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1.  FoxO4 Promotes Early Inflammatory Response upon Myocardial Infarction via Endothelial Arg1 
Circulation research  2015;117(11):967-977.
Inflammation in post-myocardial infarct (MI) is necessary for myocyte repair and wound healing. Unfortunately it is also a key component of subsequent heart failure pathology. FoxO4 regulates a variety of biological processes including inflammation. However, its role in MI remains unknown.
To test the hypothesis that FoxO4 promotes early post-MI inflammation via endothelial Arg1.
Methods and Results
We induced MI in WT and FoxO4−/− mice. FoxO4−/− mice had a significantly higher post-MI survival, better cardiac function, and reduced infarct size. FoxO4−/− hearts had significantly fewer neutrophils, reduced expression of cytokines and competitive nitric oxide synthase (NOS) inhibitor Arginase 1 (Arg1). We generated conditional FoxO4 knockout mice with FoxO4-deleted in cardiac mycoytes (cKO) or endothelial cells (ecKO). FoxO4 ecKO mice showed significant post-MI improvement of cardiac function and reduction of neutrophil accumulation and cytokine expression whereas FoxO4 cKO had no significant difference in cardiac function and post-MI inflammation from those of control littermates. FoxO4 binds the Foxo-binding site in the Arg1 promoter and activates Arg1 transcription. FoxO4-knockdown in human aortic endothelial cells upregulated nitric oxide upon ischemia and suppressed monocyte adhesion that can be reversed by ectopic-expression of Arg1. Furthermore, chemical inhibition of Arg1 in WT mice had similar cardioprotection and reduced inflammation following MI as FoxO4-inactivation and administration of NOS inhibitor to FoxO4 KO mice reversed the beneficial effects of FoxO4-deletion on post-MI cardiac function.
FoxO4 activates Arg1 transcription in endothelial cells in response to MI, leading to downregulation of nitric oxide and upregulation of neutrophil infiltration to the infarct area.
PMCID: PMC4710860  PMID: 26438688
FoxO4; Arg1; myocardial infarction; inflammation; transcription; endothelial function; endothelial cell
2.  Shaping the Murine Macrophage Phenotype: IL-4 and cAMP Synergistically Activate the Arginase I Promoter 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(5):10.4049/jimmunol.1202102.
Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP (8-Br-cAMP) individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation (ChIP) showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; i.e., differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.
PMCID: PMC3829606  PMID: 23913966
3.  Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms 
Macrophages in granulomas are both anti-mycobacterial effector and host cell for Mycobacterium tuberculosis(M.tb), yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial nitric oxide synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared to non-granulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, while epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68 and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1 and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with greater eNOS expression and ratio of iNOS:Arg1 expression in epithelioid macrophages, as compared to cells in the lymphocyte cuff. iNOS, Arg1 and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas while the inner regions were more likely to contain macrophages with pro-inflammatory, presumably bactericidal, phenotypes. Together these data support the concept that granulomas have organized microenvironments that balance anti-microbial anti-inflammatory responses to limit pathology in the lungs.
PMCID: PMC3746594  PMID: 23749634
4.  Regulation of Macrophage Arginase Expression and Tumor Growth by the Ron Receptor Tyrosine Kinase1 
M1 activation of macrophages promotes inflammation and immunity to intracellular pathogens, while M2 macrophage activation promotes resolution of inflammation, wound healing, and tumor growth. These divergent phenotypes are characterized, in part, by the expression of iNOS and arginase I (Arg1) in M1 vs. M2 activated macrophages, respectively. Here we demonstrate that the Ron receptor tyrosine kinase tips the balance of macrophage activation by attenuating the M1 phenotype while promoting expression of Arg1, through a Stat6-independent mechanism. Induction of the Arg1 promoter by Ron is mediated by an AP-1 site located 433 bp upstream of the transcription start site. Treatment of primary macrophages with MSP, the ligand for Ron, induces potent MAP kinase activation, upregulates Fos, and enhances binding of Fos to the AP-1 site in the Arg1 promoter. In vivo, Arg1 expression in tumor-associated macrophages (TAMs) from Ron−/− mice was significantly reduced compared with TAMs from control animals. Furthermore, we show that Ron is expressed specifically by Tie2-expressing macrophages (TEMs), a TAM subset that exhibits a markedly skewed M2 and pro-tumoral phenotype. Decreased Arg1 in TAMs from Ron−/− mice was associated with reduced syngeneic tumor growth in these animals. These findings indicate that Ron induces Arg1 expression in macrophages through a previously uncharacterized AP-1 site in the Arg1 promoter, and that Ron could be therapeutically targeted in the tumor microenvironment to inhibit tumor growth by targeting expression of Arg1
PMCID: PMC4042865  PMID: 21810604
5.  Arginase inhibition mediates renal tissue protection in diabetic nephropathy by a nitric oxide synthase 3-dependent mechanism 
Kidney international  2013;84(6):10.1038/ki.2013.215.
Recently we showed that pharmacological blockade or genetic deficiency of arginase-2 confers kidney protection in diabetic mouse models. Here we tested whether the protective effect of arginase inhibition is nitric oxide synthase-3 (eNOS)-dependent in diabetic nephropathy. Experiments were conducted in eNOS knockout and their wild type littermate mice using multiple low doses of vehicle or streptozotocin and treated with continuous subcutaneous infusion of vehicle or the arginase inhibitor S-(2-Boronoethyl)-L-cysteine by an osmotic pump. Inhibition of arginases for 6 weeks in diabetic wild type mice significantly attenuated albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathological changes, kidney fibronectin and TNF-α expression, kidney macrophage recruitment, and oxidative stress compared to vehicle-treated diabetic wild type mice. Arginase inhibition in diabetic eNOS knockout mice failed to affect any of these parameters but reduced kidney macrophage recruitment and kidney TNF-α expression compared to vehicle-treated diabetic eNOS knockout mice. Furthermore, diabetic wild type and eNOS knockout mice exhibited increased kidney arginase-2 protein, arginase activity and ornithine levels. Thus, arginase inhibition mediates renal tissue protection in diabetic nephropathy by an eNOS-dependent mechanism and has an eNOS-independent effect on kidney macrophage recruitment.
PMCID: PMC3783645  PMID: 23760286
6.  Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation 
European journal of immunology  2013;43(4):10.1002/eji.201242772.
Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in dendritic cells requires retinoic acid. Induction of Arg1 by retinoic acid is directly mediated by retinoic acid-responsive elements in the 5′ non-coding region of the Arg1 gene. Arg1, produced by dendritic cells in response to retinoic acid, promotes the generation of FoxP3+ regulatory T cells. Importantly, blocking the retinoic acid receptor makes dendritic cells hypo-responsive to known inducers of Arg1 such as IL-4 and GM-CSF in Arg1 expression. We found that intestinal CD103+ dendritic cells that are known to produce retinoic acid highly express Arg1. Our results establish retinoic acid as a key signal in expression of Arg1 in dendritic cells.
PMCID: PMC3826817  PMID: 23322377
arginase; dendritic cells; retinoic acid
7.  A large scale chemical screen for regulators of the arginase 1 promoter identifies the soy isoflavone, daidzein as a clinically approved, small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway 
An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1’s protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from MAG, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.
PMCID: PMC3554247  PMID: 20071539
neuroprotection; regeneration; arginase; isoflavone; SCI; stroke
8.  Arginases and Arginine Deficiency Syndromes 
Purpose of review
Many physiologic and pathophysiologic processes are modulated by arginine availability, which can be regulated by arginase. An understanding of the conditions that result in elevated arginase activity as well as the consequences of arginine deficiency is essential for design of effective nutritional support for disease. This review will emphasize recent findings regarding effects of plasma arginase and arginine deficiencies in disease.
Recent findings
Elevations in plasma arginase, derived primarily from hemolysis of red blood cells or liver damage, that are associated with arginine deficiency have been identified in an increasing number of diseases and conditions. Arginine insufficiency not only can activate a stress kinase pathway that impairs function of T lymphocytes but it also can inhibit the mitogen-activated protein kinase signaling pathway required for macrophage production of cytokines in response to bacterial endotoxin/lipopolysaccharide.
There are at least two broad categories of arginine deficiency syndromes, involving either T cell dysfunction or endothelial dysfunction, depending on the disease context in which arginine deficiency occurs. There is limited information regarding the safety and efficacy of supplementation with arginine or its precursor citrulline in ameliorating arginine deficiency in specific diseases, indicating the need for further studies.
PMCID: PMC3268370  PMID: 22037011
arginine; arginase; nitric oxide; endothelial dysfunction; T cell dysfunction
9.  Arginase-2 Mediates Diabetic Renal Injury 
Diabetes  2011;60(11):3015-3022.
To determine 1) whether renal arginase activity or expression is increased in diabetes and 2) whether arginase plays a role in development of diabetic nephropathy (DN).
The impact of arginase activity and expression on renal damage was evaluated in spontaneously diabetic Ins2Akita mice and in streptozotocin (STZ)-induced diabetic Dilute Brown Agouti (DBA) and arginase-2–deficient mice (Arg2−/−).
Pharmacological blockade or genetic deficiency of arginase-2 conferred kidney protection in Ins2Akita mice or STZ-induced diabetic renal injury. Blocking arginases using S-(2-boronoethyl)-l-cysteine for 9 weeks in Ins2Akita mice or 6 weeks in STZ-induced diabetic DBA mice significantly attenuated albuminuria, the increase in blood urea nitrogen, histopathological changes, and kidney macrophage recruitment compared with vehicle-treated Ins2Akita mice. Furthermore, kidney arginase-2 expression increased in Ins2Akita mice compared with control. In contrast, arginase-1 expression was undetectable in kidneys under normal or diabetes conditions. Arg2−/− mice mimicked arginase blockade by reducing albuminuria after 6 and 18 weeks of STZ-induced diabetes. In wild-type mice, kidney arginase activity increased significantly after 6 and 18 weeks of STZ-induced diabetes but remained very low in STZ-diabetic Arg2−/− mice. The increase in kidney arginase activity was associated with a reduction in renal medullary blood flow in wild-type mice after 6 weeks of STZ-induced diabetes, an effect significantly attenuated in diabetic Arg2−/− mice.
These findings indicate that arginase-2 plays a major role in induction of diabetic renal injury and that blocking arginase-2 activity or expression could be a novel therapeutic approach for treatment of DN.
PMCID: PMC3198072  PMID: 21926276
10.  From Inflammation to Wound Healing: Using a Simple Model to Understand the Functional Versatility of Murine Macrophages 
Bulletin of mathematical biology  2011;73(11):2575-2604.
Macrophages are fundamental cells of the innate immune system. Their activation is essential for such distinct immune functions as inflammation (pathogen-killing) and tissue repair (wound healing). An open question has been the functional stability of an individual macrophage cell: whether it can change its functional profile between different immune responses such as between the repair pathway and the inflammatory pathway. We studied this question theoretically by constructing a rate equation model for the key substrate, enzymes and products of the pathways; we then tested the model experimentally. Both our model and experiments show that individual macrophages can switch from the repair pathway to the inflammation pathway but that the reverse switch does not occur.
PMCID: PMC3131498  PMID: 21347813
Macrophage activation; Inflammation; Wound repair; Ordinary differential equation (ODE) model
11.  LXRα regulates macrophage arginase 1 via PU.1 and IRF8 
Circulation research  2011;109(5):492-501.
Activation of Liver X Receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRα levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion.
To investigate LXRα regulation of Arg1 expression in cultured macrophages and atherosclerotic regressive lesions.
Methods and Results
We found that Arg1 expression is enhanced in CD68+ cells from regressive versus progressive lesions in a murine aortic arch transplant model. In cultured macrophages, ligand-activated LXRα markedly enhances basal and IL-4-induced Arg1 mRNA and protein expression as well as promoter activity. This LXRα-enhanced Arg1 expression correlates with a reduction in nitric oxide levels. Moreover, Arg1 expression within regressive atherosclerotic plaques is LXRα-dependent as enhanced expression of Arg1 in regressive lesions is impaired in LXRα-deficient CD68+ cells. LXRα does not bind to the Arg1 promoter but instead promotes the interaction between PU.1 and IRF8 transcription factors and induces their binding of a novel composite element. Accordingly, knockdown of either IRF8 or PU.1 strongly impair LXRα regulation of Arg1 expression in macrophage cells. Finally, we demonstrate that LXRα binds the IRF8 locus and its activation increases IRF8 mRNA and protein levels in these cells.
This work implicates Arg1 in atherosclerosis regression and identifies LXRα as a novel regulator of Arg1 and IRF8 in macrophages. Furthermore it provides a unique molecular mechanism by which LXRα regulates macrophage target gene expression via PU.1 and IRF8.
PMCID: PMC3180895  PMID: 21757649
Atherosclerosis; Liver X receptor; Interferon Regulatory Factor 8; Arginase 1; Macrophages
12.  Selective Endothelial Overexpression of Arginase II Induces Endothelial Dysfunction and Hypertension and Enhances Atherosclerosis in Mice 
PLoS ONE  2012;7(7):e39487.
Cardiovascular disorders associated with endothelial dysfunction, such as atherosclerosis, have decreased nitric oxide (NO) bioavailability. Arginase in the vasculature can compete with eNOS for L-arginine and has been implicated in atherosclerosis. The aim of this study was to evaluate the effect of endothelial-specific elevation of arginase II expression on endothelial function and the development of atherosclerosis.
Methodology/Principal Findings
Transgenic mice on a C57BL/6 background with endothelial-specific overexpression of human arginase II (hArgII) gene under the control of the Tie2 promoter were produced. The hArgII mice had elevated tissue arginase activity except in liver and in resident peritoneal macrophages, confirming endothelial specificity of the transgene. Using small-vessel myography, aorta from these mice exhibited endothelial dysfunction when compared to their non-transgenic littermate controls. The blood pressure of the hArgII mice was 17% higher than their littermate controls and, when crossed with apoE −/− mice, hArgII mice had increased aortic atherosclerotic lesions.
We conclude that overexpression of arginase II in the endothelium is detrimental to the cardiovascular system.
PMCID: PMC3400622  PMID: 22829869
13.  Nitrite-generated NO circumvents dysregulated arginine/NOS signaling to protect against intimal hyperplasia in Sprague-Dawley rats 
The Journal of Clinical Investigation  2011;121(4):1646-1656.
Vascular disease, a significant cause of morbidity and mortality in the developed world, results from vascular injury. Following vascular injury, damaged or dysfunctional endothelial cells and activated SMCs engage in vasoproliferative remodeling and the formation of flow-limiting intimal hyperplasia (IH). We hypothesized that vascular injury results in decreased bioavailability of NO secondary to dysregulated arginine-dependent NO generation. Furthermore, we postulated that nitrite-dependent NO generation is augmented as an adaptive response to limit vascular injury/proliferation and can be harnessed for its protective effects. Here we report that sodium nitrite (intraperitoneal, inhaled, or oral) limited the development of IH in a rat model of vascular injury. Additionally, nitrite led to the generation of NO in vessels and SMCs, as well as limited SMC proliferation via p21Waf1/Cip1 signaling. These data demonstrate that IH is associated with increased arginase-1 levels, which leads to decreased NO production and bioavailability. Vascular injury also was associated with increased levels of xanthine oxidoreductase (XOR), a known nitrite reductase. Chronic inhibition of XOR and a diet deficient in nitrate/nitrite each exacerbated vascular injury. Moreover, established IH was reversed by dietary supplementation of nitrite. The vasoprotective effects of nitrite were counteracted by inhibition of XOR. These data illustrate the importance of nitrite-generated NO as an endogenous adaptive response and as a pathway that can be harnessed for therapeutic benefit.
PMCID: PMC3069768  PMID: 21436585
14.  Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates 
Nucleic Acids Research  2011;39(6):e39.
We describe a novel 3′-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2′-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I–dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3′-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.
PMCID: PMC3064798  PMID: 21227920
15.  Arginase Activities and Global Arginine Bioavailability in Wild-Type and ApoE-Deficient Mice: Responses to High Fat and High Cholesterol Diets 
PLoS ONE  2010;5(12):e15253.
Increased catabolism of arginine by arginase is increasingly viewed as an important pathophysiological factor in cardiovascular disease, including atherosclerosis induced by high cholesterol diets. Whereas previous studies have focused primarily on effects of high cholesterol diets on arginase expression and arginine metabolism in specific blood vessels, there is no information regarding the impact of lipid diets on arginase activity or arginine bioavailability at a systemic level. We, therefore, evaluated the effects of high fat (HF) and high fat-high cholesterol (HC) diets on arginase activity in plasma and tissues and on global arginine bioavailability (defined as the ratio of plasma arginine to ornithine + citrulline) in apoE−/− and wild-type C57BL/6J mice. HC and HF diets led to reduced global arginine bioavailability in both strains. The HC diet resulted in significantly elevated plasma arginase in both strains, but the HF diet increased plasma arginase only in apoE−/− mice. Elevated plasma arginase activity correlated closely with increased alanine aminotransferase levels, indicating that liver damage was primarily responsible for elevated plasma arginase. The HC diet, which promotes atherogenesis, also resulted in increased arginase activity and expression of the type II isozyme of arginase in multiple tissues of apoE−/− mice only. These results raise the possibility that systemic changes in arginase activity and global arginine bioavailability may be contributing factors in the initiation and/or progression of cardiovascular disease.
PMCID: PMC2997799  PMID: 21151916
16.  Recent advances in arginine metabolism: roles and regulation of the arginases 
British Journal of Pharmacology  2009;157(6):922-930.
As arginine can serve as precursor to a wide range of compounds, including nitric oxide, creatine, urea, polyamines, proline, glutamate and agmatine, there is considerable interest in elucidating mechanisms underlying regulation of its metabolism. It is now becoming apparent that the two isoforms of arginase in mammals play key roles in regulation of most aspects of arginine metabolism in health and disease. In particular, work over the past several years has focused on the roles and regulation of the arginases in vascular disease, pulmonary disease, infectious disease, immune cell function and cancer. As most of these topics have been considered in recent review articles, this review will focus more closely on results of recent studies on expression of the arginases in endothelial and vascular smooth muscle cells, post-translational modulation of arginase activity and applications of arginase inhibitors in vivo.
PMCID: PMC2737650  PMID: 19508396
arginase; arginase inhibitors; arginine; cancer; endothelial; genetic knockout; nitric oxide; polyamines; vascular
17.  Cell- and Isoform-specific Increases in Arginase Expression in Acute Silica-induced Pulmonary Inflammation 
Arginase induction was reported in several inflammatory lung diseases, suggesting that this may be a common feature underlying the pathophysiology of such diseases. As little is known regarding arginase expression in silicosis, the induction and cellular localization of arginase was elucidated in lungs of Sprague-Dawley rats 24 hr following exposure to varying doses of silica by intratracheal instillation. Arginase expression was evaluated by activity assay, quantification of arginase I and arginase II mRNA levels using real-time PCR, and immunohistochemistry. Analyses of cells and fluid obtained by bronchoalveolar lavage (BAL) showed that markers of pulmonary inflammation, tissue damage, activation of alveolar macrophages (AM) and NO production were significantly increased by all silica doses. Arginase activity was increased also in AMs isolated from BAL fluid of silica-treated rats. Silica produced 2- and 3-fold increases in arginase activity of whole lung at doses of 1 and 5 mg/100g body weight, respectively. Levels of arginase I mRNA, but not of arginase II mRNA, were similarly elevated. In control lungs, arginase I immunoreactivity was observed only in AMs sparsely dispersed throughout the lung; no iNOS immunoreactivity was detected. In silica-treated lungs, arginase I and iNOS were co-expressed in most AMs that were abundantly clustered at inflammatory foci. The rapid induction of arginase I expression in inflammatory lung cells, similar to induction of arginase in other inflammatory lung diseases, implicates elevated arginase activity as a factor in the development of lung damage following exposure to silica.
PMCID: PMC2773696  PMID: 17365572
iNOS; nitric oxide; macrophage; silicosis; inflammation; immunohistochemistry
18.  The ‘intestinal–renal’ arginine biosynthetic axis in the aging rat 
Mechanisms of ageing and development  2002;123(8):1159-1165.
It has been suggested that l-arginine availability declines with advanced age, which could contribute to the endothelial dysfunction and decreased nitric oxide (NO) production that are features of aging. l-Arginine is made in the kidney and since the aging kidney develops progressive injury there may be decreased synthesis limiting availability. In this study we investigated the impact of aging on the regulation, at the gene level, of the various enzymes that synthesize l-arginine in the kidney (argininosuccinate synthetase and argininosuccinate lyase) and citrulline, the precursor of l-arginine made in the small intestine (phosphate-dependent glutaminase, carbamyl phosphate synthetase-1 and ornithine transcarbamylase). Studies were in young (3–5 months), middle-aged (11–13 months) and old (18–22 months) male and female Sprague–Dawley rats aged under barrier conditions. The plasma, renal cortical and brain cerebellar levels of l-arginine are unchanged in the old male rat, and expression of the genes involved in renal arginine synthesis and small intestinal citrulline synthesis is unchanged or upregulated with age in both males and females. This study shows that the synthesis of l-arginine is maintained with aging despite developing kidney damage. Therefore, the reduced NO generating capacity that occurs in aging must be due to downstream changes in the NO biosynthesis pathway, such as reduced abundance of NO biosynthetic enzymes.
PMCID: PMC2745251  PMID: 12044965
Citrulline; Argininosuccinate synthetase; Argininosuccinate lyase; Phosphate-dependent glutaminase; Carbamyl phosphate synthetase-1; Ornithine transcarbamylase
Urology  2008;72(3):696-700.
We investigated the effects of an arginase inhibitor on bladder overactivity and measured bladder arginase I and II mRNA levels in rats with chronic spinal cord injury (SCI).
Awake cystometrograms were performed 3–4 weeks after spinal cord transection in female rats. Cystometric parameters such as mean amplitudes and number of non-voiding contractions (NVCs), voided volume, voiding efficiency, and micturition pressure were evaluated before and after intravenous (i.v.) injection of an arginase inhibitor (nor-NOHA: Nω-Hydroxy-nor-L-arginine) in SCI rats. The effects of a NOS inhibitor (L-NAME: Nω-Nitro-L-arginine methyl ester hydrochloride) were also examined to determine whether suppression of bladder overactivity by arginase inhibition is mediated by increased production of NO. In addition, mRNA levels of arginase I and II in SCI bladders were measured using quantitative real–time polymerase chain reaction (qRT-PCR).
nor-NOHA (10 mg/kg, i.v.) significantly decreased the amplitude and number of NVCs. There were no significant changes in other parameters before and after administration of vehicle or nor-NOHA at any dose. When L-NAME (20 mg/kg, i.v.) was administered prior to nor-NOHA injection (10 mg/kg, i.v.), nor-NOHA–induced inhibition of NVCs was prevented. The relative levels of both arginase I and II mRNA in the bladder were significantly higher in SCI rats compared to spinal cord intact rats.
These results suggest that arginase inhibition can suppress SCI-induced bladder overactivity as indicated by a reduction in NVCs. Thus, arginase inhibition could be an effective treatment for neurogenic bladder overactivity in pathological conditions such as SCI.
PMCID: PMC2574745  PMID: 18358516
Arginase inhibitor; NO; Bladder overactivity; Spinal cord injury
20.  The DNA sequence of the human X chromosome 
Ross, Mark T. | Grafham, Darren V. | Coffey, Alison J. | Scherer, Steven | McLay, Kirsten | Muzny, Donna | Platzer, Matthias | Howell, Gareth R. | Burrows, Christine | Bird, Christine P. | Frankish, Adam | Lovell, Frances L. | Howe, Kevin L. | Ashurst, Jennifer L. | Fulton, Robert S. | Sudbrak, Ralf | Wen, Gaiping | Jones, Matthew C. | Hurles, Matthew E. | Andrews, T. Daniel | Scott, Carol E. | Searle, Stephen | Ramser, Juliane | Whittaker, Adam | Deadman, Rebecca | Carter, Nigel P. | Hunt, Sarah E. | Chen, Rui | Cree, Andrew | Gunaratne, Preethi | Havlak, Paul | Hodgson, Anne | Metzker, Michael L. | Richards, Stephen | Scott, Graham | Steffen, David | Sodergren, Erica | Wheeler, David A. | Worley, Kim C. | Ainscough, Rachael | Ambrose, Kerrie D. | Ansari-Lari, M. Ali | Aradhya, Swaroop | Ashwell, Robert I. S. | Babbage, Anne K. | Bagguley, Claire L. | Ballabio, Andrea | Banerjee, Ruby | Barker, Gary E. | Barlow, Karen F. | Barrett, Ian P. | Bates, Karen N. | Beare, David M. | Beasley, Helen | Beasley, Oliver | Beck, Alfred | Bethel, Graeme | Blechschmidt, Karin | Brady, Nicola | Bray-Allen, Sarah | Bridgeman, Anne M. | Brown, Andrew J. | Brown, Mary J. | Bonnin, David | Bruford, Elspeth A. | Buhay, Christian | Burch, Paula | Burford, Deborah | Burgess, Joanne | Burrill, Wayne | Burton, John | Bye, Jackie M. | Carder, Carol | Carrel, Laura | Chako, Joseph | Chapman, Joanne C. | Chavez, Dean | Chen, Ellson | Chen, Guan | Chen, Yuan | Chen, Zhijian | Chinault, Craig | Ciccodicola, Alfredo | Clark, Sue Y. | Clarke, Graham | Clee, Chris M. | Clegg, Sheila | Clerc-Blankenburg, Kerstin | Clifford, Karen | Cobley, Vicky | Cole, Charlotte G. | Conquer, Jen S. | Corby, Nicole | Connor, Richard E. | David, Robert | Davies, Joy | Davis, Clay | Davis, John | Delgado, Oliver | DeShazo, Denise | Dhami, Pawandeep | Ding, Yan | Dinh, Huyen | Dodsworth, Steve | Draper, Heather | Dugan-Rocha, Shannon | Dunham, Andrew | Dunn, Matthew | Durbin, K. James | Dutta, Ireena | Eades, Tamsin | Ellwood, Matthew | Emery-Cohen, Alexandra | Errington, Helen | Evans, Kathryn L. | Faulkner, Louisa | Francis, Fiona | Frankland, John | Fraser, Audrey E. | Galgoczy, Petra | Gilbert, James | Gill, Rachel | Glöckner, Gernot | Gregory, Simon G. | Gribble, Susan | Griffiths, Coline | Grocock, Russell | Gu, Yanghong | Gwilliam, Rhian | Hamilton, Cerissa | Hart, Elizabeth A. | Hawes, Alicia | Heath, Paul D. | Heitmann, Katja | Hennig, Steffen | Hernandez, Judith | Hinzmann, Bernd | Ho, Sarah | Hoffs, Michael | Howden, Phillip J. | Huckle, Elizabeth J. | Hume, Jennifer | Hunt, Paul J. | Hunt, Adrienne R. | Isherwood, Judith | Jacob, Leni | Johnson, David | Jones, Sally | de Jong, Pieter J. | Joseph, Shirin S. | Keenan, Stephen | Kelly, Susan | Kershaw, Joanne K. | Khan, Ziad | Kioschis, Petra | Klages, Sven | Knights, Andrew J. | Kosiura, Anna | Kovar-Smith, Christie | Laird, Gavin K. | Langford, Cordelia | Lawlor, Stephanie | Leversha, Margaret | Lewis, Lora | Liu, Wen | Lloyd, Christine | Lloyd, David M. | Loulseged, Hermela | Loveland, Jane E. | Lovell, Jamieson D. | Lozado, Ryan | Lu, Jing | Lyne, Rachael | Ma, Jie | Maheshwari, Manjula | Matthews, Lucy H. | McDowall, Jennifer | McLaren, Stuart | McMurray, Amanda | Meidl, Patrick | Meitinger, Thomas | Milne, Sarah | Miner, George | Mistry, Shailesh L. | Morgan, Margaret | Morris, Sidney | Müller, Ines | Mullikin, James C. | Nguyen, Ngoc | Nordsiek, Gabriele | Nyakatura, Gerald | O’Dell, Christopher N. | Okwuonu, Geoffery | Palmer, Sophie | Pandian, Richard | Parker, David | Parrish, Julia | Pasternak, Shiran | Patel, Dina | Pearce, Alex V. | Pearson, Danita M. | Pelan, Sarah E. | Perez, Lesette | Porter, Keith M. | Ramsey, Yvonne | Reichwald, Kathrin | Rhodes, Susan | Ridler, Kerry A. | Schlessinger, David | Schueler, Mary G. | Sehra, Harminder K. | Shaw-Smith, Charles | Shen, Hua | Sheridan, Elizabeth M. | Shownkeen, Ratna | Skuce, Carl D. | Smith, Michelle L. | Sotheran, Elizabeth C. | Steingruber, Helen E. | Steward, Charles A. | Storey, Roy | Swann, R. Mark | Swarbreck, David | Tabor, Paul E. | Taudien, Stefan | Taylor, Tineace | Teague, Brian | Thomas, Karen | Thorpe, Andrea | Timms, Kirsten | Tracey, Alan | Trevanion, Steve | Tromans, Anthony C. | d’Urso, Michele | Verduzco, Daniel | Villasana, Donna | Waldron, Lenee | Wall, Melanie | Wang, Qiaoyan | Warren, James | Warry, Georgina L. | Wei, Xuehong | West, Anthony | Whitehead, Siobhan L. | Whiteley, Mathew N. | Wilkinson, Jane E. | Willey, David L. | Williams, Gabrielle | Williams, Leanne | Williamson, Angela | Williamson, Helen | Wilming, Laurens | Woodmansey, Rebecca L. | Wray, Paul W. | Yen, Jennifer | Zhang, Jingkun | Zhou, Jianling | Zoghbi, Huda | Zorilla, Sara | Buck, David | Reinhardt, Richard | Poustka, Annemarie | Rosenthal, André | Lehrach, Hans | Meindl, Alfons | Minx, Patrick J. | Hillier, LaDeana W. | Willard, Huntington F. | Wilson, Richard K. | Waterston, Robert H. | Rice, Catherine M. | Vaudin, Mark | Coulson, Alan | Nelson, David L. | Weinstock, George | Sulston, John E. | Durbin, Richard | Hubbard, Tim | Gibbs, Richard A. | Beck, Stephan | Rogers, Jane | Bentley, David R.
Nature  2005;434(7031):325-337.
The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
PMCID: PMC2665286  PMID: 15772651
21.  Amplified Expression Profiling of Platelet Transcriptome Reveals Changes in Arginine Metabolic Pathways in Patients With Sickle Cell Disease 
Circulation  2007;115(12):1551-1562.
In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, which suggests that pathological platelet activation may contribute to sickle cell disease vasculopathy.
Methods and Results
Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate in the present study the feasibility of comparative platelet transcriptome studies on clinical samples from single donors by the application of RNA amplification followed by microarray-based analysis of 54 000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by >10-fold increased expression compared with the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the present study included 82% or 70% of platelet-abundant genes identified in 2 previous gene expression studies on nonamplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to those of 12 black control subjects, at a 3-fold cutoff and 5% false-discovery rate, we identified ≈100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways with real-time polymerase chain reaction and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels.
The present studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology.
PMCID: PMC2225987  PMID: 17353439
platelets; genes; enzymes; metabolism; thrombolysis; polymerase chain reaction; signal transduction
22.  Dysregulated Arginine Metabolism, Hemolysis-Associated Pulmonary Hypertension and Mortality in Sickle Cell Disease 
Sickle cell disease is characterized by a state of nitric oxide (NO) resistance and limited bioavailability of L-arginine, the substrate for NO synthesis. We hypothesized that increased arginase activity and dysregulated arginine metabolism contribute to endothelial dysfunction, pulmonary hypertension and patient outcome.
To explore the role of arginase in sickle cell disease pathogenesis, pulmonary hypertension and prospective mortality.
Plasma amino acid levels, plasma and erythrocyte arginase activities, and pulmonary hypertension status as measured by Doppler-echocardiogram were prospectively obtained in outpatients with sickle cell disease. Patients were followed for survival up to 49 months.
Urban, tertiary care center and community clinics.
Two hundred twenty-eight patients with sickle cell disease aged 18 to 74 years and 36 control subjects.
Main Outcome Measures
Plasma amino acid levels, plasma and erythrocyte arginase activities, diagnosis of pulmonary hypertension, and mortality.
Plasma arginase activity was significantly elevated in patients with sickle cell disease, with highest activity found in subjects with secondary pulmonary hypertension. Arginase activity correlated with the arginine-to-ornithine ratio, and lower ratios were associated with greater severity of pulmonary hypertension and with mortality in this population (risk ratio: 2.5; 95% confidence interval [1.2, 5.2], p=0.006). Global arginine bioavailability, characterized by the arginine-to-(ornithine plus citrulline) ratio, was also strongly associated with mortality (risk ratio: 3.6; [1.5, 8.3], p<0.001). Increased plasma arginase activity was highly correlated with increased intravascular hemolytic rate and, to a lesser extent, markers of inflammation and soluble adhesion molecule levels.
These data support a novel mechanism of disease in which hemolysis contributes to reduced NO bioavailability and endothelial dysfunction, via release of erythrocyte arginase, which limits arginine bioavailability, and release of erythrocyte hemoglobin, which scavenges NO. The arginine-to-ornithine and arginine-to-(ornithine plus citrulline) ratios are independently associated with pulmonary hypertension and increased mortality in patients with sickle cell disease.
PMCID: PMC2065861  PMID: 15998894
23.  Termination of DNA synthesis by N6-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates 
Nucleic Acids Research  2007;35(19):6339-6349.
The Human Genome Project has facilitated the sequencing of many species, yet the current Sanger method is too expensive, labor intensive and time consuming to accomplish medical resequencing of human genomes en masse. Of the ‘next-generation’ technologies, cyclic reversible termination (CRT) is a promising method with the goal of producing accurate sequence information at a fraction of the cost and effort. The foundation of this approach is the reversible terminator (RT), its chemical and biological properties of which directly impact the performance of the sequencing technology. Here, we have discovered a novel paradigm in RT chemistry, the attachment of a photocleavable, 2-nitrobenzyl group to the N6-position of 2′-deoxyadenosine triphosphate (dATP), which, upon incorporation, terminates DNA synthesis. The 3′-OH group of the N6-(2-nitrobenzyl)-dATP remains unblocked, providing favorable incorporation and termination properties for several commercially available DNA polymerases while maintaining good discrimination against mismatch incorporations. Upon removal of the 2-nitrobenzyl group with UV light, the natural nucleotide is restored without molecular scarring. A five-base experiment, illustrating the exquisite, stepwise addition through a homopolymer repeat, demonstrates the applicability of the N6-(2-nitrobenzyl)-dATP as an ideal RT for CRT sequencing.
PMCID: PMC2095803  PMID: 17881370
24.  Generation of a Mouse Model for Arginase II Deficiency by Targeted Disruption of the Arginase II Gene 
Molecular and Cellular Biology  2001;21(3):811-813.
Mammals express two isoforms of arginase, designated types I and II. Arginase I is a component of the urea cycle, and inherited defects in arginase I have deleterious consequences in humans. In contrast, the physiologic role of arginase II has not been defined, and no deficiencies in arginase II have been identified in humans. Mice with a disruption in the arginase II gene were created to investigate the role of this enzyme. Homozygous arginase II-deficient mice were viable and apparently indistinguishable from wild-type mice, except for an elevated plasma arginine level which indicates that arginase II plays an important role in arginine homeostasis.
PMCID: PMC86672  PMID: 11154268

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