Anxiolytic effect of alamandine in male transgenic rats with low brain angiotensinogen is dependent on activation of MrgD receptors.

Alamandine is a peptide hormone belonging to the renin-angiotensin system (RAS). It acts through the Mas-related G-protein coupled receptor type D, MrgD, which is expressed in different tissues, including the brain. In the present study, we hypothesize that a lack of alamandine, through MrgD, could cause the anxiety-like behavior in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)680]. Adult male transgenic rats exhibited a significant increase in the latency to feeding time in the novelty suppressed feeding test and a decrease in the percentage of time and entries in the open arms in the elevated plus maze. These effects were reversed by intracerebroventricular infusion of alamandine. Pretreatment with D-Pro7-Ang-(1-7), a Mas and MrgD receptor antagonist, prevented the anxiolytic effects induced by this peptide. However, its effects were not altered by the selective Mas receptor antagonist, A779. In conclusion, our data indicates that alamandine, through MrgD, attenuates anxiety-like behavior in male TGR(ASrAOGEN)680, which reinforces the importance of the counter-regulatory RAS axis as promising target for the treatment of neuropsychiatric disorders.

The Expression of Alamandine Receptor MrgD in Clear Cell Renal Cell Carcinoma Is Associated with a Worse Prognosis and Unfavorable Response to Antiangiogenic Therapy.

Renal cell carcinoma (RCC) ranks among the most prevalent malignancies in Western countries, marked by its notable heterogeneity, which contributes to an unpredictable clinical trajectory. The insufficiency of dependable biomarkers adds complexity to assessing this tumor progression. Imbalances of several components of the intrarenal renin-angiotensin system (iRAS) significantly impact patient prognoses and responses to first-line immunotherapies. In this study, we analyzed the immunohistochemical expression of the Mas-related G-protein-coupled receptor D (MrgD), which recognizes the novel RAS peptide alamandine (ALA), in a series of 87 clear cell renal cell (CCRCCs), 19 papillary (PRCC), 7 chromophobe (ChRCC) renal cell carcinomas, and 11 renal oncocytomas (RO). MrgD was expressed in all the renal tumor subtypes, with a higher mean staining intensity in the PRCCs, ChRCCs, and ROs. A high expression of MrgD at the tumor center and at the infiltrative front of CCRCC tissues was significantly associated with a high histological grade, large tumor diameter, local invasion, and locoregional node and distant metastasis. Patients with worse 5-year cancer-specific survival and a poorer response to antiangiogenic tyrosine-kinase inhibitors (TKIs) showed higher MrgD expression at the center of their primary tumors. These findings suggest a possible role of MrgD in renal carcinogenetic processes. Further studies are necessary to unveil its potential as a novel biomarker for CCRCC prognosis and response to frontline therapies.

Effects of alamandine on monocrotaline-induced pulmonary hypertension in rats.

Objectives: Pulmonary arterial hypertension (PAH) is a severe and often fatal disease that is associated with oxidative stress and inflammation. Alamandine, a component of the renin-angiotensin system, known for its antioxidative, anti-inflammatory, and antifibrotic effects, has been investigated in this study to determine if it has protective effects against PAH induced by monocrotaline (MCT) and if these effects are associated with oxidative stress, inflammatory factors, and inducible nitric oxide synthase (iNOS). Materials and Methods: Rats were administered MCT (40 mg/kg) on day 0 and then received alamandine (50 mg/kg/day) via mini-osmotic pumps for 21 days starting one day later. Hemodynamic parameters, electrocardiograms, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), inflammatory cytokines (TNF-α, IL-1ß, and NF-κB), iNOS, and MrgD receptor expression in lung tissue were evaluated at the end of the 21-day period. The MrgD receptor was quantified through immunofluorescent staining, and the histopathology of lung tissues was evaluated using hematoxylin and eosin staining. Results: The results showed that alamandine treatment significantly improved hemodynamic parameters, oxidative stress markers, inflammatory factors, and electrocardiographic data. Furthermore, treatment with alamandine decreased the levels of iNOS. Additionally, alamandine treatment decreased the expression levels of MrgD receptors in the lung tissue of MCT-induced PAH. Conclusion: In summary, this study indicates that alamandine has protective effects against monocrotaline-induced PAH, and these effects may be attributed to the inhibition of oxidative stress, inflammatory parameters, and iNOS.

Alternative Renin-Angiotensin System.

The renin-angiotensin system is the most important peptide hormone system in the regulation of cardiovascular homeostasis. Its classical arm consists of the enzymes, renin, and angiotensin-converting enzyme, generating angiotensin II from angiotensinogen, which activates its AT1 receptor, thereby increasing blood pressure, retaining salt and water, and inducing cardiovascular hypertrophy and fibrosis. However, angiotensin II can also activate a second receptor, the AT2 receptor. Moreover, the removal of the C-terminal phenylalanine from angiotensin II by ACE2 (angiotensin-converting enzyme 2) yields angiotensin-(1-7), and this peptide interacts with its receptor Mas. When the aminoterminal Asp of angiotensin-(1-7) is decarboxylated, alamandine is generated, which activates the Mas-related G-protein-coupled receptor D, MrgD (Mas-related G-protein-coupled receptor type D). Since Mas, MrgD, and the AT2 receptor have opposing effects to the classical AT1 receptor, they and the enzymes and peptides activating them are called the alternative or protective arm of the renin-angiotensin system. This review will cover the historical aspects and the current standing of this recent addition to the biology of the renin-angiotensin system.

Novel Insights into the Cardioprotective Effects of the Peptides of the Counter-Regulatory Renin-Angiotensin System.

Currently, cardiovascular diseases are a major contributor to morbidity and mortality worldwide, having a significant negative impact on both the economy and public health. The renin-angiotensin system contributes to a high spectrum of cardiovascular disorders and is essential for maintaining normal cardiovascular homeostasis. Overactivation of the classical renin-angiotensin system is one of the most important pathophysiological mechanisms in the progression of cardiovascular diseases. The counter-regulatory renin-angiotensin system is an alternate pathway which favors the synthesis of different peptides, including Angiotensin-(1-7), Angiotensin-(1-9), and Alamandine. These peptides, via the angiotensin type 2 receptor (AT2R), MasR, and MrgD, initiate multiple downstream signaling pathways that culminate in the activation of various cardioprotective mechanisms, such as decreased cardiac fibrosis, decreased myocardial hypertrophy, vasodilation, decreased blood pressure, natriuresis, and nitric oxide synthesis. These cardioprotective effects position them as therapeutic alternatives for reducing the progression of cardiovascular diseases. This review aims to show the latest findings on the cardioprotective effects of the main peptides of the counter-regulatory renin-angiotensin system.

Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.

BACKGROUND: Alamandine is a newly characterized peptide of renin angiotensin system. Our study aims to investigate the osteo-preservative effects of alamandine, explore underlying mechanism and bring a potential preventive strategy for postmenopausal osteoporosis in the future. METHODS: An ovariectomy (OVX)-induced rat osteoporosis model was established for in vivo experiments. Micro-computed tomography and three-point bending test were used to evaluate bone strength. Histological femur slices were processed for immunohistochemistry (IHC). Bone turnover markers and nitric oxide (NO) concentrations in serum were determined with enzyme-linked immunosorbent assay (ELISA). The mouse embryo osteoblast precursor (MC3T3-E1) cells were used for in vitro experiments. The cell viability was analysed with a Cell Counting Kit­8. We performed Alizarin Red S staining and alkaline phosphatase (ALP) activity assay to observe the differentiation status of osteoblasts. Western blotting was adopted to detect the expression of osteogenesis related proteins and AMP-activated protein kinase/endothelial nitric oxide synthase (AMPK/eNOS) in osteoblasts. DAF-FM diacetate was used for semi-quantitation of intracellular NO. RESULTS: In OVX rats, alamandine alleviated osteoporosis and maintained bone strength. The IHC showed alamandine increased osteocalcin and collagen type I α1 (COL1A1) expression. The ELISA revealed alamandine decreased bone turnover markers and restored NO level in serum. In MC3T3-E1 cells, alamandine promoted osteogenic differentiation. Western blotting demonstrated that alamandine upregulated the expression of osteopontin, Runt-related transcription factor 2 and COL1A1. The intracellular NO was also raised by alamandine. Additionally, the activation of AMPK/eNOS axis mediated the effects of alamandine on MC3T3-E1 cells and bone tissue. PD123319 and dorsomorphin could repress the regulating effect of alamandine on bone metabolism. CONCLUSION: Alamandine attenuates ovariectomy-induced osteoporosis by promoting osteogenic differentiation via AMPK/eNOS axis.

Alamandine attenuates oxidative stress in the right carotid following transverse aortic constriction in mice.

OBJECTIVE: Pressure overload can result in significant changes to the structure of blood vessels, a process known as vascular remodeling. High levels of tension can cause vascular inflammation, fibrosis, and structural alterations to the vascular wall. Prior research from our team has demonstrated that the oral administration of alamandine can promote vasculoprotective effects in mice aorta that have undergone transverse aortic constriction (TAC). Furthermore, changes in local hemodynamics can affect the right and left carotid arteries differently after TAC. Thus, in this study, we aimed to assess the effects of alamandine treatment on right carotid remodeling and the expression of oxidative stress-related substances induced by TAC. METHODS AND RESULTS: Male C57BL/6 mice were categorized into three groups: Sham, TAC, and TAC treated with alamandine (TAC+ALA). Alamandine treatment was administered orally by gavage (30 µg/kg/day), starting three days before the surgery, and continuing for a period of fourteen days. Morphometric analysis of hematoxylin and eosin-stained sections revealed that TAC induced hypertrophic and positive remodeling in the right carotid artery. Picrosirius Red staining also demonstrated an increase in total collagen deposition in the right carotid artery due to TAC-induced vascular changes. Alamandine treatment effectively prevented the increase in reactive oxygen species production and depletion of nitric oxide levels, which were induced by TAC. Finally, alamandine treatment was also shown to prevent the increased expression of nuclear factor erythroid 2-related factor 2 and 3-nitrotyrosine that were induced by TAC. CONCLUSION: Our results suggest that alamandine can effectively attenuate pathophysiological stress in the right carotid artery of animals subjected to TAC.

Alamandine injection in the periaqueductal gray and rostral ventromedial medulla attenuates allodynia induced by sciatic nerve ligation in rats.

Alamandine, a peptide known to interact with Mas-related G protein-coupled receptor subtype D (MrgD), has been implicated in moderating inflammatory signals. MrgD receptors are abundantly found in pain transmission pathways, but the role of alamandine/MrgD in pain modulation has not been thoroughly explored. This study aimed to investigate the effects of alamandine (10, 40, and 100 pmol) in a rat model of allodynia induced by sciatic nerve ligation, with a specific focus on examining the involvement of MrgD receptors, NMDAR1, and serotonin transporter (SERT) in the ventrolateral periaqueductal gray (vlPAG) and rostral ventromedial medulla (RVM). Microinjection of alamandine into the vlPAG at a dose of 100 pmol and into the RVM at doses of 40 and 100 pmol resulted in a significant increase in paw withdrawal threshold (PWT). Additionally, co-administration of D-Pro7-Ang-(1-7) at 50 pmol, an MrgD receptor antagonist, effectively blocked the analgesic effects of alamandine. Immunofluorescence analysis confirmed the presence of MrgD receptors in both the vlPAG and RVM regions. Importantly, an upregulation of MrgD receptor expression was observed following allodynia induction, suggesting a potential compensatory mechanism in response to pain. Our findings support the co-localization of MrgD receptors with NMDAR1 in vlPAG neurons, suggesting their ability to initiate analgesic pathways similar to those activated by NMDA receptors in the vlPAG. Furthermore, our results underscore a significant co-localization of MrgD receptors with the SERT in the RVM, underscoring their potential impact on serotonergic neurons involved in promoting analgesic effects.

Alamandine treatment prevents LPS-induced acute renal and systemic dysfunction with multi-organ injury in rats via inhibiting iNOS expression.

Sepsis is defined as the dysregulated immune response leading to multi-organ dysfunction and injury. Sepsis-induced acute kidney injury is a significant contributor to morbidity and mortality. Alamandine (ALA) is a novel endogenous peptide of the renin-angiotensin-aldosterone system. It is known for its anti-inflammatory and anti-apoptotic effects, but its functional and vascular effects on sepsis remain unclear. We aimed to investigate the effects of ALA, as a pre- and post-treatment agent, on lipopolysaccharide (LPS)-induced systemic and renal dysfunction and injury in the LPS-induced endotoxemia model in rats via functional, hemodynamic, vascular, molecular, biochemical, and histopathological evaluation. 10 mg/kg intraperitoneal LPS injection caused both hepatic and renal injury, decreased blood flow in several organs, and renal dysfunction at 20 h in Sprague-Dawley rats. Our results showed that ALA treatment ameliorated systemic and renal inflammation, reduced inflammatory cytokines, prevented the enhancement of the mortality rate, reversed vascular dysfunction, corrected decreased blood flows in several organs, and reduced renal and hepatic injury via inhibiting iNOS (inducible nitric oxide synthase) and caspase expressions in the kidney. In addition, expressions of different ALA-related receptors showed alterations in this model, and ALA treatment reversed these alterations. These data suggest that ALA's systemic and renal protective effects are achieved through its anti-inflammatory, anti-pyroptotic, and anti-apoptotic effects on hemodynamic and vascular functions via reduced iNOS expression.

Subfatin, Asprosin, Alamandine and Maresin-1 Inflammation Molecules in Cardiopulmonary Bypass.

Purpose: Cardiopulmonary bypass (CPB) is a nonphysiological procedure in which inflammatory reactions and oxidative stress are induced, hormones and hemodynamic parameters are affected, and circulation is maintained outside the body. This study aimed to examine the effects of CPB on blood subfatin (SUB), asprossin (ASP), alamandine (ALA) and maresin-1 (MaR-1) levels. Materials and Methods: Controls and patients who underwent open-heart surgery with CPB and whose age and body mass indices were compatible with each other were included in the study. Venous blood samples were collected from CPB patients (n =19) before anesthesia induction (T1), before CPB (T2), 5 min before cross-clamp removal (T3), 5 min after cross-clamp removal (T4), when taken to the intensive care unit (T5), postoperative 24th hour (T6) and 72nd hour (T7) postoperatively. Venous blood was collected from the healthy controls (n =19). The amounts of SUB, ASP, ALA, and MaR-1 in the blood samples were measured using an Enzyme-Linked Immunosorbent Assay (ELISA). Results: The amounts of SUB and MaR-1 in the control group were significantly higher than those in CPB patients, while these parameters in T1-T3 blood gradually decreased in CPB patients (p<0.01). It was also reported that the amounts of ASP and ALA in the control group were significantly lower than those in CPB patients, whereas those parameters in the T1-T3 blood samples increased gradually in CPB patients, but started to decrease in T4-T7 blood samples. Conclusion: These hormonal changes in the organism due to CPB demonstrate that "hormonal metabolic adaptation" mechanisms may be activated to eliminate the negative consequences of surgery. According to these data, SUB, MaR-1, anti-alamandine, and anti-asprosin could be used in CPB surgeries may come to the fore in the future to increase the safety of CPB surgeries.

Nuclear import of Mas-related G protein-coupled receptor member D induces pathological cardiac remodeling.

Alamandine (Ala), a ligand of Mas-related G protein-coupled receptor, member D (MrgD), alleviates angiotensin II (AngII)-induced cardiac hypertrophy. However, the specific physiological and pathological role of MrgD is not yet elucidated. Here, we found that MrgD expression increased under various pathological conditions. Then, MrgD knockdown prevented AngII-induced cardiac hypertrophy and fibrosis via inactivating Gαi-mediacted downstream signaling pathways, including the phosphorylation of p38 (p-P38), while MrgD overexpression induced pathological cardiac remodeling. Next, Ala, like silencing MrgD, exerted its cardioprotective effects by inhibiting Ang II-induced nuclear import of MrgD. MrgD interacted with p-P38 and promoted its entry into the nucleus under Ang II stimulation. Our results indicated that Ala was a blocking ligand of MrgD that inhibited downstream signaling pathway, which unveiled the promising cardioprotective effect of silencing MrgD expression on alleviating cardiac remodeling. Video Abstract.

Alamandine/MrgD axis prevents TGF-ß1-mediated fibroblast activation via regulation of aerobic glycolysis and mitophagy.

BACKGROUND: Idiopathic pulmonary fibrosis is a chronic progressive, lethal disease in which ectopic lung fibroblast (LF) activation plays a vital part. We have previously shown that alamandine (ALA) exerts anti-fibrosis effects via the MAS-related G-protein coupled receptor D (MrgD). Here, we further investigate how it moderates transforming growth factor ß1 (TGF-ß1)-induced LF activation by regulating glucose metabolism and mitochondria autophagy (mitophagy). METHODS: In vitro, we examined glycolysis-related protein hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), and lactic acid in cells treated with TGF-ß1. The oxygen consumption rate and the extracellular acidification rate were detected using Seahorse assays. Then, mitophagy was evaluated using transmission electron microscopy, mt-Keima, and the co-localization of Parkin and COX IV with LC3 and LAMP1, respectively. The autophagic degradation of HK2 and PFKFB3 was detected by 3MA and bafilomycin A1 and assessed by their co-localization with LC3 and LAMP1, respectively. The effects of ALA on LF activation markers collagen I and α-SMA were detected. The effects of ALA on glucose metabolism, mitophagy, and the activation of LF were also investigated in vivo. RESULTS: We found that the ALA/MrgD axis improved TGF-ß1-mediated LF activation by repressing glycolysis by downregulating HK2 and PFKFB3 expression. Lactic acid sustained positive feedback between glycolysis and LF activation by maintaining the expression of HK2 and PFKFB3. We also showed that glycolysis enhancement resulted from blocking the autophagic degradation of HK2 and PFKFB3 while upregulated mRNA levels by TGF-ß1, while all of those improved by ALA adding. Importantly, we determined that moderation of Parkin/LC3-mediated mitophagy by TGF-ß1 also promotes glycolysis but is reversed by ALA. Furthermore, we proved that ALA counteracts the effects of bleomycin on HK2, PFKFB3, LC3, Parkin, and LF activation in vivo. CONCLUSION: In this study, we show that the ALA/MrgD axis prevents TGF-ß1-mediated fibroblast activation via regulation of aerobic glycolysis and mitophagy.

Brown adipose tissue transcriptome unveils an important role of the Beta-alanine/alamandine receptor, MrgD, in metabolism.

Alamandine is a recently described heptapeptide component of the renin-angiotensin system (RAS), and its effects are mediated by the receptor Mas-related G protein-coupled receptor D (MrgD) RAS represents an important link between obesity and its consequences by directly modulating the thermogenesis and brown adipose tissue (BAT) function. The alamandine/MrgD metabolic effects and signaling remain unexplored. In this context, the main goal of the present study was to assess the metabolic consequences of MrgD genetic ablation in C57BL6/J mice by evaluating brown adipose tissue RNA sequencing. The main results showed that MrgD-KO mice have diminished brown adipose tissue and that a high-glucose diet (HG) decreased both circulating alamandine levels and MrgD expression in BAT from wild-type mice (WT). BAT transcriptome reveals that MrgD-KO HG mice regulated 45 genes, while WT HG mice regulated 1,148 genes. MrgD-KO mice fed a standard diet (ST) compared with WT ST mice regulated 476 genes, of which 445 genes were downregulated. BAT uses the MrgD receptor to display a normal pattern of gene expression and to respond, like WT mice, to an HG diet. In conclusion, the MrgD signaling is important for the metabolic regulation and manutention of BAT functionality.

An overview of alamadine/MrgD signaling and its role in cardiomyocytes.

The renin-angiotensin system (RAS) is a classical hormonal system involved in a myriad of cardiovascular functions. This system is composed of many different peptides that act in the heart through different receptors. One of the most important of these peptides is angiotensin II, which in pathological conditions triggers a set of actions that lead to heart failure. On the other hand, another RAS peptide, angiotensin-(1-7) is well known to develop powerful therapeutic effects in many forms of cardiac diseases. In the last decade, two new components of RAS were described, the heptapeptide alamandine and its receptor, the Mas-related G protein-coupled receptor member D (MrgD). Since then, great effort was made to characterize their physiological and pathological function in the heart. In this review, we summarize the latest insights about the actions of alamandine/MrgD axis in the heart, with particular emphasis in the cardiomyocyte. More specifically, we focused on their antihypertrophic and contractility effects, and the related molecular events activated in the cardiomyocyte.

Activation of the Mas receptors by AVE0991 and MrgD receptor using alamandine to limit the deleterious effects of Ang II-induced hypertension.

The MrgD receptor agonist, alamandine (ALA) and Mas receptor agonist, AVE0991 have recently been identified as protective components of the renin-angiotensin system. We evaluated the effects of ALA and AVE0991 on cardiovascular function and remodeling in angiotensin (Ang) II-induced hypertension in rats. Sprague Dawley rats were subject to 4-week subcutaneous infusions of Ang II (80 ng/kg/min) or saline after which they were treated with ALA (50 µg/kg), AVE0991 (576 µg/kg), or ALA+AVE0991 during the last 2 weeks. Systolic blood pressure (SBP) and heart rate (HR) values were recorded with tail-cuff plethysmography at 1, 15, and 29 days post-treatment. After euthanization, the heart and thoracic aorta were removed for further analysis and vascular responses. SBP significantly increased in the Ang II group when compared to the control group. Furthermore, Ang II also caused an increase in cardiac and aortic cyclophilin-A (CYP-A), monocyte chemoattractant protein-1 (MCP-1), and cardiomyocyte degeneration but produced a decrease in vascular relaxation. HR, matrix metalloproteinase-2 and -9, NADPH oxidase-4, and lysyl oxidase levels were comparable among groups. ALA, AVE0991, and the drug combination produced antihypertensive effects and alleviated vascular responses. The inflammatory and oxidative stress related to cardiac MCP-1 and CYP-A levels decreased in the Ang II+ALA+AVE0991 group. Vascular but not cardiac angiotensin-converting enzyme-2 levels decreased with Ang II administration but were similar to the Ang II+ALA+AVE0991 group. Our experimental data showed the combination of ALA and AVE0991 was found beneficial in Ang II-induced hypertension in rats by reducing SBP, oxidative stress, inflammation, and improving vascular responses.

Alamandine alleviated heart failure and fibrosis in myocardial infarction mice.

Alamandine (Ala) is the newest identified peptide of the renin-angiotensin system and has protective effect on myocyte hypertrophy. However, it is still unclear whether Ala can alleviate heart failure (HF). The aim of this study was to explore the effects of Ala on HF and the related cardiac fibrosis, and to probe the mechanism. HF model was induced by myocardial infarction (MI) in mice. Four weeks after MI, Ala was administrated by intraperitoneal injection for two weeks. Ala injection significantly improved cardiac dysfunction of MI mice in vivo. The cardiac fibrosis and the related biomarkers were attenuated after Ala administration in HF mice in vivo. The increases of collagen I, alpha-smooth muscle actin and transforming growth factor-beta induced by oxygen-glucose deprivation (OGD) in neonatal rat cardiac fibroblasts (NRCFs) were inhibited by Ala treatment in vitro. The biomarkers of apoptosis were elevated in NRCFs induced by OGD, which were attenuated after treating with Ala in vitro. The enhancement of oxidative stress in the heart of MI mice or in the NRCFs treated with OGD was suppressed by treating with Ala in vivo and in vitro. These effects of Ala were reversed by tBHP, an exogenous inducer of oxidative stress in vitro. These results demonstrated that Ala could alleviate cardiac dysfunction and attenuate cardiac fibrosis via inhibition of oxidative stress.

Alamandine: A promising treatment for fibrosis.

Angiotensin (Ang) II, the main active member of the renin angiotensin system (RAS), is essential for the maintenance of cardiovascular homeostasis. However, hyperactivation of the RAS causes fibrotic diseases. Ang II has pro-inflammatory actions, and moreover activates interstitial fibroblasts and/or dysregulates extracellular matrix degradation. The discovery of new RAS pathways has revealed the complexity of this system. Among the RAS peptides, alamandine (ALA, Ala1 Ang 1-7) has been identified in humans, rats, and mice, with protective actions in different pathological conditions. ALA has similar effects to its well-known congener, Ang-(1-7), as a vasodilator, anti-inflammatory, and antifibrotic. Its protective role against cardiovascular diseases is well-reviewed in the literature. However, the protective actions of ALA in fibrotic conditions have been little explored. Therefore, in this article, we review the ability of ALA to modulate the inflammatory process and collagen deposition, to serve as an antioxidant, and to mediate protection against functional disorders. In this scenario, we also explore ALA as a promising therapy for pulmonary fibrosis after COVID-19 infection.

Phosphoproteomic studies of alamandine signaling in CHO-MrgD and human pancreatic carcinoma cells: An antiproliferative effect is unveiled.

Alamandine is a heptapeptide from the renin-angiotensin system (RAS) with similar structure/function to angiotensin-(1-7) [ang-(1-7)], but they act via different receptors. It remains elusive whether alamandine is an antiproliferative agent like ang-(1-7). The goal of this study was to evaluate the potential antiproliferative activity of alamandine and the underlying cellular signaling. We evaluated alamandine effect in the tumoral cell lines Mia PaCa-2 and A549, and in the nontumoral cell lines HaCaT, CHO and CHO transfected with the alamandine receptor MrgD (CHO-MrgD). Alamandine was able to reduce the proliferation of the tumoral cell lines in a MrgD-dependent fashion. We did not observe any effect in the nontumoral cell lines tested. We also performed proteomics and phosphoproteomics to study the alamandine signaling in Mia PaCa-2 and CHO-MrgD. Data suggest that alamandine induces a shift from anaerobic to aerobic metabolism in the tumoral cells, induces a negative regulation of PI3K/AKT/mTOR pathway and activates the transcriptional factor FoxO1; events that could explain, at least partially, the observed antiproliferative effect of alamandine. This study provides for the first time a comprehensive investigation of the alamandine signaling in tumoral (Mia PaCa-2) and nontumoral (CHO-MrgD) cells, highlighting the antiproliferative activity of alamandine/MrgD and its possible antitumoral effect.

Alamandine, a new member of the renin-angiotensin system (RAS), attenuates collagen-induced arthritis in mice via inhibiting cytokine secretion in synovial fibroblasts.

Alamandine is a novel component of the renin-angiotensin system (RAS) as well as an important biologically active peptide. It has predominantly been studied in cardiovascular context. However, its role in rheumatoid arthritis (RA) remains unknown. Here we illustrated its effects on inflammatory cytokines production by synovial fibroblasts from RA and pathological changes in collagen-induced arthritis (CIA) mice. Alamandine (0.1, 1 and 10 µg/ml) did not affect the survival of the synovial fibroblasts, but decreased the migration and proinflammatory cytokines expression in TNF-α (10 ng/ml) stimulated cells in vitro. Additionally, alamandine selectively decreased phosphorylated-JNK expression induced by TNF-a stimulation in RA FLS. DBA/1 J mice were induced arthritis by a primary injection with an emulsion of bovine type II collagen (CII) and complete Freund's adjuvant (day 0) and a booster injection of CII in incomplete Freund's adjuvant (day 21). Mice were then given alamandine intraperitoneally in saline (50 µg/kg/day) from days 21-42. Histology and multiplex immunobead assay showed that alamandine treatment inhibited the development of arthritis and reduced the joint damage. This effect was accompanied by the reduced inflammatory cytokines (IL-6, IL-23, IFN-γ) mRNA expression in local joints, the decreased TNF-α, IL-6, IL-17 and the increased IL-10 levels in the serum from alamandine administrated CIA mice. In conclusion, alamandine attenuates the development of arthritis by suppressing inflammatory cytokines expression in RA synovial fibroblasts via MAPK signaling pathway, suggesting a potential therapeutic role for RA.