated by the binding of its spike protein to the angiotensin-converting enzyme 2 (ACE2), which plays a pivotal role in the renin-angiotensin system (RAS). The study of RAS dysregulation due to SARS-CoV-2 infection is fundamentally important for a better understanding of the pathogenic mechanisms and risk factors associated with COVID-19 coronavirus disease and to design effective therapeutic strategies. In this context, we developed a mathematical model of RAS based on data regarding protein and peptide concentrations; the model was tested on clinical data from healthy normotensive and hypertensive individuals. We used our model to analyze the impact of SARS-CoV-2 infection on RAS, which we modeled through a downregulation of ACE2 as a function of viral load. We also used it to predict the effect of RAS-targeting drugs, such as RAS-blockers, human recombinant ACE2, and angiotensin 1-7 peptide, on COVID-19 patients; the model predicted an improvement of the clinical outcome for some drugs and a worsening for others. Our model and its predictions constitute a valuable framework for in silico testing of hypotheses about the COVID-19 pathogenic mechanisms and the effect of drugs aiming to restore RAS functionality.

Synergism between Angiotensin receptors ligands: Role of Angiotensin-(1-7) in modulating AT2 R agonist response on nitric oxide in kidney cells.

Angiotensin-(1-7), an endogenous agonist for the MasR, has been shown to interact with ang-II AT1 R and AT2 R. Earlier we showed a physical and functional interaction between MasR and AT2 R in response to their respective agonists ang-(1-7) and C21. Moreover, ang-(1-7) is cardio-protective via AT1 R and alters ang-II function. Such complex nature of ang-(1-7) function is not clearly understood, particularly in relation to its functional interaction with these receptors. We tested how ang-(1-7) affects AT2 R function by utilizing HK-2 cells. The HK-2 cells were treated with a wide range of concentrations of angiotensin receptor agonists. The generation of NO• in response to agonists was determined as a readout and subjected to Bliss definition (δ score) to assess the nature of functional interaction between these receptors. Preincubation with ang-(1-7) followed by incubation with endogenous AT1 R/AT2 R agonist ang-II (δ = 162) or selective AT2 R agonist C21 (δ = 304) synergized NO• formation. The synergism was also observed when the order of incubation with ang-(1-7)/C21 was reversed (δ = 484), but not when the cells were simultaneously incubated with a mixture of ang-(1-7) and C21 (δ = 76). The synergism with nonpeptidic MasR agonist AVE0991 followed by C21 (δ = 45) was minimal. Ligand binding experiment suggested the binding of ang-(1-7) with these three receptors. However, the synergism observed with ang-(1-7) and ang-II/C21 was sensitive to the antagonists of AT2 R (PD123319) and AT1 R (candesartan), but not MasR (A779). Ang-(1-7) at lower concentrations synergies the AT2 R function in an AT1 R-dependent but MasR-independent manner. This phenomenon may have a physiological significance.

Angiotensin-(1-7) Improves Integrated Cardiometabolic Function in Aged Mice.

Angiotensin (Ang)-(1-7) is a beneficial renin-angiotensin system (RAS) hormone that elicits protective cardiometabolic effects in young animal models of hypertension, obesity, and metabolic syndrome. The impact of Ang-(1-7) on cardiovascular and metabolic outcomes during aging, however, remains unexplored. This study tested the hypothesis that Ang-(1-7) attenuates age-related elevations in blood pressure and insulin resistance in mice. Young adult (two-month-old) and aged (16-month-old) male C57BL/6J mice received Ang-(1-7) (400 ng/kg/min) or saline for six-weeks via a subcutaneous osmotic mini-pump. Arterial blood pressure and metabolic function indices (body composition, insulin sensitivity, and glucose tolerance) were measured at the end of treatment. Adipose and cardiac tissue masses and cardiac RAS, sympathetic and inflammatory marker gene expression were also measured. We found that chronic Ang-(1-7) treatment decreased systolic and mean blood pressure, with a similar trend for diastolic blood pressure. Ang-(1-7) also improved insulin sensitivity in aged mice to levels in young mice, without effects on glucose tolerance or body composition. The blood pressure-lowering effects of Ang-(1-7) in aged mice were associated with reduced sympathetic outflow to the heart. These findings suggest Ang-(1-7) may provide a novel pharmacological target to improve age-related cardiometabolic risk.

Treatment with inhaled formulation of angiotensin-(1-7) reverses inflammation and pulmonary remodeling in a model of chronic asthma.

Asthma is characterized by inflammation, pulmonary remodeling and bronchial hyperresponsiveness. We have previously shown that treatment with angiotensin-(1-7) [Ang-(1-7)] promotes resolution of eosinophilic inflammation and prevents chronic allergic lung inflammation. Here, we evaluated the effect of treatment with the inclusion compound of Ang-(1-7) in hydroxypropyl ß-cyclodextrin (HPßCD) given by inhalation on pulmonary remodeling in an ovalbumin (OVA)-induced chronic allergic lung inflammation. Mice were sensitized to ovalbumin (OVA; 4 injections over 42 days, 14 days apart) and were challenged 3 times per week, for 4 weeks (days 21-46). After the 2nd week of challenge, mice were treated with Ang-(1-7) by inhalation (4.5 µg of Ang-(1-7) included in 6.9 µg of HPßCD for 14 days, i.e. days 35-48). Mice were killed 72 h after the last challenge and blood, bronchoalveolar lavage fluid (BALF) and lungs were collected. Histology and morphometric analysis were performed in the lung. Metalloproteinase (MMP)-9 and MMP-12 expression and activity, IL-5, CCL11 in the lung and plasma IgE were measured. After 2 weeks of OVA challenge there was an increase in plasma IgE and in inflammatory cells infiltration in the lung of asthmatic mice. Treatment with inhaled administration of Ang-(1-7)/HPßCD for 14 days reduced eosinophils, IL5, CCL11 in the lung and plasma IgE. Treatment of asthmatic mice with Ang-(1-7)/HPßCD by inhalation reversed pulmonary remodeling by reducing collagen deposition and MMP-9 and MMP-12 expression and activity. These results show for the first time that treatment by inhalation with Ang-(1-7) can reverse an installed asthma, inhibiting pulmonary inflammation and remodeling.

Angiotensin (1-7) through modulation of the NMDAR-nNOS-NO pathway and serotonergic metabolism exerts an anxiolytic-like effect in rats.

Although recent studies have shown that angiotensin (1-7) (Ang [1-7]) exerts anti-stress and anxiolytic-like effects, the underlying mechanisms remain elusive. The ventral hippocampus (VH) is proposed to be a critical brain region for mood and stress management through the N-methyl-d-aspartate receptor (NMDAR) signaling pathway. However, the role of VH NMDAR signaling in the effects of Ang (1-7) remains unclear. In the present study, Ang (1-7) was injected into the bilateral VH of stressed rats, or in combination with a Fyn kinase inhibitor, NMDAR antagonist, neuronal nitric oxide synthase (nNOS) inhibitor, or nitric oxide (NO) scavenger. Anxiety-like behaviors were assessed using the open field test and elevated plus maze test, while alterations in NMDAR-nNOS-NO signaling and serotonergic metabolism were examined in the VH. After 21 days of chronic restraint stress, anxiety-like behaviors were evident. Levels of phosphorylated NR2B (a key NMDAR subunit), its upstream kinase Fyn, as well as activity of nNOS and monoamine oxidase (MAO) were markedly reduced. In contrast, levels of serotonin were increased. Bilateral VH infusion of Ang (1-7) recovered NMDAR-nNOS-NO signaling and MAO-mediated serotonin metabolism, as well as reducing anxiety-like behaviors in stressed rats. These effects were diminished by blockade of MasR (Ang [1-7]-specific receptor), Fyn kinase, NMDAR, nNOS, or NO production. Altogether, these findings indicate that Ang (1-7) exerts anxiolytic effects through modulation of the NMDAR-nNOS-NO pathway and serotonergic metabolism. Future translational research should focus on the relationship between Ang (1-7), glutamatergic neurotransmission, and serotonergic neurotransmission in the VH.

The clinical impact of angiotensin-(1-7)/mitochondrial assembly receptor axis in esophageal squamous cell carcinoma patients receiving curative esophagectomy.

BACKGROUND: Mitochondrial assembly receptor (MasR), a receptor of angiotensin-(1-7), plays an important role in the anti-cancer effect of the peptide hormone. The aim of the current study was to evaluate the crucial role of angiotensin-(1-7)/MasR axis in esophageal squamous cell carcinoma (ESCC) patients who received curative esophagectomy. METHODS: The immunohistochemistry of MasR in 90 ESCC patients, including 52 patients with MasR overexpression and 38 patients with low MasR expression, was examined and correlated with their treatment outcomes. Two ESCC cell lines, TE11 and KYSE270, were treated with angiotensin-(1-7) to explore the biological function of MasR. RESULTS: A higher percentage of patients in the low MasR expression group experienced tumor recurrence than those in the MasR overexpression group (76% versus 54%, P = 0.029). Patients below 60 years of age and having early T status and negative pathologic N status were found to have significantly better disease-free survival (DFS) and overall survival (OS). Additionally, patients with MasR overexpression had higher DFS (88.1 months versus 50.0 months, p = 0.023) and OS (129.4 months versus 67.5 months, p = 0.028) relative to those with low MasR expression, although there was no significant difference in multivariable analysis. In vitro, these cell lines were treated with angiotensin-(1-7) and the results demonstrated that angiotensin-(1-7) could inhibit the growth of ESCC tumor cells in a dose-dependent manner. CONCLUSION: Low expression of MasR may be associated with poor prognosis in ESCC patients receiving curative esophagectomy. Further cohort study with larger population, or a prospective study is warranted to validate this finding.

Correlation between angiotensin 1-7-mediated Mas receptor expression with motor improvement, activated STAT3/SOCS3 cascade, and suppressed HMGB-1/RAGE/NF-κB signaling in 6-hydroxydopamine hemiparkinsonian rats.

In the current investigation, a Parkinson's disease (PD) model was established by a single direct right intrastriatal injection of the 6-hydroxydopamine (OHDA) in male Wistar rats followed by 7 daily unilateral injection of angiotensin (Ang) 1-7 in the striatum. To confirm the putative role of Mas receptor (MasR), the selective antagonist A779 was also injected intrastriatally prior to Ang 1-7 injections and a correlation analysis was performed between MasR expression and the assessed parameters. Ang 1-7 upregulated MasR expression to correlate strongly with the improved rotarod (r = 0.95, p = 0.003) and spontaneous activity task (r = 0.99, p < 0.0001). This correlation extends to involve other effects of Ang 1-7, such as the increased striatal dopamine content (r = 0.98, p = 0.0005), substantia nigra pars compacta tyrosine hydroxylase immune-reactivity (r = 0.97, p = 0.001), active pY705-STAT3 (r = 0.99, p < 0.0001) and SOCS3 (r = 0.99, p < 0.0001). Conversely, Ang 1-7 inhibited inflammatory markers to correlate negatively with NF-κBp65 (r = -0.99, p < 0.0003) and its downstream targets, high mobility group box-1 (HMGB-1; r = -0.97, p = 0.002), receptor for advanced glycation end products (RAGE; r = -0.98, p = 0.0004), and TNF-α (r = -0.99, p < 0.0003), besides poly-ADP-ribose polymerase-1 (r = -0.99, p = 0.0002). In confirmation, the pre-administration of the selective MasR antagonist, A779, partially attenuated Ang 1-7-induced alterations towards 6-OHDA neurodegeneration. Collectively, our findings support a novel role for the anti-inflammatory capacity of the MasR axis to prove potential therapeutic relevance in PD via the upregulation/activation of MasR-dependent STAT3/SOCS3 cascade to negatively control the HMGB-1/RAGE/NF-κB axis hindering PD associated neuro-inflammation along with DA depletion and motor deficits.

Addition of cyclic angiotensin-(1-7) to angiotensin-converting enzyme inhibitor therapy has a positive add-on effect in experimental diabetic nephropathy.

The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.

Central ANG-(1-7) infusion improves blood pressure regulation in antenatal betamethasone-exposed sheep and reveals sex-dependent effects on oxidative stress.

Fetal exposure to betamethasone (BMX) as a consequence of glucocorticoid administration to women threatening premature delivery may lead to long-term deleterious effects on the cardiovascular system and dysregulation of blood pressure in exposed adults. Indeed, adult offspring of BMX sheep exhibit increased mean arterial pressure (MAP) and attenuated baroreflex sensitivity (BRS) that are associated with lower medullary and cerebrospinal fluid (CSF) angiotensin-(1-7) [(ANG-(1-7)] content. Thus we determined the effects of ANG-(1-7) supplementation in the CSF on MAP, BRS, blood pressure (BPV) and heart rate variability (HRV) in conscious animals. The peptide or artificial CSF (aCSF) was infused continuously into the lateral ventricle (intracerebroventricular) of 4-mo-old male and female BMX sheep for 2 wk. Analysis of data from males and females combined revealed that intracerebroventricular ANG-(1-7) significantly lowered MAP and heart rate and improved BRS as compared with baseline; intracerebroventricular aCSF did not change these indexes. Similar patterns were observed for altered hemodynamics and autonomic function produced by intracerebroventricular ANG-(1-7) in both sexes. Oxidative stress and MAP kinase (MAPK) activation were lower in tissues from the dorsomedial medulla (DMM) of ANG-(1-7)-treated males but were unchanged in the treated females, when assessed at the end of the treatment period. We conclude that in the face of ANG-(1-7) deficiency in CSF and medullary tissue in BMX sheep intracerebroventricular supplementation of ANG-(1-7) lowers MAP and restores the impaired autonomic function to a similar degree in both males and females; however, the attenuation of MAPK and oxidative stress within the DMM was evident only in males. NEW & NOTEWORTHY We demonstrate that intracerebroventricular angiotensin-(1-7) [(ANG-(1-7)] treatment for 2 wk in antenatal betamethasone-exposed sheep provides beneficial effects on blood pressure and autonomic function. The physiological improvements are accompanied by an attenuation of oxidative stress in males but not females. The finding that ANG-(1-7) supplementation lowers blood pressure and restores the impaired autonomic function in a model of fetal programming previously shown to exhibit a deficiency in cerebrospinal fluid and brain tissue illustrates the potential for new therapeutic strategies for reducing cardiovascular dysfunction arising from prenatal events.

Chronic Angiotensin 1-7 Infusion Prevents Angiotensin-II-Induced Cognitive Dysfunction and Skeletal Muscle Injury in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly viewed as a neurological disease accompanied by a systemic disorder. Accumulating evidence supports that angiotensin II and angiotensin 1-7 exert opposite effects on various organs including the brain. However, the interaction between angiotensin II and angiotensin 1-7 in AD remains to be defined. The present study was undertaken to examine the interaction between these peptides in AD. 5XFAD mice, a useful model of AD, were separated into three groups: 1) saline-infused, 2) angiotensin II-infused, and 3) angiotensin II-infused and angiotensin 1-7-co-infused. These peptides were systemically given to 5XFAD mice via osmotic minipump for 4 weeks. Systemic angiotensin II infusion for 4 weeks induced significant hypertension in both wild-type and 5XFAD mice. Angiotensin II induced cognitive abnormality in 5XFAD mice as estimated by the Morris water maze test and the nest building test, and this effect was associated with cerebral blood flow reduction, cortical arterial amyloid-ß deposition, hippocampal inflammation, and neuron loss in 5XFAD mice. In addition, angiotensin II infusion led to gastrocnemius muscle atrophy in 5XFAD mice. Co-infusion of angiotensin 1-7 prevented the above mentioned detrimental effects of angiotensin II in the brain and gastrocnemius muscle in 5XFAD mice, without significant influence on blood pressure. The left ventricular hypertrophic response to angiotensin II was attenuated in 5XFAD mice compared with wild-type mice, which was not significantly altered by co-administration of angiotensin 1-7. Our results show that angiotensin 1-7 counteracts angiotensin II-induced cognitive impairment, brain injury, and skeletal muscle injury in AD mice.