Arginase 2 Research Articles

Deletion of Arginase 2 reduces neurodegeneration in a model of Multiple Sclerosis

PurposeVisual impairment due to optic neuritis is a major clinical feature of Multiple Sclerosis (MS), and can lead to temporary or permanent vision loss. Previous studies from our lab have demonstrated the critical involvement of arginase 2 (A2) in retinal neurodegeneration. The current study was undertaken to investigate the role of A2 signaling in MS mediated retinal neuronal damage.MethodExperimental Autoimmune Encephalomyelitis (EAE) was induced in WT mice and A2 knock out (A2 KO) mice (both on C57Bl6 background) according to established procedures. Briefly, WT and A2 KO mice were immunized by subcutaneous injections of an emulsion containing MOG35‐55 peptide (300 μg/mouse) along with Complete Freund's adjuvant (CFA, killed Mycobacterium tuberculosis H37Ra, 400 μg/ul). Each mouse additionally received 300 ng of pertussis toxin (PT, Sigma, St. Louis, MO) by i.p. injection in 200 μl of PBS on day 0 and day 2 post‐immunization. Control group will be immunized with CFA without antigen (MOG), and two doses of PT will be given on day 0 and 2. Clinical disease scores were monitored daily in all groups in a blinded fashion by measuring paralysis according to the conventional grading system. Neurodegeneration studies were performed using retinal flatmount analysis. Expression of arginase and the downstream signaling molecules were examined by Western blotting, and inflammatory molecules were analyzed by qPCR.ResultsIncreased expression A2 was observed in WT retinas in response to EAE induction. EAE‐induced motor deficits and body weight loss were markedly reduced in A2 KO mice compared to WT controls. Retinal flat mount studies demonstrated a significant reduction in the number of Brn3a positive and NeuN positive neurons in WT EAE retina (p<0.01; N=6–10) in comparison to normal control mice. A significant increase in the number of surviving neurons (NeuN‐positive and Brn3a‐positive) was evident in retinas of EAE‐induced A2KO mice compared to WT (p<0.05; N=6–10). RNA levels of proinflammatory molecules such as CCL2 ((C‐C motif) ligand 2), COX2 (Cyclooxygenase‐2), IL1a (Interleukin 1a), IL12a, and IL‐18 were significantly reduced in the A2KO EAE retinas compared to WT EAE (p<0.05, N=5–6). Signaling studies showed increased levels of phospho‐ERK1/2 and reduced levels phospho‐BAD in the WT EAE retina, while these changes were normalized in EAE‐induced A2 retinas.ConclusionOur studies establish EAE as an excellent model to study MS‐mediated retinal neuronal damage and suggest the potential value of targeting arginase 2 signaling as a therapy to prevent MS‐mediated vision loss.Support or Funding InformationThese studies supported by the National Multiple Sclerosis Society (PP‐1606‐08778 to S.P.N) and Augusta University Culver Vision Discovery Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endogenous arginase 2 as a potential biomarker for PEGylated arginase 1 treatment in xenograft models of squamous cell lung carcinoma

Depletion of arginine induced by PEGylated arginase 1 (ARG1) (BCT-100) has shown anticancer effects in arginine auxotrophic cancers that lack argininosuccinate synthetase (ASS1) and ornithine transcarbamylase (OTC). High levels of endogenous arginase 2 (ARG2) have been previously reported in human lung cancers. Although a high-ARG2 level neither causes immunosuppression nor affects disease progression, it may theoretically affect the efficacy of PEGylated ARG1 treatment. ARG2 was shown to be highly expressed in H520 squamous cell lung carcinoma (lung SCC) xenografts but undetectable in SK-MES-1 and SW900 lung SCC xenografts. We propose that high-endogenous expression of ARG2 could impede the anti-tumor effect of PEGylated ARG1 in lung SCC. The in vivo effect of PEGylated ARG1 was investigated using three xenograft models of lung SCC. PEGylated ARG1 (60 mg/kg) suppressed tumor growth in SK-MES-1 and SW900 but not H520 xenografts. ASS1 was expressed in SK-MES-1 and SW900 xenografts while OTC expression remained low in all xenografts. A high-endogenous ARG2 level was detected only in H520 xenografts. Serum arginine level was decreased significantly by PEGylated ARG1 in all xenografts. Nonetheless intratumoral arginine level was decreased by PEGylated ARG1 in SK-MES-1 and SW900, not H520 xenografts. In SK-MES-1 xenografts, PEGylated ARG1 treatment induced G1 arrest, downregulation of Ki67 and Mcl-1 and activation of apoptosis. In SW900 xenografts, upregulation of Bim and activation of apoptosis were observed upon PEGylated ARG1 treatment. Silencing of ARG2 re-sensitized the H520 xenografts to PEGylated ARG1 treatment, partially mediated through arginine depletion via G1 arrest and apoptosis. PEGylated ARG1 treatment (BCT-100) was effective in lung SCC xenografts with low-endogenous levels of ASS1/OTC and ARG2. High-endogenous ARG2 expression may cause resistance to PEGylated ARG1 treatment in lung SCC xenografts. ARG2 may serve as a third predictive biomarker in PEGylated ARG1 treatment in lung SCC.

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase

Alterations in cell-cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell-cycle phase may represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell-cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis are active during SG2M in transformed but not in normal cells, with the mitochondrial arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively use ARG2, normal epithelial cells synthesize ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduces cancer cell growth and causes G2M arrest, while not inducing compensation via OAT. In human tumors, ARG2 is highly expressed in specific tumor types, including basal-like breast tumors. This study sheds light on the interplay between metabolism and cell cycle and identifies ARG2 as a potential metabolic target.

Macrophage-Derived IL1β and TNFα Regulate Arginine Metabolism in Neuroblastoma.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)-dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1β and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)-dependent manner. IL1β and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest-derived cells. Proteomic analysis revealed that enrichment of IL1β and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. SIGNIFICANCE: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Retinal Neuroprotection From Optic Nerve Trauma by Deletion of Arginase 2.

Our previous studies have implicated expression of the mitochondrial isoform of the arginase enzyme arginase 2 (A2) in neurovascular injury during ischemic retinopathies. The aim of this study was to characterize the specific involvement of A2 in retinal injury following optic nerve crush (ONC). To accomplish this, wild-type (WT) or A2 knockout (A2-/-) mice were subjected to ONC injury. The contralateral eye served as sham control. Quantitative RT-PCR and western blot were used to evaluate mRNA and protein expression. Retinal ganglion cell (RGC) survival was assessed in retinal whole mounts. Axonal sprouting was determined by anterograde transport of Cholera Toxin B (CTB). These analyses showed increased A2 expression following ONC. Numbers of NeuN-positive neurons as well as Brn3a- and RBPMS-positive RGC were decreased in the WT retinas at 14 days after ONC as compared to the sham controls. This ONC-induced neuronal loss was diminished in the A2-/- retinas. Similarly, axonal degeneration was ameliorated by A2 deletion whereas axon sprouting was enhanced. Significant retinal thinning was also seen in WT retinas at 21 days after ONC, and this was blocked in A2-/- mice. Cell death studies showed an increase in TUNEL positive cells in the RGC layer at 5 days after ONC in the WT retinas, and this was attenuated by A2 deletion. ONC increased glial cell activation in WT retinas, and this was significantly reduced by A2 deletion. Western blotting showed a marked increase in the neurotrophin, brain derived neurotrophic factor (BDNF) and its downstream signaling in A2-/- retinas vs. WT after ONC. This was associated with increases in the axonal regeneration marker GAP-43 in A2-/- retinas. Furthermore, A2-/- retinas showed decreased NLRP3 inflammasome activation and lower interleukin (IL-) 1β/IL-18 levels as compared to WT retinas subjected to ONC. Collectively, our results show that deletion of A2 limits ONC-induced neurodegeneration and glial activation, and enhances axonal sprouting by a mechanism involving increases in BDNF and decreases in retinal inflammation. These data demonstrate that A2 plays an important role in ONC-induced retinal damage. Blockade of A2 activity may offer a therapeutic strategy for preventing vision loss induced by traumatic retinal injury.

The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target.

Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1−α and HIF2−α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω−hydroxy−nor−arginine (nor−NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor−NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor−NOHA overcame hypoxia-mediated resistance towards BCR−ABL1 kinase inhibitors. While nor−NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor−NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor−NOHA on cellular respiration. In conclusion, we show that nor−NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor−NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor−NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.

Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

Kidney cancer, one of the ten most prevalent malignancies in the world, has exhibited increased incidence over the last decade. The most common subtype is "clear cell" renal cell carcinoma (ccRCC), which features consistent metabolic abnormalities, such as highly elevated glycogen and lipid deposition. By integrating metabolomics, genomic, and transcriptomic data, we determined that enzymes in multiple metabolic pathways are universally depleted in human ccRCC tumors, which are otherwise genetically heterogeneous. Notably, the expression of key urea cycle enzymes, including arginase 2 (ARG2) and argininosuccinate synthase 1 (ASS1), is strongly repressed in ccRCC. ReducedARG2 activity promotes ccRCC tumor growth through at least two distinct mechanisms: conserving the critical biosynthetic cofactor pyridoxal phosphate and avoiding toxic polyamine accumulation. Pharmacological approaches to restore urea cycle enzyme expression would greatly expand treatment strategies for ccRCC patients, where current therapies only benefit a subset of those afflicted with renal cancer.

Mechanisms of Retinal Ischemia/Reperfusion Injury: Arginase and the Mitochondria

ObjectiveOur group has previously shown that the ureahydrolyase mitochondrial isoenzyme, arginase 2 (A2) is involved in ischemia reperfusion (I/R)‐induced retinal neurovascular degeneration. We found that A2 homozygous deletion significantly protected against necroptosis, neurovascular degeneration, retinal thinning, and impairment of retinal function. Interestingly, our group has also shown a link between hypoxia‐induced increases in A2 and impaired angiogenesis in endothelial cells. The cytosolic isoform arginase 1 (A1) was not affected which suggests distinct roles of A1 versus A2 under hypoxic conditions. However, the exact mechanisms of arginase‐induced neurovascular damage in retinal I/R are not yet known. In the current study, we investigated the involvement of arginase in retinal I/R injury in vivo and in vitro in relation to mitochondrial function.MethodsI/R insult was conducted on the right eyes of wild type (WT) and A2 homozygous knockout (A2−/−) mice. The left eyes were used as sham controls. Bovine retinal endothelial cells (BRECs) subjected to oxygen glucose deprivation (OGD/R) were used as an in vitro model. Cells were treated with the arginase inhibitor, Amino‐2‐Borono‐6‐Hexanoic Acid (ABH, 100μM), recombinant Pegylated A1 (PegA1, 1μg/ml), or vehicle. Western blot was used to evaluate different protein levels. Mitochondria structure and function were evaluated using Rhodamine 123 labeling and Seahorse XF analyzer respectively.ResultsWe found that the mitochondrial fission protein, dynamin related protein (Drp1) was significantly increased in WT I/R retinas compared to sham controls at 3 and 6 hours. With A2 deletion, the Drp1 levels were also elevated after I/R but were significantly reduced to sham control levels at 6 hours. In cell culture studies, A2 protein levels were significantly increased after 6h OGD/ 6h R. The cleavage of poly ADP‐ribose polymerase (PARP) and phosphorylation of p38‐MAPK were significantly increased after OGD/R. Arginase inhibition significantly reduced the phosphorylation of p38 and slightly reduced PARP cleavage. Western blot analysis showed increased Drp1 expression and mitochondria labeling showed more fragmented mitochondria after OGD/R. With arginase inhibition, the mitochondria were restored and resumed a more normal morphology with significantly less Drp1 expression. Seahorse XF analysis of the oxygen consumption rate (OCR) showed a significant decrease in maximal respiration and spare respiratory capacity after OGD/R indicating impaired mitochondrial function. Treatment with PegA1 significantly prevented the OGD/R‐induced mitochondrial dysfunction as reflected by increased spare respiratory capacity and maximal respiration. However, treatment with the non‐selective arginase inhibitor (ABH) did not improve mitochondrial function under OGD/R conditions.ConclusionsThis study is the first to show that A1 and A2 could be playing different roles under ischemic conditions by affecting the mitochondrial function in distinct ways. Furthermore, PegA1 therapy may offer a new strategy for limiting hypoxia‐induced impairment of angiogenic function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myeloid Arginase 1 Protects Against Retinal Ischemia‐Reperfusion Injury

PurposeWe have recently shown that the mitochondrial, ureahydrolase enzyme arginase 2 (A2) mediates retinal neurovascular degeneration after ischemia‐reperfusion (IR) injury by increasing oxidative stress and inflammation. This project aimed to examine the role of the cytosolic enzyme isoform, arginase 1 (A1) in retinal IR injury. We specifically focused on endothelial and myeloid cells because A1 competes with the nitric oxide synthase (NOS) enzymes in these cells for the common substrate L‐arginine. L‐arginine depletion reduces NO formation. This promotes vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved.MethodsWild type (WT) mice, global A1+/− knock out (KO), endothelial specific (Cdh5‐cre;A1f/f) A1 KO, and myeloid specific (LysM‐cre;A1f/f) A1 KO mice were subjected to retinal ischemia for 40 minutes through raising the intraocular pressure in the right eye followed by reperfusion to achieve IR‐injury. The left eye served as sham control. A cohort of WT mice were treated with intravitreal injection of pegylated (peg) A1 (1.7 ng) or PBS at 3 hours after IR. Retinas were collected at different time‐points for analysis. At 7 days, retinal thinning was measured using optical coherence tomography (OCT) in live mice and morphometric analysis of hematoxylin and eosin (H & E) stained tissue sections. Neurodegeneration and microglia/macrophage activation were analyzed by confocal imaging in retinal flat mounts using NeuN and Iba‐1 labeling of inner retinal neurons and microglia/macrophages, respectively. At 14 days, retina function was assessed in live mice using electroretinography (ERG) while vascular degeneration was assessed by retina trypsin digestion to quantify the loss of vascular endothelial cells and pericytes. In vitro experiments using primary peritoneal macrophages (MΦ) were conducted to examine the role of A1 in MΦ inflammatory response to lipopolysaccharide (LPS) stimulation.ResultsGlobal as well as myeloid specific A1 KO mice showed worsened IR‐induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect while treatment with peg A1 improved neuronal survival in WT mice (table). In addition to worsened neurodegeneration, A1+/− KO mice showed reduced retina function and worsened vascular injury manifested by a 50 % increase in endothelial cell/pericyte loss (p<0.05, n=4–6). Western blotting analysis of retinal tissue showed that the worsened IR outcome with A1 deletion was associated with tumor necrosis factor α (TNFα)/receptor interacting protein 3 kinase (RIP3)/dynamin‐related protein 1 (DRP1) mediated necroptosis.In vitro experiments showed that macrophage (MΦ) lacking A1 exhibit increased inflammatory response upon LPS stimulation, as demonstrated by increased iNOS expression, activation of the NLRP3 inflammasome, increased IL‐1β, TNFα and nitric oxide (NO) production. PegA1 treatment dampened this inflammatory response. Moreover, intravitreal injection of WT MΦ at 3 days after IR improved neuronal survival compared to A1 KO MΦ while systemic MΦ depletion with clodronate liposomes increased neuronal loss (table).ConclusionA1 reduces IR‐induced retinal neurovascular degeneration and necroptosis via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage‐mediated repair in the early stages of ischemic retinopathy.Support or Funding InformationThis work was supported by grants from The National Institute of Health (NIH grant R01‐EY11766 (RBC)), the Department of Veterans Affairs, Veterans Health Administration (RBC), Office of Research and Development, Biomedical Laboratory Research and Development (BX001233), and the Culver Vision Discovery Institute at Augusta University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG <sub>2</sub>M Phase

Alterations in both cell cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell cycle phase could represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M cell cycle phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis was active during SG2M in transformed but not in normal cells. An integrative data analysis implicated the arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively used ARG2, normal epithelial cells synthesized ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduced cancer cell growth and caused G2M arrest, and did not induce compensation via OAT. In human tumors, ARG2 was highly expressed in specific tumor types, including basal-like breast tumors. This study sheds new light on the interplay between metabolism and the cell cycle, and identifies ARG2 as a potential metabolic target.

Overexpression of SlARG2 or SlTD2 in Arabidopsis enhances resistance against Plutella xylostella L.

Tomato (Solanum lycopersicum L.) ARGINASE2 (ARG2) and THREONINE DEAMINASE2 (TD2) are involved in plant defense. These enzymes act in the midgut of herbivores fed on tomato plants to degrade the essential amino acids Arg and Thr, respectively. Although it has been demonstrated that overexpression of the SlARG2 gene in tomato enhanced its resistance against M. sexta larvae, knock-down the expression of SlTD2 reduced the resistance of tomato to lepidopteran herbivores; it remains unclear whether overexpression of SlTD2 could enhance the resistance of the host plants to herbivores, or whether combined overexpression of SlARG2 and SlTD2 could lead to synergistically enhanced resistance to insects. Here, we generated transgenic Arabidopsis plants overexpressing SlARG2 (SlARG2 OE) and SlTD2 (SlTD2 OE) individually as well as in combination (SlARG2-SlTD2 OE). Overexpression of these genes did not affect Arabidopsis development, seed yield, or Arg and Thr content. Insect-feeding bioassay was performed by feeding diamondback moth (Plutella xylostella L.) larvae on detached leaves of wild-type, SlARG2 OE, SlTD2 OE, and SlARG2-SlTD2 OE plants. Larvae fed on SlARG2 OE leaves showed approximately 31% to 35% reduction in weight and 6% to 10% reduction in survival rate compared to those fed on wild-type leaves. Although larvae fed on SlTD2 OE leaves showed no reduction in survival rate, they gained less weight. Whereas larvae fed on SlARG2-SlTD2 OE leaves showed neither reduction in weight nor reduction in survival rate. We further investigated the arginase enzymatic activity of the SlARG2 OE and SlARG2-SlTD2 OE transgenic plants. The SlARG2 OE line most resistant to diamondback moth larvae displayed the highest arginase activity. Our data indicate that overexpression of SlARG2 or SlTD2 in Arabidopsis can enhance its resistance against diamondback moth, whereas combined overexpression of SlARG2 and SlTD2 did not generate synergistically increased resistance to diamondback moth.

Arginine metabolic endotypes related to asthma severity.

AimsArginine metabolism via inducible nitric oxide synthase (iNOS) and arginase 2 (ARG2) is higher in asthmatics than in healthy individuals. We hypothesized that a sub-phenotype of asthma might be defined by the magnitude of arginine metabolism categorized on the basis of high and low fraction of exhaled nitric oxide (FENO).MethodsTo test this hypothesis, asthmatics (n = 52) were compared to healthy controls (n = 51) for levels of FENO, serum arginase activity, and airway epithelial expression of iNOS and ARG2 proteins, in relation to clinical parameters of asthma inflammation and airway reactivity. In parallel, bronchial epithelial cells were evaluated for metabolic effects of iNOS and ARG2 expression in vitro.ResultsAsthmatics with high FENO (≥ 35 ppb; 44% of asthmatics) had higher expression of iNOS (P = 0.04) and ARG2 (P = 0.05) in the airway, indicating FENO is a marker of the high arginine metabolic endotype. High FENO asthmatics had the lowest FEV1% (P < 0.001), FEV1/FVC (P = 0.0002) and PC20 (P < 0.001) as compared to low FENO asthmatics or healthy controls. Low FENO asthmatics had near normal iNOS and ARG2 expression (both P > 0.05), and significantly higher PC20 (P < 0.001) as compared to high FENO asthmatics. In vitro studies to evaluate metabolic effects showed that iNOS overexpression and iNOS+ARG2 co-expression in a human bronchial epithelial cell line led to greater reliance on glycolysis with higher rate of pyruvate going to lactate.ConclusionsThe high FENO phenotype represents a large portion of the asthma population, and is typified by greater arginine metabolism and more severe and reactive asthma.

Arginine auxotrophic gene signature in paediatric sarcomas and brain tumours provides a viable target for arginine depletion therapies.

Paediatric sarcomas and brain tumours, remain cancers of significant unmet need, with a poor prognosis for patients with high risk disease or those who relapse, and significant morbidities from treatment for those that survive using standard treatment approaches. Novel treatment strategies, based on the underlying tumour biology, are needed to improve outcomes. Arginine is a semi-essential amino acid that is imported from the extracellular microenvironment or recycled from intracellular precursors through the combined expression of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) enzymes. The failure to express at least one of these recycling enzymes makes cells reliant on extracellular arginine – a state known as arginine auxotrophism. Here we show in large in silico patient cohorts that paediatric sarcomas and brain tumours express predominately the arginine transporter SLC7A1 and the arginine metabolising enzyme Arginase 2 (ARG2), but have low-absent expression of OTC. The arginine metabolic pathway correlated with the expression of genes associated with tumour pathogenesis, and overall survival in paediatric sarcomas. This gene signature of arginine auxotrophism indicates paediatric sarcomas and brain tumours are a viable target for therapeutic arginase drugs under current clinical trial development.

Effect of personal exposure to black carbon on changes in allergic asthma gene methylation measured 5\xa0days later in urban children: importance of allergic sensitization

BackgroundAsthma gene DNA methylation may underlie the effects of air pollution on airway inflammation. However, the temporality and individual susceptibility to environmental epigenetic regulation of asthma has not been fully elucidated. Our objective was to determine the timeline of black carbon (BC) exposure, measured by personal sampling, on DNA methylation of allergic asthma genes 5 days later to capture usual weather variations and differences related to changes in behavior and activities. We also sought to determine how methylation may vary by seroatopy and cockroach sensitization and by elevated fractional exhaled nitric oxide (FeNO).MethodsPersonal BC levels were measured during two 24-h periods over a 6-day sampling period in 163 New York City children (age 9–14 years), repeated 6 months later. During home visits, buccal cells were collected as noninvasive surrogates for lower airway epithelial cells and FeNO measured as an indicator of airway inflammation. CpG promoter loci of allergic asthma genes (e.g., interleukin 4 (IL4), interferon gamma (IFNγ), inducible nitric oxide synthase (NOS2A)), arginase 2 (ARG2)) were pyrosequenced at the start and end of each sampling period.ResultsHigher levels of BC were associated with lower methylation of IL4 promoter CpG−48 5 days later. The magnitude of association between BC exposure and demethylation of IL4 CpG−48 and NOS2A CpG+5099 measured 5 days later appeared to be greater among seroatopic children, especially those sensitized to cockroach allergens (RR [95% CI] 0.55 [0.37–0.82] and 0.67 [0.45–0.98] for IL4 CpG−48 and NOS2A CpG+5099, respectively), compared to non-sensitized children (RR [95% CI] 0.87 [0.65–1.17] and 0.95 [0.69–1.33] for IL4 CpG−48 and NOS2A CpG+5099, respectively); however, the difference was not statistically different. In multivariable linear regression models, lower DNA methylation of IL4 CpG−48 and NOS2A CpG+5099 were associated with increased FeNO.ConclusionsOur results suggest that exposure to BC may exert asthma proinflammatory gene demethylation 5 days later that in turn may link to airway inflammation. Our results further suggest that seroatopic children, especially those sensitized to cockroach allergens, may be more susceptible to the effect of acute BC exposure on epigenetic changes.

Arginase-2 mediates renal ischemia-reperfusion injury.

Novel therapeutic interventions for preventing or attenuating kidney injury following ischemia-reperfusion injury (IRI) remain a focus of significant interest. Currently, there are no definitive therapeutic or preventive approaches available for ischemic acute kidney injury (AKI). Our objective is to determine 1) whether renal arginase activity or expression is increased in renal IRI, and 2) whether arginase plays a role in development of renal IRI. The impact of arginase activity and expression on renal damage was evaluated in male C57BL/6J (wild type) and arginase-2 (ARG2)-deficient (Arg2-/- ) mice subjected to bilateral renal ischemia for 28 min, followed by reperfusion for 24 h. ARG2 expression and arginase activity significantly increased following renal IRI, paralleling the increase in kidney injury. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection in renal IRI. Arg2-/- mice had significantly attenuated kidney injury and lower plasma creatinine and blood urea nitrogen levels after renal IRI. Blocking arginases using S-(2-boronoethyl)-l-cysteine (BEC) 18 h before ischemia mimicked arginase deficiency by reducing kidney injury, histopathological changes and kidney injury marker-1 expression, renal apoptosis, kidney inflammatory cell recruitment and inflammatory cytokines, and kidney oxidative stress; increasing kidney nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation, kidney peroxisome proliferator-activated receptor-γ coactivator-1α expression, and mitochondrial ATP; and preserving kidney mitochondrial ultrastructure compared with vehicle-treated IRI mice. Importantly, BEC-treated eNOS-knockout mice failed to reduce blood urea nitrogen and creatinine following renal IRI. These findings indicate that ARG2 plays a major role in renal IRI, via an eNOS-dependent mechanism, and that blocking ARG2 activity or expression could be a novel therapeutic approach for prevention of AKI.

Short-term exposure to PM2.5 and vanadium and changes in asthma gene DNA methylation and lung function decrements among urban children

BackgroundBoth short and long-term exposure to traffic-related air pollutants have been associated with asthma and reduced lung function. We hypothesized that short-term indoor exposure to fine particulate matter <2.5 μm (PM2.5) and vanadium (V) would be associated with altered buccal cell DNA methylation of targeted asthma genes and decreased lung function among urban children in a nested subcohort of African American and Dominican children.MethodsSix day integrated levels of air pollutants were measured from children’s homes (age 9–14; n = 163), repeated 6 months later (n = 98). Buccal samples were collected repeatedly during visits. CpG promoter loci of asthma genes (i.e., interleukin 4 (IL4), interferon gamma (IFNγ), inducible nitric oxide synthase (NOS2A), arginase 2 (ARG2)) were pyrosequenced and lung function was assessed.ResultsExposure to V, but not PM2.5, was associated with lower DNA methylation of IL4 and IFNγ. In exploratory analyses, V levels were associated with lower methylation of the proinflammatory NOS2A-CpG+5099 among asthmatic overweight or obese children but not nonasthmatics. Short-term exposure to PM2.5, but not V, appeared associated with lower lung function (i.e., reduced z-scores for forced expiratory volume in one second (FEV1, FEV1/ forced vital capacity [FEV1/FVC] and forced expiratory flow at 25–75% of FVC [FEF25–75]).ConclusionsExposure to V was associated with altered DNA methylation of allergic and proinflammatory asthma genes implicated in air pollution related asthma. However, short-term exposure to PM2.5, but not V, appeared associated with decrements in lung function among urban children.

Influence of arginase polymorphisms and arginase levels/activity on the response to erectile dysfunction therapy with sildenafil.

Arginase 1 (ARG1) and arginase 2 (ARG2) compete with nitric oxide synthases for the substrate l-arginine. Here we aim to assess whether arginase 1 and 2 plasma levels, plasma arginase activity or genetic factors are associated with altered responsiveness to sildenafil. We studied 71 post-prostatectomy erectile dysfunction (ED) patients (PED group) and 72 clinical ED patients (CED). Patients responded to the International Index of Erectile Function questionnaire before and after the treatment. We found positive and negative correlations between plasma levels of arginase 1 and sildenafil responsiveness in the PED and CED groups, respectively. PED group also presented negative correlation between plasma arginase activity and sildenafil responsiveness. Sildenafil poor responders have shown higher plasma arginase activity in PED and higher arginase 1 levels on CED groups. In addition, variant genotypes for the rs2781659, rs2781667 and rs17599586 polymorphisms were associated with reduced arginase activity, as well as the GTTT ARG1 haplotype in CED group.

Reduced bioavailable manganese causes striatal urea cycle pathology in Huntington's disease mouse model

Huntington's disease (HD) is caused by a mutation in the huntingtin gene (HTT), resulting in profound striatal neurodegeneration through an unknown mechanism. Perturbations in the urea cycle have been reported in HD models and in HD patient blood and brain. In neurons, arginase is a central urea cycle enzyme, and the metal manganese (Mn) is an essential cofactor. Deficient biological responses to Mn, and reduced Mn accumulation have been observed in HD striatal mouse and cell models. Here we report in vivo and ex vivo evidence of a urea cycle metabolic phenotype in a prodromal HD mouse model. Further, either in vivo or in vitro Mn supplementation reverses the urea-cycle pathology by restoring arginase activity. We show that Arginase 2 (ARG2) is the arginase enzyme present in these mouse brain models, with ARG2 protein levels directly increased by Mn exposure. ARG2 protein is not reduced in the prodromal stage, though enzyme activity is reduced, indicating that altered Mn bioavailability as a cofactor leads to the deficient enzymatic activity. These data support a hypothesis that mutant HTT leads to a selective deficiency of neuronal Mn at an early disease stage, contributing to HD striatal urea-cycle pathophysiology through an effect on arginase activity.

Arginase-2, a miR-1299 target, enhances pigmentation in melasma by reducing melanosome degradation via senescence-induced autophagy inhibition.

Expression profiles revealed miR-1299 downregulation concomitant with arginase-2 (ARG2) upregulation in hyperpigmented skin of melasma patients. Opposite regulation of tyrosinase and PMEL17 by miR-1299 and inverse relationship between miR-1299 and ARG2 expression denoted a role of miR-1299 in pigmentation with ARG2 as a miR-1299 target. ARG2 overexpression or knock-down in keratinocytes, the main source of ARG2 in epidermis, positively regulated tyrosinase and PMEL17 protein levels, but not mRNA levels or melanosome transfer. ARG2 overexpression in keratinocytes reduced autophagy equivalent to 3-MA, an autophagy inhibitor which also increased tyrosinase and PMEL17 protein levels, whereas ARG2 knock-down induced opposite results. Autophagy inducer rapamycin reduced ARG2-increased tyrosinase and PMEL17 protein levels. Also, autophagy was reduced in late passage-induced senescent keratinocytes showing ARG2 upregulation. ARG2, but not 3-MA, stimulated keratinocyte senescence. These results suggest that ARG2 reduces autophagy in keratinocytes by stimulating cellular senescence, resulting in skin pigmentation by reducing degradation of transferred melanosomes.

Arginase 2 promotes neurovascular degeneration during ischemia/reperfusion injury.

Retinal ischemia is a major cause of visual impairment and blindness and is involved in various disorders including diabetic retinopathy, glaucoma, optic neuropathies and retinopathy of prematurity. Neurovascular degeneration is a common feature of these pathologies. Our lab has previously reported that the ureahydrolase arginase 2 (A2) is involved in ischemic retinopathies. Here, we are introducing A2 as a therapeutic target to prevent neurovascular injury after retinal ischemia/reperfusion (I/R) insult. Studies were performed with mice lacking both copies of A2 (A2−/−) and wild-type (WT) controls (C57BL6J). I/R insult was conducted on the right eye and the left eye was used as control. Retinas were collected for analysis at different times (3 h–4 week after injury). Neuronal and microvascular degeneration were evaluated using NeuN staining and vascular digests, respectively. Glial activation was evaluated by glial fibrillary acidic protein expression. Necrotic cell death was studied by propidium iodide labeling and western blot for RIP-3. Arginase expression was determined by western blot and quantitative RT-PCR. Retinal function was determined by electroretinography (ERG). A2 mRNA and protein levels were increased in WT I/R. A2 deletion significantly reduced ganglion cell loss and microvascular degeneration and preserved retinal morphology after I/R. Glial activation, reactive oxygen species formation and cell death by necroptosis were significantly reduced by A2 deletion. ERG showed improved positive scotopic threshold response with A2 deletion. This study shows for the first time that neurovascular injury after retinal I/R is mediated through increased expression of A2. Deletion of A2 was found to be beneficial in reducing neurovascular degeneration after I/R.