Activity Of Arg Research Articles

Fenugreek extract improves diabetes-induced endothelial dysfunction via the arginase 1 pathway.

Endothelial dysfunction (ED) is an initiating trigger and key factor in vascular complications, leading to disability and mortality in individuals with diabetes. The research concerning therapeutic interventions for ED has gained considerable interest. Fenugreek, a commonly used edible plant in dietary consumption, has attracted significant attention due to its management of diabetes and its associated complications. The research presented in this study examines the potential therapeutic benefits of fenugreek in treating ED and investigates the underlying mechanism associated with its effects. The analysis on fenugreek was performed using 70% ethanol extract, and its chemical composition was analyzed using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). In total, we identified 49 compounds present in the fenugreek extract. These compounds encompass flavonoids, saponins, and phospholipids. Then, the models of ED in streptozotocin-induced diabetic mice and high glucose-induced isolated rat aortas were established for research. Through vascular function testing, it was observed that fenugreek extract effectively improved ED induced by diabetes or high glucose. By analyzing the protein expression of arginase 1 (Arg1), Arg activity, Arg1 immunohistochemistry, nitric oxide (NO) level, and the protein expression of endothelial nitric oxide synthase (eNOS), p38 mitogen-activated protein kinase (p38 MAPK), and p-p38 MAPK in aortas, this study revealed that the potential mechanism of fenugreek extract in anti-ED involves the downregulation of Arg1, leading to enhanced NO production. Furthermore, analysis of serum exosomes carrying Arg activity indicates that fenugreek may decrease the activity of Arg transported by serum exosomes, potentially preventing the increase in Arg levels triggered by the uptake of serum exosomes by vascular endothelial cells. In general, this investigation offers valuable observations regarding the curative impact of fenugreek extract on anti-ED in diabetes, revealing the involvement of the Arg1 pathway in its mechanism.

Response of meat chickens to different sources of arginine in low-protein diets.

Arginine activity in broiler diets can be supplied by L-arginine (Arg), guanidinoacetic acid (GAA) and L-citrulline (Cit), all of which are commercially available. This study was conducted to assess the effects of Arg source and level on broiler performance, nutrient digestibility and carcass parameters. Day-old Ross 308 cockerels (n=768) were assigned to one of eight dietary treatments using a completely randomized design: normal protein (NP), low protein deficient in Arg (LP) and LP with two levels of either Arg (0.238% and 0.476%), GAA (0.309% and 0.618%) or Cit (0.238 and 0.476%). The LP was 5 percentage points lower in protein level than the NP. Wheat, sorghum, soya bean meal, canola meal, and meat and bone meal-based diets were fed over three feeding phases to 6 replicate floor pens with 16 birds each. Compared to NP, birds fed LP had reduced feed intake (FI, p<0.001), reduced body weight gain (BWG, p<0.001) and increased feed conversion ratio (FCR, p<0.001) from day 0 to day 35. Additions of Arg or Cit to the LP at both levels resulted in increased BWG and reduced FCR (p<0.05). Birds fed LP with GAA added had lower FCR (p<0.05) but not higher BWG (p>0.05) compared with the LP observed from day 0 to day 35. Supplementation of Arg, Cit and the low level of GAA to LP resulted in increased carcass yield, bone length, diameter and ash (p<0.05) but did not increase ileal energy or nitrogen digestibility (p>0.05). The findings indicate that Cit is an efficacious source of Arg activity in Arg-deficient diets.

Influence of bacterial lectin on key regulatory links of functional activity of macrophages in mice with Ehrlich carcinoma.

Recent studies have shown the potential of using different approaches for immunotherapy in cancer treatment. Macrophages (Mph) are one of the promising targets for immunotherapy. To investigate changes in the functional activity of Mph in mice with Ehrlich carcinoma by nitric oxide (NO)/arginase (Arg), IRF4/IRF5and STAT1/STAT6ratios caused by administration of lectin from B. subtilis IMV-7724. From the 2nd day after Ehrlich carcinoma inoculation into female Balb/c mice, lectin from B. subtilis IMV B-7724 (0.02mg/mouse) was administered for 10days. The peritoneal Mph were isolated on days 14, 21, and 28after tumor transplantation and their functional state (NO production, Arg activity and cytotoxic activity) was examined. The levels of mRNA expression of transcription factors STAT-1, STAT-6, IRF5, IRF4were evaluated. In lectin-treated animals with Ehrlich carcinoma, the functional state of Mph (NO/Arg ratio, index of cytotoxic activity) was maintained at the level of intact mice exceeding the values in untreated animals with Ehrlich carcinoma at late terms of tumor growth (21, 28days). Analysis of mRNA expression levels of transcription factors in these animals showed a significant increase (p< 0.05) in the ratio of STAT1/STAT6on the day 21 and IRF5/IRF4on day 28 of tumor growth compared to that in untreated mice. Administration of lectin from B. subtilis IMV B-7724to mice with Ehrlich carcinoma led to the prevalence of Mph exhibiting the functional properties of M1type at late-term tumor growth. The transcription factors of the STAT and IRF signaling pathways are involved in the process of Mph polarization induced by lectin from B. subtilis IMV B-7724.

Abstract B067: L-Arginine in the regulation of NOS2 expression to overcome renal cell carcinoma tumor growth

Abstract Renal cell carcinoma (RCC) is a cancer that is hard to treat because of its evasive nature, and its resistance to chemotherapy, radiotherapy, and immunotherapy. There is only a 10% survival rate in humans if RCC is not caught at early stages. L-Arginine metabolism is a highly regulated process that can produce global effects in tumors and in the immune system. Arginase 1 (ARG1) and ARG2 can metabolize L-arginine to induce the synthesis of polyamines necessary for tumor growth, whereas inducible nitric oxide synthase (NOS2) can metabolize L-arginine to produce nitric oxide (NO) which has been shown to possess antitumor activity. In addition, L-citrulline, another metabolite of NOS2, may play an important role in cancer, since it acts as a substrate for the de novo synthesis of L-arginine. Previous data have shown that certain RCC cell lines respond to IFNy or IFNa treatments by activating the NOS2 protein to produce NO, which inhibits tumor growth. However, in several other RCC cell lines there is a lack of NOS2 protein induction, suggesting that these RCC cells have developed a mechanism that blocks NOS2 expression. Therefore, treatments that could activate NOS2 expression to induce tumor regression are much needed. We believe that the lack of NOS2 is due to L-arginine deprivation, which is highly depleted in ARG2-RCC tumors. It is possible that decreased extracellular L-arginine limits its intracellular availability, deactivating downstream translational proteins, then blocking NOS2 expression. We hypothesize that in some RCC, the competition of ARG and NOS2 for L-arginine regulates the expression of NOS2 as a mechanism to increase tumor growth. To test our hypothesis, increasing concentrations of L-arginine of 1,000, 2,000 and 4,000 uM were added to RPMI media and cells were cultured for 24 and 48 hours. At each time point cell lysates were tested for ARG2, NOS2, GCN2 and eIF2a proteins, as well as for intracellular levels of L-arginine. Supernatants were tested for L-arginine, L-glutamine, L-citrulline and nitrite production. Our results show that after 48 hours in culture, there was a decrease in ARG activity as compared to untreated controls (p=0.004) in cells treated with 4,000 uM of L-arginine; a slight increase in NOS2 protein was observed with no significant increases in nitrites. More experiments using closer L-arginine concentration increments are under way in lieu to determine the expression of GNC2 and eIF2a. The levels of L-citrulline were significantly higher at 48 hours in cells cultured in 4,000 uM of L-arginine. One striking observation was that the cells consumed L-glutamine at higher rates as early as 24 hours. This finding is very important because these cells are possibly using L-glutamine as a source for polyamine synthesis, bypassing the L-arginine-ARG pathway. Understanding the mechanisms by which RCC tumors grow could help us to develop new and more effective immune-therapeutic strategies to overcome RCC growth and resistance. Citation Format: Charity F. Sylvester, Paula Datri, Arnold Zea. L-Arginine in the regulation of NOS2 expression to overcome renal cell carcinoma tumor growth [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B067.

Changes in arginase isoforms in a murine model of neonatal brain hypoxia-ischemia.

Background:Arginases (ARG-isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair, however their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown.Methods:C57BL/6-mice subjected to the Vannucci procedure on postnatal day 9 were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically.Results:ARG-isoforms expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 d. ARG activity during neurodevelopment remained unchanged but increased at 1 d with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons.Conclusions:ARG-isoforms expression increases with age from P9-P17 but is suppressed by injury specifically in the hippocampus and not in the cortex. Both, levels and activity of ARG-isoforms increase with H while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG-isoforms in brain differ in spatiotemporal localization, expression and activity during neurodevelopment and after injury.

Antimicrobial activity of Arg–Ser–Ser against the food‐borne pathogen Pseudomonas aeruginosa

SummaryWe had previously reported a peptide sequence, Arg–Ser–Ser (RSS), which was identified after coincubation of an edible orchid species, Dendrobium aphyllum, with Lactobacillus amylolyticus. In this study, we investigated the potential mechanism of the antibacterial effect of RSS on the pathogen Pseudomonas aeruginosa. The minimum inhibitory concentration of RSS for P. aeruginosa was 6.3%, and the peptide was shown to induce morphological damage to bacterial cells. RSS treatment increased the cell surface zeta potential, hydrophobicity and cell membrane permeability, indicating that the positively charged RSS likely interacted with the negatively charged bacterial membrane. Moreover, leakage of potassium ions and proteins from bacterial cells was increased by RSS by 30 ppm and 13.5 μg mL−1, respectively. In conclusion, RSS treatment caused leakage of cellular components from P. aeruginosa cells, mainly through damaging the cell membrane by electrostatic action. Thus, RSS may be a potent new antimicrobial agent.

Arginase II activity regulates cytosolic Ca2+ level in a p32-dependent manner that contributes to Ca2+-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells

Although arginase II (ArgII) is abundant in mitochondria, Ca2+-accumulating organelles, the relationship between ArgII activity and Ca2+ translocation into mitochondria and the regulation of cytosolic Ca2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca2+]m as measured by CoCl2-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII−/− mice. Conversely, [Ca2+]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca2+]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE−/− +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca2+]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE−/− +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE−/− +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.

Association of the Arginase Ι with Bronchial Asthma

Objective The aim of this study was to detect the arginase Ι (ARG I) enzyme in asthma patients, clarify its role, in addition to examining the relationship of this enzyme with bronchial asthma.&#x0D; Methods:&#x0D; Blood samples were collected from 100 patients from the Department of Medicine in Rizgary Hospital in Erbil City, in addition to intact 100 volunteers; the introduced questionnaire was filled out on the basis of type-induced asthma, duration of the disease, age of the patients, gender, family history, and allergy condition, Serum was separated to perform Enzyme-linked immunosorbent assay (ELISA) in Medical Research Center to examine the association of ARGΙ with bronchial asthma. By ARG activity we can measure the conversion of arginine to ornithine and urea. By using a quantitative colorimetric assay at 490 nm, employing a QuantiChrom arginase assay kit (Bioassay Systems).&#x0D; Results: Our results depicted the association between ARGΙ and bronchial asthma: based on their age, significant elevation of serum arginase level was observed in the patients with ≥81 years old, which mean value (100.16±19.77c), p-value (0.000); also the duration of asthma ≥20 years (82.48±38.81c) , p-value (0.01) were remarkably affected; this sign was found in those with types of induced asthma and with allergy condition. But the non- significant difference in the frequency of abnormal serum arginase level was observed in those patients that have a family history of asthma disease and gender of the patients. This finding demonstrated a remarkable association of ARG Ι in the development of asthma at p ˂ 0.05.

Identification of an Arginase II Inhibitor via RapidFire Mass Spectrometry Combined with Hydrophilic Interaction Chromatography

Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-scale high-throughput screening to identify other structural types of Arg II inhibitors.

Arginine reverses growth hormone resistance through the inhibition of toll-like receptor 4-mediated inflammatory pathway

ObjectiveGrowth hormone stimulates growth by increasing insulin-like growth factor 1 expression and secretion. In the presence of insufficient nutrients, GH increases, whereas IGF-1 expression becomes severely suppressed, leading to GH resistance. This study aimed to explore the effect of arginine (Arg) on GH resistance during malnutrition and to describe its underlying mechanism. MethodsC57BL/6J mice were injected intraperitoneally with Arg for 1h or subjected to caloric restriction with Arg supplement in drinking water for 18days. HepG2 cells were exposed to different Arg concentrations for 24h. Signaling pathway agonists/inhibitors, siRNA, and overexpression plasmids were used to investigate the underlying molecular mechanism. Liver-specific toll-like receptor (TLR4) knockout mice were utilized to clarify the role of TLR4 in Arg-induced IGF-I expression and secretion. ResultsArg inhibited the TLR4 downstream pathway by binding to TLR4 and consequently activated Janus kinase 2/signal transducer and activator of transcription 5 signaling pathway. As a result, IGF-1 transcription and secretion increased. Arg activity was absent in liver-specific TLR4 knockout mice and was greatly suppressed in liver with overexpressed TLR4, suggesting that hepatic TLR4 was required and sufficient to induce GH resistance. By contrast, the mammalian target of rapamycin pathway was unnecessary for Arg activity. Arg not only significantly increased IGF-1 expression and secretion under acute fasting and chronic CR conditions but also attenuated body weight loss. ConclusionsOur results demonstrate a previously unappreciated pathway involving Arg that reverses GH resistance and alleviates malnutrition-induced growth restriction through the inhibition of TLR4-mediated inflammatory pathway.

Pyk2 and FAK differentially regulate invadopodia formation and function in breast cancer cells.

The nonreceptor tyrosine kinase Pyk2 is highly expressed in invasive breast cancer, but the mechanism by which it potentiates tumor cell invasiveness is unclear at present. Using high-throughput protein array screening and bioinformatic analysis, we identified cortactin as a novel substrate and interactor of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 colocalizes with cortactin to invadopodia of invasive breast cancer cells, where it mediates epidermal growth factor-induced cortactin tyrosine phosphorylation both directly and indirectly via Src-mediated Abl-related gene (Arg) activation, leading to actin polymerization in invadopodia, extracellular matrix degradation, and tumor cell invasion. Both Pyk2 and the closely related focal adhesion kinase (FAK) regulate tumor cell invasion, albeit via distinct mechanisms. Although Pyk2 regulates tumor cell invasion by controlling invadopodium-mediated functions, FAK controls invasiveness of tumor cells by regulating focal adhesion-mediated motility. Collectively, our findings identify Pyk2 as a unique mediator of invadopodium formation and function and also provide a novel insight into the mechanisms by which Pyk2 mediates tumor cell invasion.

Abstract P287: Endothelial Dysfunction in Small Resistance Arteries of Patients with Severe Obesity: Role of Arginase

Nitric oxide (NO) is produced by endothelial NO synthase (eNOS) using the aminoacid L-Arginine. Arginase (Arg) also uses L-Arginine as substrate, converting it in L-Ornitine and urea. An increased Arg activity causes a progressive L-Arginine depletion, which in turn determines a lower NO bioavailability. Studies in murine models of obesity identify Arg as a determinant of endothelial dysfunction. In this study, we evaluated whether Arg might play a role in determining the lower bioavailability of NO in small resistance arteries isolated from subcutaneous tissue of patients with severe obesity (Ob), split in age groups (younger than 30 aa, range 21-29, n=5; older than 30 aa, range 35-56, n=5) vs normoweight controls (Ctrl younger 30 years, range 20-29, n=5; older than 30 yrs, range 36-58, n=5). Each patient underwent a subcutaneous biopsy during a laparoscopic surgical procedure. Small arteries, isolated from periadvential fat, were evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation (VD) was assessed by acetylcholine (Ach, 0,001-100μM). NO availability was assessed by repeating Ach with L-NAME (100μM). Ach was also infused in the presence of norNOHA (10μM, Arg inhibitor). In Ctrl, VD induced by Ach was inhibited by L-NAME and not modified by norNOHA. Ob younger exhibited a reduced VD induced by Ach vs Ctrl of the same age, a reduced inhibition by L-NAME, and a potentiating effect by norNOHA, which also normalized the inhibitory effect of L-NAME on Ach. In Ob older, VD induced by Ach was reduced vs Ob younger, resistant to L-NAME and not modified by norNOHA. In conclusions, in small arteries from younger Ob, the Arg inhibition improves endothelial function by increasing the NO availability, while in older Ob Arg does not seem to play any role in endothelial dysfunction.

Caffeine plus nicotine improves motor function, spatial and non-spatial working memory and functional indices in BALB/c male mice

There is a greater prevalence of cigarette smoking among caffeine dependent individuals. This study therefore sought to assess the effect of nicotine and/or caffeine on some key biochemical indices and neurobehavioural parameters associated with brain function in male mice. Forty male BALB/c mice were divided into 4 groups of 10 animals each; Group A serve as the control and received normal saline (s.c), Group B received 2mg/kg body weight of nicotine (s.c), Group C received 2mg/kg body weight of caffeine (s.c) and Group D received 2mg/kg of nicotine and 2mg/kg of caffeine (s.c). The experiment lasted for 21 days, and then the animals were subjected to behavioral test. Thereafter the animals were sacrificed and their brain isolated for the determination of endothelial nitric oxide (NO) level, acetylcholinesterase (AChE), arginase (Arg) and adenosine deaminase (ADA) activities; as well as some antioxidant indices. Administration of nicotine or caffeine caused a significant (P<0.05) inhibition on AChE, ADA and Arg activities as well as a significant increase in NO level when compared with the control. However, it was observed from this study that nicotine enhances caffeine cognitive properties through a significant increase in non-spatial working memory whereas; it was otherwise on the spatial working memory and motor coordination. Therefore, we can suggest from our present study that caffeine enhances the effect of nicotine either synergistically or additively on memory and motor function and some key biochemical indices associated with brain function in male mice.

Myeloid-derived suppressor cells reveal radioprotective properties through arginase-induced l-arginine depletion

Background and purposeHigh arginase-1 (Arg) expression by myeloid-derived suppressor cells (MDSC) is known to inhibit antitumor T-cell responses through depletion of l-arginine. We have previously shown that nitric oxide (NO), an immune mediator produced from l-arginine, is a potent radiosensitizer of hypoxic tumor cells. This study therefore examines whether Arg+ overexpressing MDSC may confer radioresistance through depleting the substrate for NO synthesis. Material and methodsMDSC and Arg expression were studied in preclinical mouse CT26 and 4T1 tumor models and further validated in rectal cancer patients in comparison with healthy donors. The radioprotective effect of MDSC was analyzed in hypoxic tumor cells with regard to l-arginine depletion. ResultsIn both mouse tumors and cancer patients, MDSC expansion was associated with Arg activation causing accelerated l-arginine consumption. l-Arginine depletion in turn profoundly suppressed the capacity of classically activated macrophages to synthesize NO resulting in impaired tumor cell radiosensitivity. In advanced cT3-4 rectal cancer, circulating neutrophils revealed Arg overexpression approaching that in MDSC, therefore mounting a protumor compartment wherein Arg+ neutrophils increased from 17% to over 90%. ConclusionsProtumor Arg+ MDSC reveal a unique ability to radioprotect tumor cells through l-arginine depletion, a common mechanism behind both T-cell and macrophage inhibition.

The Src kinases Hck, Fgr and Lyn activate Arg to facilitate IgG-mediated phagocytosis and Leishmania infection.

Leishmaniasis is a devastating disease that disfigures or kills nearly two million people each year. Establishment and persistence of infection by the obligate intracellular parasite Leishmania requires repeated uptake by macrophages and other phagocytes. Therefore, preventing uptake could be a novel therapeutic strategy for leishmaniasis. Amastigotes, the life cycle stage found in the human host, bind Fc receptors and enter macrophages primarily through immunoglobulin-mediated phagocytosis. However, the host machinery that mediates amastigote uptake is poorly understood. We have previously shown that the Arg (also known as Abl2) non-receptor tyrosine kinase facilitates L. amazonensis amastigote uptake by macrophages. Using small-molecule inhibitors and primary macrophages lacking specific Src family kinases, we now demonstrate that the Hck, Fgr and Lyn kinases are also necessary for amastigote uptake by macrophages. Src-mediated Arg activation is required for efficient uptake. Interestingly, the dual Arg and Src kinase inhibitor bosutinib, which is approved to treat cancer, not only decreases amastigote uptake, but also significantly reduces disease severity and parasite burden in Leishmania-infected mice. Our results suggest that leishmaniasis could potentially be treated with host-cell-active agents such as kinase inhibitors.

Leishmanicidal Galloylquinic Acids are Noncompetitive Inhibitors of Arginase

Leishmanicidal galloylquinic acids were isolated from the ethyl acetate extract of Byrsonima coccolobifolia. These phenols and gallic acid showed to be a new class of potent noncompetitive inhibitors of arginase ARG (Ki ranging from 0.10 to 0.68 µmol L -1 ) from Leishmania amazonensis. Quinic acid did not exhibit significant inhibition of ARG, indicating that galloyl moiety has important features that allows the enzyme-inhibitor interactions. The significant inhibitory activity of gallic acid on ARG can be a clue to understand the immune response previously observed on L. donovani, since ARG activity is associated with the decrease of the levels of NO in Leishmania infection.

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity In CML Patients At Diagnosis

IntroductionIn some solid tumors it has been demonstrated that a subpopulation of myeloid cells, defined as “myeloid-derived suppressor cells” (MDSCs), plays an important role in inducing T cell tolerance by production of arginase 1 (arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function. Since chronic myeloid leukemia (CML) patients have high levels of immature myeloid cells it is of interest to investigate if these cells have MDSCs phenotype and activity. The aim of this study was to analyze MDSCs and investigate their activity in CML patients. MethodsMDSCs were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis. In 21 patients MDSCs were also measured during TKI treatment. Granulocytic MDSCs (G-MDSCs) were identified as CD11b+CD33+CD14-HLADR- cells, while the monocytic MDSCs (Mo-MDSCs) as CD14+HLADR by cytofluorimetric analysis. Arg1 expression was assessed using real time PCR and Western Blot. Arg activity was measured in granulocyte lysates using a colorimetric test after enzymatic activation and arginine hydrolysis. Microvesicles (MV) were isolated from CML serum at diagnosis (n=5) by sequential ultracentrifugation. ResultsCML patients showed high levels of Mo- and G-MDSCs at diagnosis in comparison to HD (41±8 and 82,5±12,2% respectively for CML vs 9±2,1 and 55±5,3% for HD; p<0.001), while after TKIs therapy both subpopulations decreased, returning to normal values. T-reg (CD4+ CD25high Foxp3+ cells) were also significantly increased in CML patients at diagnosis in respect to HD (9±2% vs 6,1±0,8%, p<0.001) with a significant correlation with the percentage of Gr-MDSCs (r=0,6254; p<0.001). Both in PB and purified granulocytic cells, Arg1 expression showed a 30 fold increase in CML at diagnosis compared to HD (p<0.001) and decreased after therapy. The same data were confirmed by Western Blot analysis. Arg enzymatic activity in granulocytes resulted also increased in CML (n=10) compared to HD (n=10) (p<0.001). The suppressive function of CML G-MDSCs was demonstrated by their ability to inhibit the proliferation of CFSE+ HD T cells (p<0.001). In addition, an increase of Mo-MDSCs in vitro was observed after incubation of HD monocytes with CML sera (29±13%; p<0.0001) or MV (8±2,8%; p<0.05). ConclusionsMDSCs are increased in CML patients at diagnosis and decrease during TKIs treatment. CML granulocytes have high arg1 activity and immunosuppressive activity. Moreover, CML serum as well as CML microvesicles increase the percentage of HD Mo-MDSCs. Disclosures:No relevant conflicts of interest to declare.

Local Arginase 1 Activity Is Required for Cutaneous Wound Healing

Chronic nonhealing wounds in the elderly population are associated with a prolonged and excessive inflammatory response, which is widely hypothesized to impede healing. Previous studies have linked alterations in local L-arginine metabolism, principally mediated by the enzymes arginase (Arg) and inducible nitric oxide synthase (iNOS), to pathological wound healing. Over subsequent years, interest in Arg/iNOS has focused on the classical versus alternatively activated (M1/M2) macrophage paradigm. Although the role of iNOS during healing has been studied, Arg contribution to healing remains unclear. Here, we report that Arg is dynamically regulated during acute wound healing. Pharmacological inhibition of local Arg activity directly perturbed healing, as did Tie2-cre-mediated deletion of Arg1, revealing the importance of Arg1 during healing. Inhibition or depletion of Arg did not alter alternatively activated macrophage numbers but instead was associated with increased inflammation, including increased influx of iNOS+ cells and defects in matrix deposition. Finally, we reveal that in preclinical murine models reduced Arg expression directly correlates with delayed healing, and as such may represent an important future therapeutic target.

Contribution of arginase activation to vascular dysfunction in cigarette smoking

BackgroundCigarette smoke increases the risk of several cardiovascular diseases and has synergistic detrimental effects when present with other risks that contribute to its pathogenesis. Oxidative injury to the endothelium via reactive oxygen species (ROS) and nitric oxide (NO) dysregulation is a common denominator of smoking-induced alterations in vascular function. However, the mechanisms underlying ROS and NO dysregulation due to smoking remain unclear. We tested if arginase (Arg) activation/upregulation contributes to this phenomenon by constraining nitric oxide synthase (NOS) activity. MethodsArg2 knockout (Arg2−/−) and control C57BL/6J mice were either exposed to cigarette smoke, 6 h/day/2 weeks (Second Hand Smoking; SHS) or housed in normal environment (Non Smoking; NS). Arg activity, NO and ROS levels were determined by measuring urea production, fluorescent dye (DAF), and dihydroethedium (DHE) respectively in isolated mouse aorta. ResultsArg activity and ROS levels were higher NO lower in SHS compared to NS mice. SHS failed to lower NO levels in Arg2−/− mice. Endothelial dependent vasodilation (EDV) was attenuated in SHS mice as compared to controls (78.80% ± 8 vs 46.58% ± 5). This impaired EDV was abolished in Arg2−/− mice (67.48% ± 7 in SHS vs. 78.80% ± 8 in NS). Vascular stiffness was increased in SHS mice as compared to NS controls but remained unchanged in Arg2−/− mice. ConclusionEndothelial NOS is uncoupled by smoking exposure, leading to endothelial dysfunction and vascular stiffness, a process that is prevented by Arg2 deletion. Hence, we identify Arg2 upregulation as a critical pathogenic factor and target for therapy in oxidative injury following smoking exposure through reciprocal regulation of endothelial NOS.

Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

BackgroundElevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice.Methods and ResultsEndothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser1177 (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks.ConclusionsOver 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.