Mas Receptor Research Articles

Transgenic rat with ubiquitous expression of angiotensin-(1-7)-producing fusion protein: a new tool to study the role of protective arm of the renin-angiotensin system in the pathophysiology of cardio-renal diseases.

The aim of the present study was to assess systemic circulatory and tissue activities of both the classical arm and of the alternative arm of the renin-angiotensin system (RAS) in a new transgenic rat line (TG7371) that expresses angiotensin-(1-7) (ANG 1-7)-producing fusion protein; the results were compared with the activities measured in control transgene-negative Hannover Sprague-Dawley (HanSD) rats. Plasma and tissue concentrations of angiotensin II (ANG II) and ANG 1-7, and kidney mRNA expressions of receptors responsible for biological actions of ANG II and ANG 1-7 [i.e. ANG II type 1 and type 2 (AT1 and AT2) and Mas receptors] were assessed in TG7371 transgene-positive and in HanSD rats. We found that male TG7371 transgene-positive rats exhibited significantly elevated plasma, kidney, heart and lung ANG 1-7 concentrations as compared with control male HanSD rats; by contrast, there was no significant difference in ANG II concentrations and no significant differences in mRNA expression of AT1, AT2 and Mas receptors. In addition, we found that in male TG7371 transgene-positive rats blood pressure was lower than in male HanSD rats. These data indicate that the balance between the classical arm and the alternative arm of the RAS was in male TGR7371 transgene-positive rats markedly shifted in favor of the latter. In conclusion, TG7371 transgene-positive rats represent a new powerful tool to study the long-term role of the alternative arm of the RAS in the pathophysiology and potentially in the treatment of cardio-renal diseases.

Downregulation of Neuronal Nitric Oxide Synthase (nNOS) within the Paraventricular Nucleus in Ins2Akita-type-1 Diabetic Mice Contributes to Sympatho-excitation

Activation of both renin-angiotensin system (RAS) and the sympathetic system is the primary etiologic event in developing cardiovascular complications in diabetes mellitus (DM). However, the precise mechanisms for sympathetic activation in DM have not been elucidated. Here we attempted to investigate diabetes-linked cardiovascular dysregulation due to angiotensin II (Ang II)-mediated reduction in neuronal nitric oxide (NO) synthase (nNOS) within the paraventricular neuleus (PVN). In the present study, we used Ins2+/-Akita (a spontaneous, insulin-dependent genetic diabetic non-obese murine model) and wild-type (WT) littermates mice as controls. At 14 weeks of age, we found the Akita mice had increased renal sympathetic nerve activity and elevated levels of plasma norepinephrine. There was decreased expression of nNOS protein (Akita 0.43 ± 0.11 vs. WT 0.75 ± 0.05, P < 0.05) in the PVN of Akita mice. Akita mice had increased expression of angiotensin-converting enzyme (ACE) (Akita 0.58 ± 0.05 vs. WT 0.34 ± 0.04, P < 0.05) and Ang II type 1 receptor (Akita 0.49 ± 0.03 vs. WT 0.29 ± 0.09, P < 0.05), decreased expressions of ACE2 (Akita 0.17 ± 0.05 vs. WT 0.27 ± 0.03, P < 0.05) and angiotensin (1-7) Mas receptor (Akita 0.46 ± 0.02 vs. WT 0.77 ± 0.07, P < 0.05). Futher, there were increased protein levels of protein inhibitor of nNOS (PIN) (Akita 1.75 ± 0.08 vs. WT 0.71 ± 0.09, P < 0.05) with concomitantly decreased catalytically active dimers of nNOS (Akita 0.11 ± 0.04 vs. WT 0.19 ± 0.02, P < 0.05) in the PVN in Akita mice. Our studies suggest that activation of the excitatory arm of RAS, leads to a decrease NO, causing an over-activation of the sympathetic drive in DM.

Alamandine, a protective component of the renin-angiotensin system, reduces cellular proliferation and interleukin-6 secretion in human macrophages through MasR–MrgDR heteromerization

Alamandine (ALA) exerts protective effects similar to angiotensin (Ang) (1–7) through Mas-related G protein-coupled receptor type D receptor (MrgDR) activation, distinct from Mas receptor (MasR). ALA induces anti-inflammatory effects in mice but its impact in human macrophages remains unclear. We aimed to investigate the anti-inflammatory effects of ALA in human macrophages. Interleukin (IL)-6 and IL-1β were measured by ELISA in human THP-1 macrophages and human monocyte-derived macrophages exposed to lipopolysaccharide (LPS). Consequences of MasR–MrgDR heteromerization were investigated in transfected HEK293T cells. ALA decreased IL-6 and IL-1β secretion in LPS-activated THP-1 macrophages. The ALA-induced decrease in IL-6 but not in IL-1β was prevented by MasR blockade and MasR downregulation, suggesting MasR–MrgDR interaction. In human monocyte-derived M1 macrophages, ALA decreased IL-1β secretion independently of MasR. MasR–MrgDR interaction was confirmed in THP-1 macrophages, human monocyte-derived macrophages, and transfected HEK293T cells. MasR and MrgDR formed a constitutive heteromer that was not influenced by ALA. ALA promoted Akt and ERK1/2 activation only in cells expressing MasR–MrgDR heteromers, and this effect was prevented by MasR blockade. While Ang-(1–7) reduced cellular proliferation in MasR −but not MrgDR- expressing cells, ALA antiproliferative effect was elicited in cells expressing MasR–MrgDR heteromers. ALA also induced an antiproliferative response in THP-1 cells and this effect was abolished by MasR blockade, reinforcing MasR–MrgDR interaction. MasR–MrgDR heteromerization is crucial for ALA-induced anti-inflammatory and antiproliferative responses in human macrophages. This study broaden our knowledge of the protective axis of the RAS, thus enabling novel therapeutic approaches in inflammatory-associated diseases

Angiotensin-(1-7) relieves behavioral defects and α-synuclein expression through NEAT1/miR-153-3p axis in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, whose characteristic pathology involves progressive deficiency of dopaminergic neurons and generation of Lewy bodies (LBs). Aggregated and misfolded α-synuclein (α-syn) is the major constituent of LBs. As the newly discovered pathway of renin-angiotensin system (RAS), Angiotensin-(1-7) (Ang-(1-7)) and receptor Mas have attracted increasing attentions for their correlation with PD, but underlying mechanisms remain not fully clear. Based on above, this study established PD models of mice and primary dopaminergic neurons with AAV-hα-syn(A53T), then discussed the effects of Ang-(1-7)/Mas on α-syn level and neuronal apoptosis for these models combined with downstream long non-coding RNA (lncRNA) and microRNA (miRNA). Results showed that Ang-(1-7) alleviated behavioral impairments, rescued dopaminergic neurons loss and lowered α-syn expression in substantia nigra of hα-syn(A53T) overexpressed PD mice. We also discovered that Ang-(1-7) decreased level of α-syn and apoptosis in the hα-syn(A53T) overexpressed dopaminergic neurons through lncRNA NEAT1/miR-153-3p axis. Moreover, miR-153-3p level in peripheral blood is found negatively correlated with that of α-syn. In conclusion, our work not only showed neuroprotective effect and underlying mechanisms for Ang-(1-7) on α-syn in vivo and vitro, but also brought new hope on miR-153-3p and NEAT1 for diagnosis and treatment in PD.

α-Mangostin Attenuates Blood Pressure and Reverses Vascular Remodeling by Balancing ACE/AT1R and ACE2/Ang-(1-7)/MasR Axes in Ang II-Infused Hypertensive Mice.

Hyperuricemia is a common comorbidity of hypertension and probably has a causal relationship with hypertension. Alpha-mangostin (α-MG) has been reported to have uric acid lowering effect. This study aimed to investigate the dual effects of α-MG on blood pressure (BP) and uric acid levels in angiotensin II (Ang II)-infused hypertensive mice. Male C57BL/6 mice were randomized into five groups: control, Ang II infusion (500 ng/kg/min for 2 weeks), Ang II infusion with gavage administration of α-MG 4.0 and 8.0 mg/kg and benzbromarone (25 mg/kg) respectively. BP, uric acid levels, vascular structure and function, and renin-Ang II system expressions in the aorta were assessed. Treatment with α-MG reduced BP, improved endothelial relaxation, and reversed aortic wall thickening and collagen deposition in Ang II-induced hypertensive mice. It also downregulated Ang II receptor 1 (AT1R) and angiotensin converting enzyme (ACE) expression, while upregulating ACE2, Mas receptor (MasR), and angiotensin (1-7) in the aorta. Moreover, α-MG demonstrated a significant enhancement in uric acid clearance and reduction in serum uric acid levels. Conversely, benzbromarone did not result in a decrease in BP, indicating that the hypotensive effect of α-MG may not be necessarily dependent on its urate-lowering properties. α-MG can attenuate Ang II-induced hypertension and reverse vascular remodeling, potentially by balancing the ACE/Ang II/AT1R axis and the ACE2/Ang-(1-7)/MasR axis. Our findings provide insights into α-MG as a novel anti-hypertensive drug especially in patients with hyperuricemia.

Organ-protective effects of the alternative renin-angiotensin system and prospects for therapeutic treatment. Review

The article is devoted to review the data regarding the identification of biochemical and functional features of the components of the renin‑angiotensin system using the tools of bibliometric analysis of scientific literature. A modern interpretation by the dividing of the multifunctional complex into two systems, namely the classical renin‑angiotensin system and alternative renin‑angiotensin system, in accordance with the definition of biological effects, is given. The enzymatic cascade involved in the production and metabolism of peptides in both classical and non‑classical pathways of renin‑angiotensin systems is shown from the point of view of the evolution of scientific concepts. The triggering role of components of the classical renin‑angiotensin system in pathophysiological processes with transformation into cardiovascular diseases and metabolic dysfunction has been proven. The history of the creation of pharmacological drugs for the correction of the classic renin‑angiotensin system is presented. In view of the achievement of recent years, the revision of the alternative renin‑angiotensin system is emphasized due to the discovery of the counter‑regulatory action of the angiotensin‑converting enzyme 2, angiotensin‑(1—7), the Mas receptor in relation to their correction of metabolic processes, regulation of blood pressure, and the presence of anti‑fibrotic, anti‑inflammatory and antioxidant properties. The positive effect of the components of the non‑classical renin‑angiotensin system on the protection of the cardiovascular system and kidney is presented based on the results of experimental studies on animals. Pharmacokinetic and pharmacodynamics characteristics of recombinant angiotensin‑converting enzyme 2 in experiments on healthy volunteers are highlighted. The potential protective effects of angiotensin‑converting enzyme 2 and angiotensin‑(1—7) concerning cardiometabolic risk are presented. The available data indicate the need for further comprehensive scientific investigations of the components of alternative renin‑angiotensin system to create a new class of pharmacological agents with their implementation in medical practice.

ACE2 alleviates sepsis-induced cardiomyopathy through inhibiting M1 macrophage via NF-κB/STAT1 signals

Angiotensin-converting enzyme 2 (ACE2), a crucial element of the renin-angiotensin system (RAS), metabolizes angiotensin II into Ang (1–7), which then combines with the Mas receptor (MasR) to fulfill its protective role in various diseases. Nevertheless, the involvement of ACE2 in sepsis-induced cardiomyopathy (SIC) is still unexplored. In this study, our results revealed that CLP surgery dramatically impaired cardiac function accompanied with disruption of the balance between ACE2-Ang (1–7) and ACE-Ang II axis in septic heart tissues. Moreover, ACE2 knockin markedly alleviated sepsis induced RAS disorder, cardiac dysfunction and improved survival rate in mice, while ACE2 knockout significantly exacerbates these outcomes. Adoptive transfer of bone marrow cells and in vitro experiments showed the positive role of myeloid ACE2 by mitigating oxidative stress, inflammatory response, macrophage polarization and cardiomyocyte apoptosis by blocking NF-κB and STAT1 signals. However, the beneficial impacts were nullified by MasR antagonist A779. Collectively, these findings showed that ACE2 alleviated SIC by inhibiting M1 macrophage via activating the Ang (1–7)-MasR axis, highlight that ACE2 might be a promising target for the management of sepsis and SIC patients.

The angiotensin-(1-7)/MasR axis improves pneumonia caused by Pseudomonas aeruginosa: Extending the therapeutic window for antibiotic therapy.

Pseudomonas aeruginosa is a frequent cause of antimicrobial-resistant hospital-acquired pneumonia, especially in critically ill patients. Inflammation triggered by P. aeruginosa infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Emerging data have shed light on the pro-resolving actions of angiotensin-(1-7) [Ang-(1-7)] signaling through the G protein-coupled receptor Mas (MasR) during infections. Herein, we investigated the role of the Ang-(1-7)/Mas axis in pneumonia caused by P. aeruginosa by using genetic and pharmacological approach and found that Mas receptor-deficient animals developed a more severe form of pneumonia showing higher neutrophilic infiltration into the airways, bacterial load, cytokines, and chemokines production and more severe pulmonary damage. Conversely, treatment of pseudomonas-infected mice with Ang-(1-7) was able to decrease neutrophilic infiltration in airways and lungs, local and systemic levels of pro-inflammatory cytokines and chemokines, and increase the efferocytosis rates, mitigating lung damage/dysfunction caused by infection. Notably, the therapeutic association of Ang-(1-7) with antibiotics improved the survival rates of mice subjected to lethal inoculum of P. aeruginosa, extending the therapeutic window for imipenem. Mechanistically, Ang-(1-7) increased phagocytosis of bacteria by neutrophils and macrophages to accelerate pathogen clearance. Altogether, harnessing the Ang-(1-7) pathway during infection is a potential strategy for the development of host-directed therapies to promote mechanisms of resistance and resilience to pneumonia.

Abstract P213: Mechanisms of Renin-Angiotensin System-Mediated Nitric Oxide Signaling in Podocytes

Introduction: The renin-angiotensin system (RAS) is a crucial cardiovascular and renal function regulator. Podocytes, glomerular epithelial cells that play a pivotal role in maintaining the renal filtration barrier, are key targets for RAS peptides. Our findings showed that RAS can promote calcium (Ca 2+ ) and nitric oxide (NO) signaling in podocytes. Moreover, a decrease in NO bioavailability directly correlates with glomerular filtration barrier damage, Ca 2+ overload, and the development of salt-sensitive hypertension. We hypothesized that RAS-mediated NO production is crucial for Ca 2+ control and cytoskeletal rearrangements in podocytes, contributing to glomerular filtration regulation. Methods: We performed live confocal imaging on human podocytes and freshly isolated rat glomeruli to detect the production of NO (DAF-FM) and Ca 2+ (Fluo-8). RAS peptides (Ang II, III, IV, 1-7, 1-9, and TRV120027) were used to stimulate cells, and NO and Ca 2+ production were measured. Blockers for Ang II Type 1 (AT1R), Ang II Type 2 (AT2R), and MAS receptors were used to identify the pathways involved in NO production. The dynamic changes in podocyte cytoskeleton mediated by RAS-NO intracellular signaling were detected using rhodamine-phalloidin staining and scanning ion microscopy. The NOS isoform-specific blockers were used to determine the source of NO in podocytes. ANOVA with post-hoc was used for comparisons. Results: Angiotensins II and III generate the most profound increases in NO levels in podocytes. Ang II, Ang III, and Ang 1-7 promote NO production by activating AT2R (n ≥ 20, p &lt; 0.01). Interestingly, the increase in NO modulates AT1R- and β-arrestin-mediated Ca 2+ signaling (n ≥ 20, p &lt; 0.05), suggesting the role of the NO/AT2R axis in Ca 2+ control in podocytes. NO production correlates with podocyte volume increase, and scanning ion microscopy experiments further confirmed that AT2R activation and corresponding NO elevation are responsible for the protrusion of podocyte's lamellipodia. Finally, we found that in podocytes, NOS1 is responsible for NO bioavailability, accounting for 69±5% of the total NO response, while the remaining NO levels are generated by NOS2 (39±5%). Conclusion: Our data show that RAS activation promotes NO production in podocytes, mainly via the AT2R pathway. NO is crucial for Ca 2+ control in podocytes and lamellipodia formation, suggesting the essential role of NO in glomerular filtration barrier function.

P181 AGE-RELATED CHANGES IN THE PROTECTIVE AXIS OF THE RENIN-ANGIOTENSIN SYSTEM IN THE BRAIN OF NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS

Background and Objective. Aging is a major risk factor for the development of hypertension, that leads to end organ damage and cognitive dysfunction. The axis of the renin-angiotensin system composed of angiotensin (Ang) (1-7) the Mas receptor (MasR) and angiotensin converting enzyme 2 (ACE2) plays a cerebroprotective role by increasing blood flow, angiogenesis and improving cognitive function among others. Evidence on the expression of this axis with age is limited. We investigated Ang-(1-7)/Mas/ACE2 axis in different areas of the brain from normotensive and spontaneously hypertensive rats (SHR). Methods. Hypothalamus (HT), brainstem (BS), hippocampus (HC) and cerebral cortex (CC) were isolated from Wistar normotensive rats and SHR of 1, 3 and 12 months. Ang-(1-7) levels were measured by radioimmunoassay and ACE2 and MasR abundance was determined by immunoblotting. Results. Ang-(1-7) levels decreased with age in all the areas from normotensive rats and in CC from SHR, while it increased from 1 to 3 months but then decreased at 12 months in BS, HC and HT from SHR. There was no difference in Ang-(1-7) levels between normotensive and SHR except at 1 month of age where Ang-(1-7) levels were lower in BS, HC and HT but the same in CC from SHR compared to normotensive rats. MasR and ACE2 expression did not change with age in all the investigated areas from normotensive rats and in CC from SHR. However, MasR and ACE2 increased with age in HC from SHR and they were higher than in normotensive rats. Conclusions. Our results showed changes in the expression of the brain Ang-(1-7)/Mas/ACE2 axis which may contribute to deleterious functions observed in the brain with age.

Abstract P430: Angiotensin-(1-7) Induces A Decrease In Proinflammatory Genes Expression Through A Mas Receptor-Mediated Translocation To Brainstem Nuclei Of Spontaneously Hypertensive Rats

Mas receptor (MasR) displays differential trafficking in brainstem neurons from spontaneously hypertensive rats (SHRs): the fraction of dynamin-mediated internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in Wistar neurons. Surprisingly, in SHR neurons but not in Wistar neurons, Ang-(1-7) induced MasR translocation to the nucleus. Our aim was to investigate the biological response elicited by MasR in the nucleus and whether MasR translocation was due to interaction with other receptors. We evaluated: 1) nitric oxide (NO) generation by DAF (diaminofluorescein) fluorescence; 2) interleukin (IL) 6, IL-1β, cyclooxygenase 2 (COX2) and NFkB expression by real time PCR; and 3) MasR interaction with AT1R and the dopamine type 2 receptor (D2R) by proximity ligation assay in brainstem neuronal primary cultures from Wistar and SHR rats incubated in the presence or absence of Ang-(1-7) or Ang-(1-7) + dynasore (DYN) (dynamin inhibitor) or Ang-(1-7) + A779 (MasR antagonist) during 30 min. Ang-(1-7) (100 nM) induced a decrease in the pro-inflammatory factors IL-6, IL-1β, COX2 and NFkB expression in brainstem neurons from Wistar and SHR rats which was blocked by A779, suggesting a MasR-mediated response. The effect of Ang-(1-7) was prevented when MasR internalization was prevented by DYN only in SHRs neurons but not in Wistar neurons, suggesting that the effect of Ang-(1-7) in SHR neurons is mediated by MasR translocation to the nucleus. Treatment of neurons with Ang-(1-7) induced an increase in NO in the nucleus of SHR but not of Wistar neurons and this effect was prevented by DYN and A779, suggesting that the Ang-(1-7)-induced NO production in the nucleus was due to MasR translocation to this compartment. MasR stimulation with Ang-(1-7) induced an increase in MasR-D2R heteromerization in SHR but not in Wistar neurons, and no change in MasR-AT1R heteromerization. Ou study showed that MasR translocation to the nucleus resulted in a reduction in pro-inflammatory genes expression and an increase in NO generation in SHR neurons. A fraction of MasR was translocated through the interaction with D2Rs, suggesting that this heteomer may be involved in the anti-inflammatory responses elicited by Mas translocation to the nucleus.

O48 CHRONIC EXPOSURE TO POLLUTED URBAN AIR ALTERS MOUSE SYSTEMIC AND TISSUE-SPECIFIC COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM (RAS)

Background and Objective: Chronic exposure to air pollution induces pulmonary, cardiovascular, and renal diseases. In the current study, we investigated the hypothesis that postulates that a dysregulation of the RAS may contribute to these pathologies. Methods: Male Balb/C mice were exposed to urban air from Buenos Aires City, in whole-body exposure chambers for 14 weeks. Control animals were exposed to filtered air. The levels of main RAS components including angiotensin-converting enzyme (ACE) and ACE2; AT1, AT2, and Mas receptors (R) were determined in mouse lungs, kidneys, and heart, through western blotting (WB) and RT-qPCR, respectively. The tissue content of cytokine mARN was determined by RT-qPCR. The tissue content of nitrotyrosine and 4-hydroxynonenal (4-HNE) levels, were determined by WB. Circulating angiotensin (Ang) II levels were determined by radioimmunoassay. Systolic blood pressure was measured by the tail-cuff method. Results: Exposure to air pollution resulted in a) Increased levels of ACE and AT1R mRNA in the lungs and augmented the abundance markers of inflammation (TNF-α, IL-6, and IL-1β) and oxidative stress (nitrotyrosine and 4-HNE) in this tissue, b) increased levels of MasR mRNA (all analyzed tissues) and MasR protein levels (lungs and heart), c) upregulated mRNA and protein abundance of cardiac and renal AT2Rs, and d) Increased levels of circulating Ang II and e) augmented systolic blood pressure. Conclusions: Our findings indicate that chronic exposure to air pollution alters the expression of both tissue and systemic components of the RAS. Since both AT2R and MasRs have been shown to participate in tissue-repair mechanisms, the upregulation of these receptors detected in mouse lungs, heart, and kidney after chronic exposure to polluted urban air could represent a mechanism of tissue protection against damage induced by air pollution.

O98 NEW APPROACHES TARGETING THE RENIN-ANGIOTENSIN SYSTEM: BRAIN ACE2 ACTIVATION FOLLOWING BRAIN AMINOPEPTIDASE A BLOCKADE BY FIRIBASTAT IN SALT-DEPENDENT HYPERTENSION

Background and Objective: In the brain, aminopeptidase A (APA), a membrane-bound zinc metalloprotease, generates angiotensin III from angiotensin II. Brain angiotensin III exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive rats and increases vasopressin release. Blocking brain angiotensin III formation by the APA inhibitor prodrug RB150/firibastat normalizes arterial BP in hypertensive deoxycorticosterone acetate (DOCA)-salt rats. Ang II and Ang III have similar affinities for AT1 receptors and blocking brain AngIII formation by RB150 decreases arterial BP without inducing Ang II accumulation. Methods: We therefore hypothesized that another metabolic pathway of brain angiotensin II, such as the conversion of angiotensin II into angiotensin 1-7 (Ang 1-7) by angiotensin-converting enzyme 2 (ACE2) might be activated following brain APA inhibition. To answer this question, we use surgical animal models that could allow us to monitor awake blood pressure responses to intracerebroventricular (icv) injections and measure enzymatic activity. Results: We found that the intracerebroventricular administration of RB150/firibastat in conscious DOCA-salt rats both inhibited brain APA activity and induced an increase in brain ACE2 activity. Then, we showed that the decreases in BP and vasopressin release resulting from brain APA inhibition with RB150/firibastat were reduced if ACE2 was concomitantly inhibited by MLN4760, a potent ACE2 inhibitor, or if the Mas receptor (MasR) was blocked by A779, a MasR antagonist. Conclusions: Our findings suggest that in the brain, the increase in ACE2 activity resulting from APA inhibition by RB150/firibastat treatment, subsequently increasing Ang 1-7 and activating the MasR while blocking angiotensin III formation, contributes to the antihypertensive effect and the decrease in vasopressin release induced by RB150/firibastat. RB150/firibastat treatment constitutes an interesting therapeutic approach to improve BP control in hypertensive patients by inducing in the brain renin-angiotensin system, hyperactivity of the beneficial ACE2/Ang 1-7/MasR axis while decreasing that of the deleterious APA/Ang II/Ang III/ATI receptor axis.

Effect of Diminazene Aceturate, an ACE2 activator, on platelet CD40L signaling induced glial activation in rat model of hypertension

Hypertension causes platelet activation and adhesion in the brain resulting in glial activation and neuroinflammation. Further, activation of Angiotensin-Converting Enzyme 2/Angiotensin (1–7)/Mas Receptor (ACE2/Ang (1–7)/MasR) axis of central Renin-Angiotensin System (RAS), is known to reduce glial activation and neuroinflammation, thereby exhibiting anti-hypertensive and anti-neuroinflammatory properties. Therefore, in the present study, the role of ACE2/Ang (1–7)/MasR axis was studied on platelet-induced glial activation and neuroinflammation using Diminazene Aceturate (DIZE), an ACE2 activator, in astrocytes and microglial cells as well as in rat model of hypertension. We found that the ACE2 activator DIZE, independently of its BP-lowering properties, efficiently prevented hypertension-induced glial activation, neuroinflammation, and platelet CD40-CD40L signaling via upregulation of ACE2/Ang (1–7)/MasR axis. Further, DIZE decreased platelet deposition in the brain by reducing the expression of adhesion molecules on the brain endothelium. Activation of ACE2 also reduced hypertension-induced endothelial dysfunction by increasing eNOS bioavailability. Interestingly, platelets isolated from hypertensive rats or activated with ADP had significantly increased sCD40L levels and induced significantly more glial activation than platelets from DIZE treated group. Therefore, injection of DIZE pre-treated ADP-activated platelets into normotensive rats strongly reduced glial activation compared to ADP-treated platelets. Moreover, CD40L-induced glial activation, CD40 expression, and NFкB-NLRP3 inflammatory signaling are reversed by DIZE. Furthermore, the beneficial effects of ACE2 activation, DIZE was found to be significantly blocked by MLN4760 (ACE2 inhibitor) as well as A779 (MasR antagonist) treatments. Hence, our study demonstrated that ACE2 activation reduced the platelet CD40-CD40L induced glial activation and neuroinflammation, hence imparted neuroprotection.

Acupuncture modulation of the ACE/Ang II/AT1R and ACE2/Ang(1-7)/MasR pathways in the rostral ventrolateral medulla reduces sympathetic output and prevents cardiac injury caused by SHR hypertension.

Acupuncture can reduce blood pressure, heart rate (HR), and ameliorate cardiac damage by modulating the excitability of the sympathetic nervous system, but the exact mechanism of this effect remains unclear. This study investigated the potential mechanisms of acupuncture in the treatment of cardiac damage in hypertension. Spontaneously hypertensive rats (SHR) were used as the hypertension model with Wistar-Kyoto rats as the control. Manual acupuncture, electroacupuncture, and metoprolol were used as interventions. Systolic and diastolic blood pressure (SBP, DBP) plus HR were monitored with cardiac structure determined using Masson staining. Angiotensin II (Ang II) and norepinephrine in myocardium were detected with ELISA as was Ang(1-7) and gamma aminobutyric acid (GABA) in the rostral ventrolateral medulla (RVLM). Expression of mRNA for collagen type I (Col-I), Col-III, actin α1 (ACTA1), and thrombospondin 4 (THBS4) in myocardium was detected using real-time PCR. Expression of angiotensin converting enzyme (ACE), Ang II, angiotensin II type 1 receptor (AT1R), ACE2, and Mas receptor (MasR) proteins in RVLM was monitored using western blot. After manual acupuncture and electroacupuncture treatment, SHRs showed decreased SBP, DBP and HR, reduced myocardial damage. There was decreased expression of the ACE/Ang II/AT1R axis, and increased expression of the ACE2/Ang(1-7)/MasR axis within the RVLM. GABA levels were increased within the RVLM and norepinephrine levels were decreased in myocardial tissue. Metoprolol was more effective than either manual acupuncture or electroacupuncture. Acupuncture directed against hypertensive cardiac damage may be associated with regulation of ACE/Ang II/AT1R and the ACE2/Ang(1-7)/MasR pathway within the RLVM to reduce cardiac sympathetic excitability.

Effect of exercise duration on toluene-induced locomotor sensitization in mice: a focus on the Renin Angiotensin System.

Exercise attenuates addictive behavior; however, little is known about the contribution of exercise duration to this positive effect. The Renin Angiotensin System (RAS) has been implicated both in addictive responses and in the beneficial effects of exercise; though, its role in the advantageous effects of exercise on toluene-induced addictive responses has not been explored. To evaluate the impact of different exercise regimens in mitigating the expression of toluene-induced locomotor sensitization and to analyze changes in RAS elements' expression at the mesocorticolimbic system after repeated toluene exposure and following voluntary wheel running in toluene-sensitized animals. Toluene-induced addictive-like response was evaluated with a locomotor sensitization model in mice. Toluene-sensitized animals had access to running wheels 1, 2, 4 or 24 h/day for 4 weeks; thereafter, locomotor sensitization expression was evaluated after a toluene challenge. RAS elements (ACE and ACE2 enzymes; AT1, AT2 and Mas receptors) expression was determined by Western blot in the VTA, NAc and PFCx of toluene-sensitized mice with and without exercise. Individual differences in toluene-induced locomotor sensitization development were observed. Access to wheel running 1 and 2 h/day reduced but 4 and 24 h/day completely blocked locomotor sensitization expression. Repeated toluene exposure changed RAS elements' expression in the VTA, NAc and PFCx, while exercise mainly modified ACE and AT1 in air-exposed and toluene-sensitized mice. Inhalant-exposed animals show different sensitization phenotypes. Exercise duration determined its efficacy to attenuate the addictive-like response. Toluene exposure and exercise each modified RAS, the latter also modifying toluene-induced changes.

Plasma angiotensin-converting enzyme 2 (ACE2) is a marker for renal outcome of diabetic kidney disease (DKD) (U-CARE study 3)

IntroductionACE cleaves angiotensin I (Ang I) to angiotensin II (Ang II) inducing vasoconstriction via Ang II type 1 (AT1) receptor, while ACE2 cleaves Ang II to Ang (1–7) causing vasodilatation...

Renin angiotensin system-induced muscle wasting: putative mechanisms and implications for clinicians.

Renin angiotensin system (RAS) alters various mechanisms related to muscle wasting. The RAS system consists of classical and non-classical pathways, which mostly function differently. Classical RAS pathway, operates through angiotensin II (AngII) and angiotensin type 1 receptors, is associated with muscle wasting and sarcopenia. On the other hand, the non-classical RAS pathway, which operates through angiotensin 1-7 and Mas receptor, is protective against sarcopenia. The classical RAS pathway might induce muscle wasting by variety of mechanisms. AngII reduces body weight, via reduction in food intake, possibly by decreasing hypothalamic expression of orexin and neuropeptide Y, insulin like growth factor-1 (IGF-1) and mammalian target of rapamycin (mTOR), signaling, AngII increases skeletal muscle proteolysis by forkhead box transcription factors (FOXO), caspase activation and muscle RING-finger protein-1 transcription. Furthermore, AngII infusion in skeletal muscle reduces phospho-Bad (Ser136) expression and induces apoptosis through increased cytochrome c release and DNA fragmentation. Additionally, Renin angiotensin system activation through AT1R and AngII stimulates tumor necrosis factor-α, and interleukin-6 which induces muscle wasting, Last but not least classical RAS pathway, induce oxidative stress, disturb mitochondrial energy metabolism, and muscle satellite cells which all lead to muscle wasting and decrease muscle regeneration. On the contrary, the non-classical RAS pathway functions oppositely to mitigate these mechanisms and protects against muscle wasting. In this review, we summarize the mechanisms of RAS-induced muscle wasting and putative implications for clinical practice. We also emphasize the areas of uncertainties and suggest potential research areas.

MRGPRX2 antagonist GE1111 attenuated DNFB-induced atopic dermatitis in mice by reducing inflammatory cytokines and restoring skin integrity.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches. We developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111. Our in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD.

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with β-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.