Arginase Inhibitor Research Articles

Arginase: Biological and Therapeutic Implications in Diabetes Mellitus and Its Complications.

Arginase is a ubiquitous enzyme in the urea cycle (UC) that hydrolyzes L-arginine to urea and L-ornithine. Two mammalian arginase isoforms, arginase1 (ARG1) and arginase2 (ARG2), play a vital role in the regulation of β-cell functions, insulin resistance (IR), and vascular complications via modulating L-arginine metabolism, nitric oxide (NO) production, and inflammatory responses as well as oxidative stress. Basic and clinical studies reveal that abnormal alterations of arginase expression and activity are strongly associated with the onset and development of diabetes mellitus (DM) and its complications. As a result, targeting arginase may be a novel and promising approach for DM treatment. An increasing number of arginase inhibitors, including chemical and natural inhibitors, have been developed and shown to protect against the development of DM and its complications. In this review, we discuss the fundamental features of arginase. Next, the regulatory roles and underlying mechanisms of arginase in the pathogenesis and progression of DM and its complications are explored. Furthermore, we review the development and discuss the challenges of arginase inhibitors in treating DM and its related pathologies.

Thin-cap fibroatheroma and increased coronary intima-media thickness are associated with an altered balance of arginine metabolites.

Arginase inhibition increases plasma citrulline and citrulline / ornithine (C/O) ratio, and reduces plasma ornithine and ornithine / arginine (O/A) ratio in an animal model of myocardial infarction (MI). We hypothesized that the presence of thin‑cap fibroatheroma (TCFA) in the culprit lesion and increased non‑culprit intima‑media thickness of an infarct‑related artery (IRA) are associated with an altered balance of arginine metabolites. Arginine and its metabolites were measured using liquid chromatography and tandem mass spectrometry in 100 consecutive MI patients upon admission and at 6‑month follow‑up. TCFA and adjacent to culprit lesion proximal and distal 10‑mm segments were assessed with optical coherence tomography in the acute phase. Twenty five patients without coronary lesions on angiography served as controls. The C/O ratio increased 5.33 times (P <0.001), while the O/A ratio decreased 2.53 times (P <0.001) at the 6‑month follow‑up, as compared with the acute phase of MI. The patients with (n = 75) vs without (n = 25) TCFA had lower C/O ratio by 29% (P = 0.003), while the mean intima‑media diameter of adjacent non‑culprit region correlated with the follow‑up O/A ratio (R = 0.337; P = 0.003). In a multivariable analysis, a higher acute phase C/O ratio was associated with a lower risk of TCFA presence (odds ratio, 0.978; 95% CI, 0.962-0.994; P = 0.006), whereas a higher follow‑up O/A ratio correlated with larger intima‑media diameter of the adjacent segments (β coefficient, 0.227; 95% CI for β coefficient, 0.045-0.409; P = 0.018). Enhanced arginase activity over nitric oxide synthase following ischemia was associated with the presence of TCFA in the culprit lesion, while a similar metabolic shift in the chronic phase correlated with a greater thickness of the intima‑media in the adjacent non‑culprit IRA segments.

Arginase 1 in erythrocytes is a critical modulator of cardioprotective signalling by nitric oxide and soluble guanylyl cyclase

Abstract Background Arginase is involved in the development of ischemia-reperfusion injury by regulating nitric oxide (NO) bioactivity via competition with NO synthase (NOS) for their common substrate L-arginine. Erythrocytes are known to contain high levels of arginase that may reduce export of cardioprotective NO bioactivity. Aim To determine the role of arginase 1 in erythrocytes for cardiac protection against myocardial ischemia-reperfusion injury. Methods A tie2 cre-flox mouse model, in which arginase 1 was deleted (Arg 1-KO) in hematopoietic and endothelial cells, was developed. In vivo ischemia-reperfusion was performed in anaesthetized mice by left anterior descending coronary artery ligation (30 min ischemia and 120 min reperfusion). To determine the specific role of erythrocytes, an isolated perfused mouse heart model was applied. Erythrocytes from Arg 1-KO and wild type (WT) mice were given to isolated hearts from WT mice at the onset of global ischemia. After 40 min ischemia, the recovery of left ventricular developed pressure (LVDP) during 60 min reperfusion was recorded as an indicator of cardiac functional recovery. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996) Results Following in vivo ischemia-reperfusion, infarct size was smaller in Arg 1-KO mice than in WT mice (Fig. A). A similar degree of infarct size reduction was obtained by i.v. administration of an arginase inhibitor in WT mice. The cardioprotective effect observed in Arg 1-KO mice was abolished by the NOS inhibitor L-NMMA (Fig. A). In isolated buffer-perfused hearts, there was no difference in the post-ischemic recovery of LVDP between Arg 1-KO and WT hearts. When erythrocytes from Arg 1-KO mice were administrated to isolated WT mouse hearts, the post-ischemic recovery of LVDP was significantly improved compared to hearts given erythrocytes from WT mice. Also, the cardioprotective effect of erythrocytes from Arg 1-KO mice was abolished by pre-incubation of the erythrocytes with the NOS inhibitor L-NAME (Fig. B) or the inhibitor of soluble guanylyl cyclase ODQ (Fig. C). Conclusion Arginase 1 in erythrocyte plays an important role in regulating cardioprotection mediated via the NO-soluble guanylyl cyclase pathway. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation (20190266),The Swedish Research Council (2020-01372)

There is no direct competition between arginase and nitric oxide synthase for the common substrate l-arginine

AimsExtrahepatic arginases are postulated to be involved in cardiovascular-related pathologies by competing with nitric oxide synthase (NOS) for the common substrate l-arginine, subsequently decreasing nitric oxide production. However, previous models used to study arginase and NOS competition did not account for steady state level of l-arginine pool, which is dependent on conditions of l-arginine supply and utilization pathways. This work aimed at revisiting the concept of NOS and arginase competition while considering different conditions of l-arginine supply and l-arginine utilization pathways. Methods and resultsMouse macrophage-like RAW cells and human vascular endothelial cells co-expressing NOS and arginase were used to reevaluate the concept of substrate competition between arginase and NOS under conditions of l-arginine supply that mimicked either a continuous (similar to in vivo conditions) or a limited supply (similar to previous in vitro models). Enzyme kinetics simulation models were used to gain mechanistic insight and to evaluate the tenability of a substrate competition between the two enzymes. In addition to arginase and NOS, other l-arginine pathways such as transporters and utilization towards protein synthesis were considered to understand the intricacies of l-arginine metabolism. Our results indicate that when there is a continuous supply of l-arginine, as is the case for most cells in vivo, arginase does not affect NOS activity by a substrate competition. Furthermore, we demonstrate that l-arginine pathways such as transporters and protein synthesis are more likely to affect NOS activity than arginase. ConclusionsArginase does not outcompete NOS for the common substrate l-arginine. Findings from this study should be considered to better understand the role of arginase in certain pathologies and for the interpretation of in vivo studies with arginase inhibitors.

Preclinical evidence of a direct pro-survival role of arginine deprivation in multiple myeloma.

Multiple myeloma grows by establishing multiple interactions with bone marrow cells. These include expansion of myeloid-derived suppressor cells, which drive immunoevasion via mechanisms that include arginase-1-driven depletion of L-arginine, thus indirectly promoting myeloma cell survival and tumor progression. The peculiar biology of malignant plasma cells postulates that arginine depletion may benefit their fitness also directly, e.g., by engaging the integrated stress response, or by stimulating autophagy through mTORC1 inhibition. We thus investigated the direct impact of arginine deprivation on myeloma cells and challenged its pathophysiological relevance in vitro and in vivo. First, we found that partial arginine depletion spared proliferation of human multiple myeloma cells at concentrations that arrest human T cells. Next, we asked if arginine shortage activates putative adaptive pathways in myeloma cells. Low arginine failed to activate the integrated stress response, as indicated by unmodified phosphorylation of the eukaryotic initiation factor 2α, but sizably inhibited mTORC1, as revealed by reduced phosphorylation of ribosomal protein S6. Notably, depressed mTORC1 activity was not sufficient to increase autophagy, as assessed by the lysosomal digestion rate of the autophagosome-associated protein, LC3-II. Rather, it stimulated mTORC2, resulting in increased phosphatidylinositol-3 kinase-dependent AKT phosphorylation and activity, leading to heightened inhibitory phosphorylation of the pro-apoptotic BAD protein. We then tested whether arginine depletion-activated AKT may protect malignant plasma cells from cell death. Indeed, culturing myeloma cells in low arginine medium significantly reduced the apoptotic effect of the first-in-class proteasome inhibitor, bortezomib, an outcome prevented by pharmacological inhibition of AKT phosphorylation. Finally, we challenged the relevance of the identified circuit in vivo. To gauge the pathophysiologic relevance of low arginine to myeloma growth independently of immunoevasion, we xenotransplanted human myeloma cells subcutaneously into T cell-deficient Rag2–/–γc–/– recipient mice and treated palpable tumor-bearing mice with the clinical-grade arginase inhibitor CB1158. Arginase inhibition significantly raised serum arginine concentration, reduced tumor growth by caliper assessment, and decreased intra-tumor AKT phosphorylation in vivo. Altogether, our results reveal a novel direct pro-survival effect of arginine deprivation on myeloma cells, with potential therapeutic implications.

In Vitro and In Silico Analyses of New Cinnamid and Rosmarinic Acid-Derived Compounds Biosynthesized in Escherichia coli as Leishmania amazonensis Arginase Inhibitors.

Arginase is a metalloenzyme that plays a central role in Leishmania infections. Previously, rosmarinic and caffeic acids were described as antileishmanial agents and as Leishmania amazonensis arginase inhibitors. Here, we describe the inhibition of arginase in L. amazonensis by rosmarinic acid analogs (1–7) and new caffeic acid-derived amides (8–10). Caffeic acid esters and amides were produced by means of an engineered synthesis in E. coli and tested against L. amazonensis arginase. New amides (8–10) were biosynthesized in E. coli cultured with 2 mM of different combinations of feeding substrates. The most potent arginase inhibitors showed Ki(s) ranging from 2 to 5.7 μM. Compounds 2–4 and 7 inhibited L. amazonensis arginase (L-ARG) through a noncompetitive mechanism whilst compound 9 showed a competitive inhibition. By applying an in silico protocol, we determined the binding mode of compound 9. The competitive inhibitor of L-ARG targeted the key residues within the binding site of the enzyme, establishing a metal coordination bond with the metal ions and a series of hydrophobic and polar contacts supporting its micromolar inhibition of L-ARG. These results highlight that dihydroxycinnamic-derived compounds can be used as the basis for developing new drugs using a powerful tool based on the biosynthesis of arginase inhibitors.

Vanillic Acid Attenuates Monocrotaline-Induced Pulmonary Arterial Hypertension by Enhancing NO Signaling Pathways

Pulmonary arterial hypertension (PAH) is a severe progressive disease characterized by elevated mean pulmonary arterial pressure, right ventricular hypertrophy, and eventual progression to right heart failure and death. This study aimed to examine the effect of the natural product vanillic acid (VA) on monocrotaline (MCT)-induced PAH in rats. The arginase inhibitory activity and enzyme kinetic reaction of VA were also investigated. The results showed that VA could improve pulmonary arterial pressure, pulmonary artery vascular remodeling, and right ventricular remodeling induced by MCT in rats and reduce the degree of pulmonary tissue fibrosis. Moreover, VA downregulated the gene and protein expression levels of Hif-2α, Hif-1β, and Arg2 and increased the P-eNOS/eNOS levels, thus increasing nitric oxide (NO) levels in PAH rats. Furthermore, VA was determined to be a mixed competitive arginase inhibitor with an IC50 of 26.1 μM. In conclusion, the arginase inhibitor VA exerted protective effects on MCT-induced PAH and pulmonary vascular remodeling by enhancing NO signaling pathways.

1689P OATD-02 validates the benefits of pharmacological inhibition of arginase 1 and 2 in cancer

Arginases play important roles in the metabolic pathways determining the fitness of both immune and tumour cells. ARG1 expressed by the myeloid cells in the tumour microenvironment (TME) contributes to the suppression of the effector functions of T and NK cells by a local L-arginine depletion, while ARG2 constitutes a cell-autonomous and regulator of cytotoxic and regulatory T lymphocytes. ARG2 is highly expressed in various cancers and its significant role in cancer has recently emerged as ARG2 levels correlate with malignant phenotype and poor prognosis.

OATD-02 Validates the Benefits of Pharmacological Inhibition of Arginase 1 and 2 in Cancer.

Simple SummaryArginase 1 and 2 are drivers of multiple immunosuppressive mechanisms and tumour-specific metabolic adaptations. Pharmacological inhibition of extracellular ARG1 has shown antitumour efficacy in various syngeneic tumour models, however, the importance of ARG2 as a therapeutic target has only been demonstrated by genetic deletion studies. This is the first study validating the benefits of pharmacological inhibition of ARG2 in cancer. Our work describes OATD-02 as a potent dual ARG1/ARG2 inhibitor with a cellular activity (necessary for targeting ARG2) exhibiting immunomodulatory and direct antitumour efficacy in animal models. Our results present OATD-02 as an attractive option for combination with other immunotherapeutics, such as PD-1/PD-L1 antibodies or IDO1 inhibitors, especially in the therapy of particularly resistant hypoxic tumours. The presented findings provided the rationale for planning first-in-human clinical trials for OATD-02 in cancer patients.Background: Arginases play essential roles in metabolic pathways, determining the fitness of both immune and tumour cells. Along with the previously validated role of ARG1 in cancer, the particular significance of ARG2 as a therapeutic target has emerged as its levels correlate with malignant phenotype and poor prognosis. These observations unveil arginases, and specifically ARG2, as well-validated and promising therapeutic targets. OATD-02, a new boronic acid derivative, is the only dual inhibitor, which can address the benefits of pharmacological inhibition of arginase 1 and 2 in cancer. Methods: The inhibitory activity of OATD-02 was determined using recombinant ARG1 and ARG2, as well as in a cellular system using primary hepatocytes and macrophages. In vivo antitumor activity was determined in syngeneic models of colorectal and kidney carcinomas (CT26 and Renca, respectively), as well as in an ARG2-dependent xenograft model of leukaemia (K562). Results: OATD-02 was shown to be a potent dual (ARG1/ARG2) arginase inhibitor with a cellular activity necessary for targeting ARG2. Compared to a reference inhibitor with predominant extracellular activity towards ARG1, we have shown improved and statistically significant antitumor efficacy in the CT26 model and an immunomodulatory effect reflected by Treg inhibition in the Renca model. Importantly, OATD-02 had a superior activity when combined with other immunotherapeutics. Finally, OATD-02 effectively inhibited the proliferation of human K562 leukemic cells both in vitro and in vivo. Conclusions: OATD-02 is a potent small-molecule arginase inhibitor with optimal drug-like properties, including PK/PD profile. Excellent activity against intracellular ARG2 significantly distinguishes OATD-02 from other arginase inhibitors. OATD-02 represents a very promising drug candidate for the combined treatment of tumours, and is the only pharmacological tool that can effectively address the benefits of ARG1/ARG2 inhibition. OATD-02 will enter clinical trials in cancer patients in 2022.

Virtual Screening for FDA-Approved Drugs That Selectively Inhibit Arginase Type 1 and 2.

Arginases are often overexpressed in human diseases, and they are an important target for developing anti-aging and antineoplastic drugs. Arginase type 1 (ARG1) is a cytosolic enzyme, and arginase type 2 (ARG2) is a mitochondrial one. In this study, a dataset containing 2115-FDA-approved drug molecules is virtually screened for potential arginase binding using molecular docking against several ARG1 and ARG2 structures. The potential arginase ligands are classified into three categories: (1) Non-selective, (2) ARG1 selective, and (3) ARG2 selective. The evaluated potential arginase ligands are then compared with their clinical use. Remarkably, half of the top 30 potential drugs are used clinically to lower blood pressure and treat cancer, infection, kidney disease, and Parkinson’s disease thus partially validating our virtual screen. Most notable are the antihypertensive drugs candesartan, irbesartan, indapamide, and amiloride, the antiemetic rolapitant, the anti-angina ivabradine, and the antidiabetic metformin which have minimal side effects. The partial validation also favors the idea that the other half of the top 30 potential drugs could be used in therapeutic settings. The three categories greatly expand the selectivity of arginase inhibition.

Epithelial arginase-1 is a key mediator of age-associated delayed healing in vaginal injury.

Pelvic organ prolapse is a disorder that substantially affects the quality of life of millions of women worldwide. The greatest risk factors for prolapse are increased parity and older age, with the largest group requiring surgical intervention being post-menopausal women over 65. Due to ineffective healing in the elderly, prolapse recurrence rates following surgery remain high. Therefore, there is an urgent need to elucidate the cellular and molecular drivers of poor healing in pelvic floor dysfunction to allow effective management and even prevention. Recent studies have uncovered the importance of Arginase 1 for modulating effective healing in the skin. We thus employed novel in vitro and in vivo vaginal injury models to determine the specific role of Arginase 1 in age-related vaginal repair. Here we show, for the first time, that aged rat vaginal wounds have reduced Arginase 1 expression and delayed healing. Moreover, direct inhibition of Arginase 1 in human vaginal epithelial cells also led to delayed scratch-wound closure. By contrast, activation of Arginase 1 significantly accelerated healing in aged vaginal wounds in vivo, to rates comparable to those in young animals. Collectively, these findings reveal a new and important role for Arginase 1 in mediating effective vaginal repair. Targeting age-related Arginase 1 deficiency is a potential viable therapeutic strategy to promote vaginal healing and reduce recurrence rate after surgical repair of pelvic organ prolapse.

An Update on Arginase Inhibitors and Inhibitory Assays.

Arginase, which converts arginine into ornithine and urea, is a promising therapeutic target. Arginase is involved in cardiovascular diseases, parasitic infections and through a critical role in immunity, in some cancers. There is a need to develop effective arginase inhibitors and therefore efforts to identify and optimize new inhibitors are increasing. Several methods of evaluating arginase activity are available, but few directly measure the product. Radiometric assays need to separate urea and dying reactions require acidic conditions and sometimes heating. Hence, there are a variety of different approaches available, and each approach has its own limits and benefits. In this review, we provide an update on arginase inhibitors, followed by a discussion on available arginase assays and alternative methods, focusing on the intrinsic biases and parameters that are likely to impact results.

Effects of arginase inhibition on myocardial Ca2+ and contractile responses.

Nitric oxide (NO) is thought to increase cardiac contractility by increasing cytosolic Ca2+ concentration ([Ca2+]cyt) during excitation. Alternatively, NO could increase the sensitivity of the contractile response to [Ca2+]cyt (Ca2+ sensitivity). Arginase regulates NO production by competing with NO synthase (NOS), and thus, arginase inhibition should increase cardiac contractility by increasing NO production. We hypothesized that arginase inhibition increases cardiac contractility by increasing both [Ca2+]cyt and Ca2+ sensitivity. [Ca2+]cyt and contractile (sarcomere length [SL] shortening) responses to electrical stimulation were measured simultaneously in isolated rat cardiomyocytes using an IonOptix system. In the same cardiomyocytes, measurements were obtained at baseline, following 3‐min exposure to an arginase inhibitor (S‐[2‐boronoethyl]‐l‐cysteine; BEC) and following 3‐min exposure to BEC plus a NOS inhibitor (N G‐nitro‐l‐arginine‐methyl ester; l‐NAME). These responses were compared to time‐matched control cardiomyocytes that were untreated. Compared to baseline, BEC increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, whereas addition of l‐NAME mitigated these effects. The [Ca2+]cyt at 50% contraction and relaxation were not different across treatment groups indicating no effect of BEC on Ca2+ sensitivity. The [Ca2+]cyt and SL shortening responses in time‐matched controls did not vary with time. Arginase inhibition by BEC significantly increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, but did not affect Ca2+ sensitivity. These effects of BEC were mitigated by l‐NAME. Together, these results indicate an effect of NO on [Ca2+]cyt responses that then increase the contractile response of cardiomyocytes.

Catalytic mechanism of DcsB: Arginase framework used for hydrolyzing its inhibitor.

DcsB, an enzyme produced from the d-cycloserine biosynthetic gene cluster, displays moderate similarity to arginase in the sequence and three-dimensional structure. Arginase is a ubiquitous enzyme hydrolyzing l-arginine to generate l-ornithine and urea, whereas DcsB hydrolyzes Nω -hydroxy-l-arginine (l-NOHA), an arginase inhibitor, to generate l-ornithine and hydroxyurea. We determined the crystal structure of DcsB associated with l-ornithine and that with the tetrahedral derivative of 2(S)-amino-6-boronohexanoic acid, whose boron atom forms a covalent bond with an oxygen atom bridging two manganese ions at the active center. The substrate-binding pocket of DcsB is narrower than that of arginase, suggesting that DcsB is unsuitable for the binding of l-NOHA in an inhibitory manner. The transition state-like structure demonstrated that Asp210 and Glu241 have a role to trap a positively charged ion near the dimanganese cluster. Kinetic analysis using the mutated DcsB showed that the enzyme employs different catalytic mechanisms under the neutral and alkaline pH conditions. Glu241 in DcsB is likely involved in the recognition of the hydroxyguanidino group of l-NOHA, whereas Asp210, in cooperation with Glu241, seems to contribute to the reactivity toward the protonated l-NOHA, which is a preferable species under the neutral pH conditions. After entering of the protonated l-NOHA to the substrate-binding pocket of DcsB, a hydronium ion may be trapped at the positive ion-binding site. Then, the ion serves as a specific acid catalyst to facilitate the collapse of the tetrahedral intermediate of l-NOHA.

In vitro Inhibition of Dipeptidyl Peptidase-4 (DPP-4) and Arginase by a Dietary Supplement and Its Potential for Improving Quality of Life Factors in Adults

Aim: Somatopause is the progressive and age-related decline of growth hormone (GH) production due to changes in hypothalamic-pituitary axis signaling and results in typical physiological changes associated with the aging process. The purpose of our study was to investigate the anti-aging potential of several botanical ingredients, including noni fruit juice, as well as a dietary supplement formulation.&#x0D; Methodology: Dipeptidyl peptidase-4 (DPP-4) influences hypothalamic-pituitary axis signaling and reduces GH production and secretion. Since DPP-4 concentrations increase with age, we evaluated the ability of several botanical ingredients to inhibit DPP-4 activity in vitro. We subsequently formulated an anti-aging dietary supplement with this blend of botanical ingredients by adding amino acids (L-arginine, L-Lysine, citrulline and beta-alanine), gamma-aminobutyric acid (GABA), flax seed oil and several nutrients. The supplement was evaluated for anti-arginase activity in vitro, as the expression of this enzyme also increases with age. The dietary supplement was also consumed by three men and one woman, ages 56 to 62, with relevant but disparate medical histories. Following short-term ingestion of the supplement, these volunteers were interviewed to ascertain any changes in health status.&#x0D; Results: We discovered that a blend of the botanical ingredients synergistically inhibited DPP-4 in vitro by up to approximately 99%. A concentration-dependent reduction in arginase activity was also observed for the anti-aging dietary supplement. Further, all volunteers reported positive improvements in at least one quality of life factor, which included more restful sleep, reduced musculoskeletal discomfort, increased energy, improved physical stamina and better skin quality.&#x0D; Conclusion: These initial findings suggest that the anti-aging dietary supplement can mitigate declines in quality of life associated with aging by reducing DPP-4 and arginase activities, thereby supporting healthy GH production and endothelial nitric oxide production.

The Impact of Long‐Duration Exposure to Microgravity and Galactic Cosmic Radiation on Corpus Cavernosum Function

During spaceflight missions, astronauts are exposed to an extreme environment with high levels of radiation and microgravity, which negatively influence the cardiovascular system. With an increased development in space exploration and growing interest in manned missions to mars, it is of high importance to evaluate the health risks associated with long‐duration spaceflights. The increased risk of oxidative stress and inflammatory damage post‐spaceflight has been associated with cardiovascular dysfunction, but little is known about the influence of space exposure on erectile function, which is a critical component for quality of life in men. The purpose of this investigation was to determine the influence of long‐duration spaceflight on corpus cavernosum (CC) function. 86 adult male Sprague‐Dawley rats were randomized into 6 different groups with half of them enduring 4‐weeks of hindlimb unloading (HLU), and exposed to sham, 0.75Gy or 1.5Gy of simulated galactic cosmic radiation at the Ground‐based GCR Simulator at the NASA Space Radiation Laboratory (NSRL). Following a 6–9‐month recovery the rats were sacrificed, and CC tissue segments were harvested and mounted into a muscle strip myograph system for ex vivo functional assessment. CC reactivity to six 10‐s,30V‐electric field stimulations with progressive frequencies in the range of 1‐32 Hz targeting the non‐adrenergic non‐cholinergic (NANC) stimulation was assessed. The effects of HLU and radiation were determined by two‐way repeated measures ANOVA. High levels of radiation significantly decreased NANC‐mediated relaxation of the CC. Treatment with nitric oxide synthase (NOS) inhibitor L‐NAME completely inhibited tissue response to electric stimulation, showing that the relaxation was mainly driven by nitric oxide. Incubation of the CC tissue with the xanthine oxidase inhibitor allopurinol, the mitochondria‐targeted antioxidant mito‐TEMPO, and the superoxide dismutase (SOD) mimetic TEMPOL increased the relaxation response of the erectile tissue for the groups exposed to radiation, with little or opposite effect on the groups under no radiation exposure. Upon treatment with the Arginase inhibitor S‐(2‐boronoethyl)‐l‐cysteine (BEC), CC tissue for the groups under radiation exposure showed a significant improvement in relaxation under NANC electric field stimulation. Comparably, western blot analysis showed increased levels of oxidative stress and lipid peroxidation in the corpus cavernosum on the groups under radiation exposure by assessment of 4‐hydroxynonenal (HNE), as well as higher levels of arginase 1 and 2. This information suggests that increased oxidative stress from radiation exposure in long spaceflight trips impairs corpus cavernosum vasoreactivity and exposes new factors to consider with space exploration to ensure that astronauts can return to normal life after long trips in space.

Mitochondrial damage-associated molecular patterns trigger arginase-dependent lymphocyte immunoregulation.

Tissue damage leads to loss of cellular and mitochondrial membrane integrity and release of damage-associated molecular patterns, including those of mitochondrial origin (mitoDAMPs). Here, we describe the lymphocyte response to mitoDAMPs. Using primary cells from mice and human donors, we demonstrate that natural killer (NK) cells and Tcells adopt regulatory phenotypes and functions in response to mitoDAMPs. NK cell-mediated cytotoxicity, interferon gamma (IFN-γ) production, Tcell proliferation, and invivo anti-viral Tcell activation are all interrupted in the presence of mitoDAMPs or mitoDAMP-rich irradiated cells in invitro and invivo assays. Mass spectrometry analysis of mitoDAMPs demonstrates that arginase and products of its enzymatic activity are prevalent in mitoDAMP preparations. Functional validation by arginase inhibition and/or arginine add-back shows that arginine depletion is responsible for the alteration in immunologic polarity. We conclude that lymphocyte responses to mitoDAMPs reflect a highly conserved mechanism that regulates inflammation in response to tissue injury.

H1N1 Influenza Virus-Infected Nasal Mucosal Epithelial Progenitor Cells Promote Dendritic Cell Recruitment and Maturation

The barrier function of nasal mucosal epithelial cells plays an irreplaceable role in the spread and expansion of viruses in the body. This study found that influenza A virus H1N1 could induce apoptosis of nasal mucosal epithelial progenitor cells, cause an inflammatory response, and trigger the maturation and recruitment of nasal submucosal dendritic cells (DCs), but the mechanism remained unclear. Therefore, we used RNA sequencing and high-resolution untargeted metabolomics to sequence and perform combined bioinformatic analysis of H1N1 virus-infected nasal mucosal epithelial cells from 6 different patients. The abnormal arginine metabolism signaling pathway caused by H1N1 virus infection was screened out, and arginase inhibitors were used to interfere with the abnormal arginine metabolism and the maturation and recruitment of submucosal DCs caused by the H1N1 virus in vitro and in vivo. We conclude that H1N1 influenza virus promotes the recruitment and maturation of submucosal DCs by causing abnormal arginine metabolism in nasal mucosal epithelial cells, thereby triggering respiratory mucosal immunity.

Antihyperglycemic activity of L-norvaline and L-arginine in high-fat diet and streptozotocin-treated male rats

BackgroundA decrease in nitric oxide (NO) bioavailability has been shown to cause hyperglycemia, type II diabetes mellitus (DM), and chronic cardio-metabolic complications. In turn, hyperglycemia and hypercholesterolemia are associated with increased oxidative stress that leads to reduced nitric oxide bioavailability through disruption of L-arginine transport into cells, inactivation of nitric oxide synthase, and activation of arginase. Upregulation of arginase has been demonstrated in both diabetic patients and animal models of hyperglycemia and type 2 diabetes. L-norvaline is a nonselective inhibitor of arginase that increases NO production and promotes the normal functioning of the vascular endothelium. Another means of increasing NO bioavailability in the cardiovascular system is L-arginine supplementation. Whether L-norvaline and L-arginine have antihyperglycemic effects has not been studied. HypothesisWe hypothesized that inhibition of arginase will provide an antihyperglycemic effect and, as a result of the recovery of NO bioavailability, will protect against oxidative stress and hypercholesterolemia. MethodsRats were fed a high-fat diet (HFD) for three weeks concomitant with the two-time injection of 30 mg/kg of streptozotocin (STZ) to induce stable hyperglycemia. We studied the antihyperglycemic properties of arginase inhibition (via L-norvaline) and its combination with NOS substrate supplementation (via L-arginine). ResultsTreatment of HFD/STZ mice with L-norvaline and L-arginine reduced fasting blood glucose levels by 27.1% vs. untreated HFD/STZ rats (p < 0.001). Blood levels of total cholesterol, low-density lipoprotein (LDL), and malondialdehyde (MDA), a marker for oxidative stress, were significantly decreased in both L-norvaline- and L-norvaline+L-arginine-treated HFD/STZ rats when compared with untreated rats. In addition, administration of L-norvaline and L-arginine reversed the progression of pancreatic and kidney pathology in HFD/STZ rats as assessed by histology (p < 0.001). ConclusionsBoth L-norvaline and L-arginine act as potent antihyperglycemic agents and can represent alternative therapeutic tools in individuals with hyperglycemia and pre-diabetes.

Significance of the activity of liver arginase and Kupffer cells in the detoxification processes and the thyroid status formation in rats under alcoholic intoxication of different severity

Modern medicine faces the problem of a steady growth of alcoholic pathology. As you know, morbidity and mortality with a regular consumption of alcoholic beverages is associated with the toxic effects of ethanol on the most important human organs and, first of all, on the liver. To date, a sufficient number of facts are accumulated, indicating the importance of liver arginase and Kupffer cells in vital processes in health and disease. The aim of the study was to elucidate the significance of the activity of liver arginase and Kupffer cells in the detoxification processes and the thyroid status formation in rats with chronic ethanol intoxication of different severity. In rat experiments using modern physiological, biochemical research methods and a pharmacological approach, it was found that liver arginase and Kupffer cells participate in changes in the liver detoxification function and the thyroid status formation induced by chronic ethanol intoxication. The activity of liver arginase and Kupffer cells determines the severity of detoxification processes and the thyroid status formation in chronic alcohol intoxication. The direction and severity of changes in the arginase activity and the liver detoxification function during chronic alcoholism depends on the severity of chronic alcohol intoxication. Under the influence of daily intragastric administration for 60 days, a 30 % aqueous solution of ethanol (3.5 g 92 % ethanol per kg of body weight) in animals inhibited the activity of liver arginase and the detoxification function, but the introduction of a 10 % aqueous solution of ethanol (1.0 g 92 % ethanol per kg of body weight) within 2 months leads to an increase in the activity of liver arginase and detoxification processes. Kupffer cells depression by GdCl3 as the action in the body of the NO-synthase inhibitor methyl ester NG-nitro-L-arginine weakens and the arginase inhibitor Nω-hydroxy-nor-L-arginine contributes to the development of characteristic changes in the processes of detoxification and triiodothyronine level in plasma during chronic alcohol intoxication caused by intragastric introduction of ethanol at a dose of 3.5 g/kg for 60 days.