Inhibition Of Arginase Activity Research Articles

Arginase inhibitor reduces fungal dissemination in murine pulmonary cryptococcosis by promoting anti-cryptococcal immunity

Elevation of arginase enzyme activity in the lung contributes to the pathogenesis of various chronic inflammatory diseases and infections. Inhibition of arginase expression and activity is able to alleviate those effects. Here, we investigated the immunomodulatory effect of arginase inhibitor in C. neoformans infection. In the pulmonary cryptococcosis model that was shown to recapitulate human infection, we found arginase expression was excessively induced in the lung during the late stage of infection. To inhibit the activity of arginase, we administered a specific arginase inhibitor, nor-NOHA, during C. neoformans infection. Inhibition of arginase reduced eosinophil infiltration and level of IL-13 secretion in the lungs. Whole lung transcriptome RNA-sequencing analysis revealed that treatment with nor-NOHA resulted in shifting the Th2-type gene expression patterns induced by C. neoformans infection to the Th1-type immune profile, with higher expression of cytokines Ifng, Il6, Tnfa, Csf3, chemokines Cxcl9 and Cxcl10 and transcription factor Stat1. More importantly, mice treated with arginase inhibitor had more infiltrating brain leukocytes and enhanced gene expression of Th1-associated cytokines and chemokines that are known to be essential for protection against C. neoformans infection. Inhibition of arginase dramatically attenuated spleen and brain infection, with improved survival. Taken together, these studies demonstrated that inhibiting arginase activity induced by C. neoformans infection can modulate host immune response by enhancing protective type-1 immune response during C. neoformans infection. The inhibition of arginase activity could be an immunomodulatory target to enhance protective anti-cryptococcal immune responses.

Enhanced infectivity of bovine viral diarrhoea virus (BVDV) in arginase-producing bovine monocyte-derived macrophages

ABSTRACT Macrophages are important cells of the innate immunity that play a major role in Bovine Viral Diarrhea Virus (BVDV) pathogenesis. Macrophages are not a homogenous population; they exist in different phenotypes, typically divided into two main categories: classically (pro-inflammatory) and alternatively activated (anti-inflammatory) or M1 and M2, respectively. The role of bovine macrophage phenotypes on BVDV infection is still unclear. This study characterized the interaction between BVDV, and monocyte-derived macrophages (Mo-Mφ) collected from healthy cattle and polarized to an M1 or M2 state by using LPS, INF-γ, IL-4 or azithromycin. Arginase activity quantitation was utilized as a marker of the M2 Mo-Mφ spectrum. There was a significant association between arginase activity and the replication rate of BVDV strains of different genotypes and biotypes. Inhibition of arginase activity also reduced BVDV infectivity. Calves treated with azithromycin induced Mo-Mφ of the M2 state produced high levels of arginase. Interestingly, azithromycin administered in vivo increased the susceptibility of macrophages to BVDV infection ex vivo. Mo-Mφ from pregnant dams and calves produced higher arginase levels than those from non-pregnant adult animals. The increased infection of arginase-producing alternatively activated bovine macrophages with BVDV supports the need to delve into a possible leading role of M2 macrophages in establishing the immune-suppressive state during BVDV convalescence.

Changes in the Metabolism of Nitric Oxide, Hydrogen Sulfide of the Oral Fluid Taking into Account Age and Dental Status

The relevance of the study is conditioned by the prevalence of dental pathology, its complicated course in senior age and the search for the age-associated metabolic markers of oral fluid.
 Objective. The aim of the study is: to determine the age peculiarities of the content of nitric oxide and hydrogen sulfide metabolism in the oral fluid of patients, taking into account their dental status.
 Materials and Methods. Indicators of nitric oxide metabolism, L-arginine/arginase, hydrogen sulfide content in oral fluid, dental status of different aged people under conditions of intact dentitions, their defects and against the background of correction with different types of dentures, were studied.
 Results. It was determined that with age, changes in oral fluid are characterized by an increase in nitrite ion, the amount of nitrites and nitrates, increasing the formation of peroxynitrite against the background of lower levels of L-arginine and hydrogen sulfide, inhibition of arginase activity. The results obtained are confirmed by the presence of close correlations between the concentrations of hydrogen sulfide and peroxynitrite (reverse, strong), caries intensity and: the total level of nitrites and nitrates (direct, strong), peroxynitrite content (direct, medium strength).
 Conclusion. The studied biomarkers of oral fluid depend more on age, hygienic condition of the oral cavity, intensity of dental caries and defects of dentitions than types of dentures. Therefore, age-related changes in the biochemical composition of oral fluid, metabolic, somatic, comorbid pathologies are likely to be a trigger for structural-functional disorders of the dental-maxillofacial system.

A39 TARGETING IL-4 TO TREAT CROHN’S DISEASE ASSOCIATED INTESTINAL FIBROSIS IN SHIP DEFICIENT MICE

Abstract Background Crohn’s disease (CD) is an immune-mediated disease characterized by chronic, relapsing, or progressive inflammation along the gastrointestinal tract. One in 3 people with CD will develop intestinal fibrosis requiring surgery within 10 years of diagnosis. Biological therapy is very effective at reducing inflammation in CD; however, biologics do not reduce the incidence of fibrosis or surgery in people with CD, suggesting that a distinct mechanism(s) may exist that drives intestinal fibrosis. Treatments for intestinal fibrosis are urgently needed as currently, no treatment exists that target intestinal fibrosis directly. Mice deficient in the Src homology 2 domain-containing inositolphosphate 5’-phosphatase (SHIP-/-) develop spontaneous CD-like ileal inflammation and fibrosis. Fibrosis is dependent on PI3Kp110δ activation, which leads to induction of the enzyme arginase I (argI). Genetic ablation or pharmacologic inhibition of PI3Kp110δ or inhibition of arginase activity blocked the development of intestinal fibrosis in SHIP-/- mice. Both IL-4 and IL-13 can activate PI3Kp110δ but intestinal pathology in SHIP-/- mice is characterized by high levels of IL-4. Thus, we hypothesize that SHIP-/- mice develop CD-like intestinal fibrosis due to increased IL-4 signalling. Aims Aim 1: To determine whether genetic ablation of IL-4 prevents ileal fibrosis in SHIP-/- mice Aim 2 To determine whether inhibition of IL-4 activity can block or reverse ileal fibrosis in SHIP-/- mice Methods SHIP-/- mice were crossed with mice deficient in IL-4 (IL-4-/-) to generate wild type, SHIP+/+IL-4-/- SHIP-/-IL-4+/+, and SHIP-/-IL4-/- mice. Fibrosis was assessed in mice at 8 weeks of age. SHIP-/- mice (8-week-old) were treated with an anti-IL-4 antibody, IgG isotype control or PBS (injection control), for 2 weeks. Ileal fibrosis was assessed in SHIP-/- mice and compared to mice treated with anti-IL-4 antibody or controls. Measurements of ileal fibrosis include muscle thickening, and collagen accumulation by Masson’s trichrome staining. Results SHIP-/-IL4-/- mice do not have less ileal fibrosis than their SHIP-/- littermates. Inhibition of IL-4 signalling in SHIP-/- mice did not reduce ileal fibrosis. Conclusions Blocking IL-4 is not sufficient to block the development of ileal fibrosis in SHIP-/- mice. In future experiments, we will examine whether blocking IL-13, alone or in combination with IL-4, can ameliorate the development of intestinal fibrosis in SHIP-/- mice. Funding Agencies CIHR

Метаболизм L-аргинина у гипертензивных крыс при угнетении аргиназы L-норвалином

The hypotensive effect of arginase inhibition in the experiment [1] can be realized in two ways: by relaxation of blood vessels by an increase in the concentration of nitric oxide (NO) and/or a decrease in the amount of circulating fluid. The first effect occurs with an increase in the availability of the substrate of NO synthase (NOS) - arginine (ARG). The second - with an increase in diuresis. To clarify the ways in which arginase affects the regulation of blood pressure (BP) we have analyzed the metabolic characteristics of the amino acid L-arginine in hyper- and normotonic rats during inhibition of arginase activity by the administration of its inhibitor L-norvaline. The concentration of arginine and its metabolite ornithine in blood, urine, and in the homogenate of kidney tissue was measured using high-performance liquid chromatography with the separation on a reversed-phase sorbent and with a fluorescence detection. Norvaline was injected intraperitoneally (ip) once a day at a dose of 30 mg/kg for 7 days. In experiments under the influence of norvaline, no significant increase in the concentration of amino acids in the blood plasma was found either in hypertensive rats (ISIAH strain) or in normotensive control (WAG strain). The concentration of arginine in the urine of ISIAH animals treated with norvaline was found doubled, while in WAG rats it did not change. The daily excretion of arginine under norvaline increased in normotonics only slightly, and in hypertensive rats it increased almost threefold. At the same time, norvaline increased diuresis only in hypertensive animals. Under the influence of norvaline, the content of arginine per 1 g of kidney weight increased almost twofold in hypertensive rats, while in normotensive rats it did not change. The studies performed allow us to conclude, that inhibition of arginase by norvaline had a stronger hypotensive effect in hypertensive rats than in normotensive rats. At the same time, the hypotensive effect of norvaline in normotonic rats can be provided by the nitric oxide system, whereas in hypertensive rats this function is taken over by diuresis.

L-Arginine metabolism alteration by L-Lysine intervention increased cell death in triple negative breast cancer cell

Abstract Background L-Arginine effectiveness cancer therapy is a matter of controversy. L-Arginine is a substrate for different metabolites influencing cancer cells. Arginase and nitric oxide synthase (NOS) are two important enzymes in the L-Arginine metabolic pathway. Nitric oxide (NO) is produced by NOS and the main products of arginase activity are polyamines. NO can decrease cancer cells viability dependent on its concentration but, polyamines shown to promote cancer cell growth/metastasis. Due to Arginase and NOS competition over L-Arginine binding, inhibition of arginase activity seems to increase NOS activity as well as NO production. Thus, the aim of the present study is to affect L-Arginine metabolic pathway through L-Lysine induced arginase inhibition in MDA-MB-231 and MDA-MB-468 Methods MDA-MB-231 and MDA-MB-468 were used as triple negative breast cancer cells with different arginase activity. Both cell lines were treated with different concentrations of L-Arginine and L-Lysine. Cell viability was assessed by MTT assay 24 and 48h after treatment. Arginase activity, ROS level and NO concentration was examined. Cell cycle was assessed using flowcytometry. Real-time PCR was performed to assess apoptotic and metabolic genes expression Results Our results indicated that cell viability and arginase activity were decreased in MDA-MB-468 with high arginase level more than MDA-MB-231 and studying the cell cycle also showed G2/M arrest in both cell lines following combination treatment with L-Lysine with L-Arginine. L-Lysine treatment resulted in increased apoptotic genes and decreased metabolic related genes expression in both cell lines Conclusion Altogether, our findings indicated that alternation in L-Arginine metabolic pathway using L-Lysine causes higher NO concentration which consequently increases cell death in triple negative breast cancer cells with different arginase level, which in turn can increase L-Arginine effect on cell lines with higher arginase activity

Concomitant type I IFN and M-CSF signaling reprograms monocyte differentiation and drives pro-tumoral arginase production.

BackgroundType I IFN-based therapies against solid malignancies have yielded only limited success. How IFN affects tumor-associated macrophage (TAM) compartment to impact the therapeutic outcomes are not well understood.MethodsThe effect of an IFN-inducer poly(I:C) on tumor-infiltrating monocytes and TAMs were analyzed using a transplantable mouse tumor model (LLC). In vitro culture systems were utilized to study the direct actions by poly(I:C)-IFN on differentiating monocytes.ResultsWe found that poly(I:C)-induced IFN targets Ly6C+ monocytes and impedes their transition into TAMs. Such an effect involves miR-155-mediated suppression of M-CSF receptor expression, contributing to restricting tumor growth. Remarkably, further analyses of gene expression profile of IFN-treated differentiating monocytes reveal a strong induction of Arg1 (encoding arginase-1) in addition to other classical IFN targets. Mechanistically, the unexpected Arg1 arm of IFN action is mediated by a prolonged STAT3 signaling in monocytes, in conjunction with elevated macrophage colony-stimulating factor (M-CSF) signaling. Functionally, induction of ARG1 limited the therapeutic effect of IFN, as inhibition of arginase activity could strongly synergize with poly(I:C) to enhance CD8+ T cell responses to thwart tumor growth in mice.ConclusionsTaken together, we have uncovered two functionally opposing actions by IFN on the TAM compartment. Our work provides significant new insights on IFN-mediated immunoregulation that may have implications in cancer therapies.

Arginase Inhibition Activity of Stem Bark Extract of Caesalpinia pulcherrima

: Background: Inhibition of arginase activity can ameliorate endothelial dysfunction in vascular system which can cause some cardiovascular diseases. This study was aimed to test the arginase activity inhibition of n-hexane, ethyl acetate, and methanol extract of C. pulcherrima (L.) Sw. stem bark and determine total flavonoid content of the most active extract. Methods: C. pulcherrima (L.) Sw. stem bark was extracted using multistage reflux. In vitro arginase activity inhibition was done using 1 U/mL enzyme concentration and 570 mM substrate concentration. The product was measured by microplate reader of wavelength 430 nm. Results: Methanolic extract showed to be the most active extract in the test. IC50 value of methanolic extract was 21.969 μg/mL which was higher than standard N(omega)-hydroxy-nor-L-arginine acetate (nor-NOHA acetate) that had IC50 value of 3.994 μg/mL. The result of total flavonoid content determination of methanol extract by colorimetric AlCl3 was 3.943 mgQE/gram extract. Phytochemical screening shows that methanol extract of C. pulcherrima (L.) Sw. stem bark contains flavonoids, tannins, saponins, and triterpenoids. Conclusion: Methanol extract of C. pulcherrima (L.) Sw. had significant inhibitory activity of arginase and can be used as a potential source for the development of arginase inhibitor.Key words: Arginase, Caesalpinia pulcherrima (L.) Sw. stem bark, Total flavonoid content, Phytochemical screening.

Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1.

We have recently found that diabetes-induced premature senescence of retinal endothelial cells is accompanied by NOX2-NADPH oxidase-induced increases in the ureohydrolase enzyme arginase 1 (A1). Here, we used genetic strategies to determine the specific involvement of A1 in diabetes-induced endothelial cell senescence. We used A1 knockout mice and wild type mice that were rendered diabetic with streptozotocin and retinal endothelial cells (ECs) exposed to high glucose or transduced with adenovirus to overexpress A1 for these experiments. ABH [2(S)-Amino-6-boronohexanoic acid] was used to inhibit arginase activity. We used Western blotting, immunolabeling, quantitative PCR, and senescence associated β-galactosidase (SA β-Gal) activity to evaluate senescence. Analyses of retinal tissue extracts from diabetic mice showed significant increases in mRNA expression of the senescence-related proteins p16INK4a, p21, and p53 when compared with non-diabetic mice. SA β-Gal activity and p16INK4a immunoreactivity were also increased in retinal vessels from diabetic mice. A1 gene deletion or pharmacological inhibition protected against the induction of premature senescence. A1 overexpression or high glucose treatment increased SA β-Gal activity in cultured ECs. These results demonstrate that A1 is critically involved in diabetes-induced senescence of retinal ECs. Inhibition of arginase activity may therefore be an effective therapeutic strategy to alleviate diabetic retinopathy by preventing premature senescence.

The kinetic properties of arginase in sperm cells of inferile men

Nowadays the role of NO in the development of male infertility is actively studied. Arginase (EC 3.5.3.1) is a manganese metalloenzyme which converts L-arginine to L-ornithine and urea and reciprocally regulates NO production. Although arginase activity has usually been detected in the reproductive tract, including spermatozoa, no data relating to the kinetic properties of the enzyme in ejaculated spermatozoa has been reported. This study was designed to study the kinetic parameters of arginase of spermatozoa of infertile men. Spermatozoa arginase activity was measured by determining levels of urea production. Kinetic analysis of the enzyme reaction was performed in a standard incubation system with modified physical and chemical characteristics or the respective components (the substrate concentration, Mn2+ concentration, incubation time and protein content). Pathobiochemical and kinetic properties of sperm arginase obtained from human normozoo- and pathospermic samples were compared. The maximum rate of L-arginine hydrolysis (detrermined by L-arginine) for arginase of spermatozoa obtained from men with preserved fertility was 2.0, 1.8 and 1.9 times greater than this value for oligo-, astheno- and oligoasthenozoospermic samples respectively. However, affinity constants for L-arginine was not significantly different between fertile and infertile men. The maximum rate of L-arginine hydrolysis (deretmined by Mn2+) for arginase of spermatozoa obtained from men with preserved fertility was 1.6, 1.7 and 1.7 times greater than this value for oligo-, astheno- and oligoasthenozoospermic samples respectively. However, affinity constants for Mn2+ were not significantly different between fertile and infertile men. In the whole range of time, the urea production by arginase in sperm cells obtained from oligozoospermic samples is much lower compared to value in healthy donors. The results of kinetic analysis indicate that urea production by arginase is much more intense in the control group than in patients with various forms of pathospermia. The initial (instantaneous) reaction rate of arginase reaction was lower for oligozoospermic samples compared to normozoospermic samples. It has been found that inhibition of arginase activity in sperm cells of infertile men occurs by non-competitive type and was related to marked decrease in maximum reaction rate while affinity of arginase to L-arginine and Mn2+ was unaffected.

P1657Pharmacological inhibition of arginase activity in a rat model of monocrotaline-induced pulmonary hypertension

P1657Pharmacological inhibition of arginase activity in a rat model of monocrotaline-induced pulmonary hypertension

In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum

With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects.

Exogenous l-Valine Promotes Phagocytosis to Kill Multidrug-Resistant Bacterial Pathogens.

The emergence of multidrug-resistant bacteria presents a severe threat to public health and causes extensive losses in livestock husbandry and aquaculture. Effective strategies to control such infections are in high demand. Enhancing host immunity is an ideal strategy with fewer side effects than antibiotics. To explore metabolite candidates, we applied a metabolomics approach to investigate the metabolic profiles of mice after Klebsiella pneumoniae infection. Compared with the mice that died from K. pneumoniae infection, mice that survived the infection displayed elevated levels of l-valine. Our analysis showed that l-valine increased macrophage phagocytosis, thereby reducing the load of pathogens; this effect was not only limited to K. pneumoniae but also included Escherichia coli clinical isolates in infected tissues. Two mechanisms are involved in this process: l-valine activating the PI3K/Akt1 pathway and promoting NO production through the inhibition of arginase activity. The NO precursor l-arginine is necessary for l-valine-stimulated macrophage phagocytosis. The valine-arginine combination therapy effectively killed K. pneumoniae and exerted similar effects in other Gram-negative (E. coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. Our study extends the role of metabolism in innate immunity and develops the possibility of employing the metabolic modulator-mediated innate immunity as a therapy for bacterial infections.

Immunostimulated Arginase II Expression in Intestinal Epithelial Cells Reduces Nitric Oxide Production and Apoptosis

Increased production of nitric oxide (NO) and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as a putative mechanism involved in the development of necrotizing enterocolitis (NEC). Intestinal epithelial cells (IECs) metabolize L-arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell proliferation, while NO production can lead to apoptosis. We hypothesized that in IECs immunostimulation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were immunostimulated by either incubation with lipopolysaccharide (LPS) alone for 24 h or by incubation with conditioned media (CM) for 24 h. CM was obtained from RAW 264.7 cells (a macrophage cell line) treated with LPS (E. coli 0127:B8; 1 μg/ml) for 4 h. The rIEC-6 stimulated with LPS or with CM had significantly higher levels of inducible NOS (iNOS) protein, NO production, and arginase II protein than did the control cells. Direct LPS stimulation of rIEC-6 produced a less robust increase in iNOS expression and NO (represented as nitrite percent of control) than did CM stimulation. Inhibition of arginase using Nω hydroxyl-L-arginine (NOHA) further increased stimulated NO production in rIEC-6. Viable cell numbers were significantly lower in CM stimulated cells after 24 h than in controls, and inhibition of arginase activity with NOHA resulted in a further significant decrease in viable cell numbers. We conclude that immunostimulated arginase expression of rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Abstract 170: Arginase Contributes to Delayed Constriction of Large Cerebral Arteries in a Model of Subarachnoid Hemorrhage

Introduction: Increased arginase activity can limit nitric oxide synthase activity and contribute to age-related increase in aortic stiffness. Hypothesis: Subarachnoid hemorrhage (SAH) produces a delayed increase in arginase activity that contributes to delayed decreases in diameter of major cerebral arteries. Methods: Male rats underwent injection of blood into the cisterna magna on day 0 and again on day 2. Shams had double injection of artificial CSF. Measurements of arginase activity on vessels in the Circle of Willis and pia matter were made with an assay based on the conversion of radiolabeled arginine to urea. Measurements of diameter of basilar, posterior (PCA), middle (MCA), and anterior (ACA) cerebral arteries were made ex vivo after perfusion with paraformaldehyde and black latex casting. Results: Arginase activity (nmol of urea/min/mg of protein) increased from the control value of 13±3 (±SE; n=17) to 24±6 (n=6) at 3 days, 36±14 (n=4) at 5 days, and 48±16 (n=9) at 7 days after SAH and then recovered at 10 days (14±5; n=4) and 14 days (18±6; n=5) after SAH. Infusion of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) for 7 days after SAH with an ip osmotic pump blocked the increase in arginase activity (10±2; n=4). Assessment of arterial diameter at 3, 5, 7, 10, and 14 days after SAH revealed the smallest diameters occurring at 7 days (except for MCA which occurred at 5 days). Continuous ip infusion of 10 mg/kg/day ABH significantly attenuated the decrease in diameter (μm) 7 days after SAH in PCA (sham = 249±9, n=8; SAH = 209±12, n=10; SAH+ABH = 255±9, n=6) and ACA (sham = 178±11; SAH = 141±11; SAH+ABH = 198±10). Effects on basilar artery were of marginal significance (P=0.065). Conclusion: SAH produces an increase in vascular arginase activity that is temporally related to delayed decreases in diameter of cerebral arteries. Inhibition of arginase activity prevents the decrease in diameter at 7 days after SAH, thereby indicating a contribution of arginase to delayed arterial constriction/remodeling in post-fixed arteries.

Abstract 18885: CTRP1, an Adipokine Causing Endothelial Dysfunction Through Upregulation of Arginase 1, is Associated With the Severity of Coronary Artery Disease in Patients and Promotes Atherogenesis in Mice

Background: C1q/TNF-related protein (CTRP) 1 was initially identified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins. However, vascular functions of CTRP1 remain largely undetermined. Here, we sought to investigate whether CTRP1 influences the pathogenesis of atherosclerosis. Methods and Results: We analyzed CTRP1 levels in sera of coronary artery disease (CAD) patients (n=451) and non-CAD controls (n=686), in coronary endarterectomy specimens (n=26) and non-atherosclerotic internal mammary arteries (n=32), and in peripheral blood mononuclear cells (PBMCs) from severe CAD patients (n=58) and non-CAD controls (n=25). CTRP1 levels were higher in sera, endarterectomy specimens and PBMCs from CAD patients compared with controls, and were related to CAD severity. Immunohistochemistry of human femoral arteries exhibited VE-cadherin loci-associated increased CTRP1 expression in atherosclerotic plaques. In cultured endothelial cells, CTRP1 stimulation resulted in reduced nitric oxide (NO) bioavailability and promotion of inflammatory reactions (increased production of inflammatory cytokines and induction of adhesion molecules). Intravital microscopy exhibited impaired endothelium-dependent arteriole dilation and enhanced leukocyte-endothelium interactions after injection of recombinant CTRP1, whereas improved arteriole dilation and decreased leukocyte-endothelium interactions were observed in CTRP1 knockout mice. Furthermore, we found that arginase 1 was significantly increased by CTRP1 in a dose-dependent fashion, thereby leading to eNOS-uncoupling and reactive oxygen species generation. Inhibition of arginase activity by synthetic chemicals markedly improved CTRP1-dependent endothelial dysfunction. Finally, intraperitoneal injection of recombinant CTRP1 promoted atherogenesis in apoE-/- mice. In contrast, proatherogenic effects were significantly attenuated in CTRP1-/-/apoE-/- double knockout mice. Conclusions: Our findings have demonstrated that CTRP1 is a pro-atherogenic adipokine, likely by modulating arginase 1 expression and enzyme activity. CTPP1 may represent a crucial therapeutic target in atherosclerosis.

The effects of arginase inhibitor on lung oxidative stress and inflammation caused by pneumoperitoneum in rats.

BackgroundPneumoperitoneum-induced oxidative stress and organ injury are known to be associated with nitric oxide (NO) inactivation. Because arginase competes with NO synthase (NOS) for a common substrate, L-arginine, arginase inhibition may increase NO bioavailability. Therefore, we evaluated the ability of the arginase inhibitor, 2 (S)-amino-6-boronohexanoic acid (ABH), to attenuate pneumoperitoneum-induced decrease of NO bioavailability and lung injury.MethodsThirty rats were randomly divided into the following groups: 1) the PP-ABH group received a subcutaneous injection of ABH (5 mg/kg) 1 h before induction of pneumoperitoneum (insufflation to intraperitoneal pressure of 15 mmHg for 60 min); 2) the PP group received saline by subcutaneous injection 1 h before induction of pneumoperitoneum; and 3) the control group received saline by subcutaneous injection before a sham procedure with no gas insufflation. After desufflation, blood was collected to determine levels of plasma nitrite, NOS, inflammatory cytokines, and malondialdehyde, a marker of oxidative stress. Lung tissue was obtained for histological evaluation.ResultsWe found that plasma nitrite levels were lower in the PP group and higher in the PP-ABH group, compared with controls (P <0.01 and P <0.05, respectively). In the PP group, endothelial NOS activity was decreased and inducible NOS activity was increased compared with the PP-ABH and control groups. Malondialdehyde levels increased 3-fold in the PP group and 2-fold in the PP-ABH group compared with controls. Tumor necrosis factor-α, interleukin-6, and interleukin-1ß levels were elevated in the PP group compared to the control group, but the increase in cytokine production was attenuated or blocked in the PP-ABH group. Lung injury scores were 4.8-fold higher in the PP group and 2-fold higher in the PP-ABH group compared with controls (P <0.001 and P <0.01, respectively).DiscussionPneumoperitoneum decreases NO bioavailability and increases the inflammation cytokines, resulting in organ injuries. Inhibition of arginase activity could maintain NO bioavailability by attenuating pneumoperitoneum-induced changes in NOS activity. In addition, arginase inhibition attenuated the oxidative stress and inflammation and decreased the severity of lung injury caused by pneumoperitoneum.ConclusionsBy increasing NO bioavailability and suppressing oxidative stress and inflammation, pretreatment with an arginase inhibitor may protect against lung injury caused by pneumoperitoneum.

Cytokine‐Induced Arginase Expression in Intestinal Epithelial Cells Reduce Nitric Oxide Production and Apoptosis

Intestinal epithelial cells are involved in disease progression of inflammatory bowel diseases such as necrotizing entercolitis in preterm neonates. Intestinal epithelial cells metabolize arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell viability, while NO production can lead to apoptosis. The hypothesis of this study is that in intestinal epithelial cells inflammation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were incubated with conditioned media (CM) from RAW 264.7 cells (a macrophage cell line) treated with LPS (E.coli 0127:B8; 1μg/ml) for 4 hours. rIEC-6 cells incubated with CM had significantly higher levels of iNOS, cleaved caspase-3, and arginase II protein levels than did control cells. Levels of arginase I were not different between controls and CM treated cells. rIEC-6 incubated in CM had significantly greater NO production than did untreated rIEC-6, and NO production in CM-treated cells was further increased when the arginase inhibitor nor-NOHA was added to the media. Viable cell number was significantly lower after 24 hours with CM than in controls, and inhibition of arginase activity with nor-NOHA resulted in a further significant decrease in viable cell numbers. These data suggest that cytokine-induced arginase expression in rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Dietary supplementation of ginger and turmeric improves reproductive function in hypertensive male rats.

Ginger [Zingiber officinale Roscoe (Zingiberaceae)] and turmeric [Curcuma longa Linn (Zingiberaceae)] rhizomes have been reportedly used in folk medicine for the treatment of hypertension. However, the prevention of its complication such as male infertility remains unexplored. Hence, the aim of the present study was to investigate the preventive effects of ginger and turmeric rhizomes on some biomarkers of male reproductive function in L-NAME-induced hypertensive rats. Male Wistar rats were divided into seven groups (n = 10): normotensive control rats; induced (L-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of L-NAME (40 mg/kg/day). The results revealed significant decrease in serum total testosterone and epididymal sperm progressive motility without affecting sperm viability in hypertensive rats. Moreover, increased oxidative stress in the testes and epididymides of hypertensive rats was evidenced by significant decrease in total and non-protein thiol levels, glutathione S-transferase (GST) activity with concomitant increase in 2′,7′-dichlorofluorescein (DFCH) oxidation and thiobarbituric acid reactive substances (TBARS) production. Similarly, decreased testicular and epididymal NO level with concomitant elevation in arginase activity was observed in hypertensive rats. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations in biomarkers of reproductive function in hypertensive rats. The inhibition of arginase activity and increase in NO and testosterone levels by both rhizomes could suggest possible mechanism of action for the prevention of male infertility in hypertension. Therefore, both rhizomes could be harnessed as functional foods to prevent hypertension-mediated male reproductive dysfunction.

In vitro studies to assess the antioxidative, radical scavenging and arginase inhibitory potentials of extracts from Artocarpus altilis, Ficus exasperate and Kigelia africana

To justify the use of Artocarpus altilis (A. altilis), Ficus exasperata (F. exasperata) and Kigelia africana (K. africana) in ethnomedicine for the treatment of several ailments and to evaluate the in vitro antioxidant, radical scavenging and arginase inhibitory potentials of these herbs and compared with catechin (Standard). Antioxidant activities were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide, hydrogen peroxide (H2O2) and hydroxyl (OH) radicals scavenging methods. The flavonoids and phenolics content, inhibition of arginase activity, Fe(2+)/ascorbate-induced lipid peroxidation (LPO) and reducing power were also determined. The A. altilis, F. exasperata and K. africana showed dose-dependent and significant scavenging of DPPH, H2O2 and OH radicals in vitro relative to catechin. The A. altilis and F. exasperata effectively scavenged DPPH radical with IC50 of 593 and 635 µg/mL and, OH radical with IC50 of 487 and 514 µg/mL, respectively. The DPPH and OH radicals scavenging activities followed the order A. altilis>F. exasperata>K. africana. In addition, A. altilis and F. exasperata significantly (P<0.05) inhibited LPO in a dose-dependent manner. The A. altilis extract had the most potent inhibitory activity against LPO with 79% relative to catechin (28%) at 750 µg/mL. The reducing power followed the order: A. altilis>Catechin>F. exasperata>K. africana at 1 000 µg/mL. The A. altilis at 500 and 750 µg/mL significantly (P<0.05) inhibited arginase activity by 63% and 67%, respectively. The flavonoids contents were found to be highest in A. altilis. Extracts of A. altilis and F. exasperata are potent antioxidative agents with strong radical scavenging activity and inhibition of lipid peroxidation.