Am J Physiol Gastrointest Liver Physiol 307: G347–G354, 2014. First published June 5, 2014; doi:10.1152/ajpgi.00403.2013.
Argininosuccinate lyase in enterocytes protects from development of necrotizing enterocolitis M. H. Premkumar,1 G. Sule,2 S. C. Nagamani,2 S. Chakkalakal,2 A. Nordin,2 M. Jain,2 M. Z. Ruan,2 T. Bertin,2 B. Dawson,2 J. Zhang,2 D. Schady,3 N. S. Bryan,4 P. M. Campeau,2 A. Erez,5 and B. Lee2,6 1
Division of Neonatology, Texas Children’s Hospital, Baylor College of Medicine; 2Department of Molecular and Human Genetics, Baylor College of Medicine; 3Department of Pathology, Texas Children’s Hospital, Houston; 4University of Texas Health Science Center at Houston, Texas; 5Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel; and 6Howard Hughes Medical Institute, Houston, Texas Submitted 21 November 2013; accepted in final form 3 June 2014
Premkumar MH, Sule G, Nagamani SC, Chakkalakal S, Nordin A, Jain M, Ruan MZ, Bertin T, Dawson B, Zhang J, Schady D, Bryan NS, Campeau PM, Erez A, Lee B. Argininosuccinate lyase in enterocytes protects from development of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 307: G347–G354, 2014. First published June 5, 2014; doi:10.1152/ajpgi.00403.2013.— Necrotizing enterocolitis (NEC), the most common neonatal gastrointestinal emergency, results in significant mortality and morbidity, yet its pathogenesis remains unclear. Argininosuccinate lyase (ASL) is the only enzyme in mammals that is capable of synthesizing arginine. Arginine has several homeostatic roles in the gut and its deficiency has been associated with NEC. Because enterocytes are the primary sites of arginine synthesis in neonatal mammals, we evaluated the consequences of disruption of arginine synthesis in the enterocytes on the pathogenesis of NEC. We devised a novel approach to study the role of enterocyte-derived ASL in NEC by generating and characterizing a mouse model with enterocyte-specific deletion of Asl (Aslflox/flox; VillinCretg/⫹, or CKO). We hypothesized that the presence of ASL in a cell-specific manner in the enterocytes is protective in the pathogenesis of NEC. Loss of ASL in enterocytes resulted in an increased incidence of NEC that was associated with a proinflammatory state and increased enterocyte apoptosis. Knockdown of ASL in intestinal epithelial cell lines resulted in decreased migration in response to lipopolysaccharide. Our results show that enterocyte-derived ASL has a protective role in NEC. NEC; ASL; arginine
is a critical step in conversion of waste nitrogen into urea, whereas, in most other tissues, this is the sole pathway for synthesis of endogenous arginine. We recently showed that ASL is an essential regulator in the synthesis of nitric oxide (NO) (6). We demonstrated that ASL is required to maintain a NO synthesis complex containing NO synthase (NOS), argininosuccinate synthase, HSP90, and the arginine transporter SLC7A1. In the absence of ASL, this complex is assembled less efficiently, despite unaltered levels of the remaining components. Loss of ASL not only leads to loss of arginine recycling from citrulline, that is, intracellular de novo arginine synthesis, but also to loss of channeling of extracellular arginine to NOS for NO production. Hence, loss of ASL leads to deficiency of NOS-dependent NO production at the cellular level. Moreover, we have shown that this structural requirement for ASL in NO production is conserved, because humans with structural and catalytic deficiency of ASL are also NO deficient. To study the cell-autonomous role of ASL in NEC, we generated a novel mouse model with enterocyte-specific deletion of Asl. We show that the loss of enterocyte-derived ASL results in increased incidence of NEC, and hence manipulating ASL expression or activity could be of potential benefit for treatment of NEC. METHODS
(NEC), the most common gastrointestinal emergency in extremely low birth weight newborn infants, is caused by complex pathogenic mechanisms (12, 16). The incidence of NEC in North American neonatal intensive care units is reported to be 3–11% (8 –10). A recent study from over 600 hospitals in the United States demonstrated a significant increase in the incidence of NEC between 1999 and 2009, while all the other neonatal morbidities demonstrated a stable or declining trend (8, 10, 11). Observational studies have shown decreased levels of plasma arginine in the immediate weeks preceding the onset of NEC (4, 21), and arginine supplementation has been shown to have some beneficial effects in the prevention of NEC (1, 19). Enterocytes are the main sites of arginine synthesis in neonatal mammals. The synthesis of arginine is dependent on argininosuccinate lyase (ASL), which catalyzes the conversion of argininosuccinate to arginine and fumarate. In the liver, this
NECROTIZING ENTEROCOLITIS
Address for reprint requests and other correspondence: B. Lee, Dept. of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, R814, MS225 Houston, TX 77030 (e-mail: [email protected]). http://www.ajpgi.org
The experimental procedures were approved by the Animal Care and Use Committee of Baylor College of Medicine, Houston, TX. Generation of enterocyte-specific conditional ASL knockout mice. Aslflox[FRT-neo-FRT]/⫹ mice carrying the neomycin phosphotransferase (neo) gene were generated as previously described (6). The Aslflox[FRT-neo-FRT]/⫹ mice were crossed with transgenic mice expressing Flp recombinase to remove the neo cassette, leaving behind the loxP sites flanking exons 7–9 of Asl . The resulting mice with the Aslflox/⫹ allele were interbred to generate Aslflox/flox mice, which were further crossed with transgenic mice bearing cre-recombinase expressed under the control of the enterocyte-specific mouse villin 1 promoter [B6.SJL-Tg(Vil-cre)997 Gum/J; Jackson Laboratory, Bar Harbor, ME] to generate mice specifically lacking ASL within the enterocytes, Aslflox/flox, VillinCretg/⫹, or CKO. Induction of NEC. NEC was induced by techniques previously described by Barlow et al. (3) and Caplan et al. (5). Briefly, newborn mice pups were delivered at 18.5 days of gestation by cesarean section and were maintained in an incubator with constant temperature of 36°C and humidity of 55%. These pups were gavage fed infant formula (Similac PM60/40, Abbott Nutrition, Columbus, OH) every 3 h for 72 h using a 2F polystyrene catheter. Infant formula was prepared at a concentration of 1 kcal/ml and fed at a volume of 200 ml·kg⫺1·day⫺1. Lipopolysaccharide (LPS; L-1887; Sigma-Aldrich,
0193-1857/14 Copyright © 2014 the American Physiological Society
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St. Louis, MO) was introduced into the feed on day 1 at 2 mg/kg. Pups were exposed to hypoxia (99% N2) for 1 min in a hypoxic chamber, followed by hypothermia (4°C) for 10 min twice daily for 3 days (Fig. 1A) (20). The animals were killed when they appeared to be severely ill with signs such as abdominal distension, lethargy, cyanosis, or at the end of 72 h. Terminal ilea were obtained for histological and biochemical analysis. The diagnosis of NEC was made based on the assessment of two independent observers, including a pathologist, both of whom were blinded to the genotype. NEC was graded based on the severity of the damage from the mucosal to serosal surface as follows: Grade 0 ⫽ intact villi, Grade 1⫽ sloughing of cells on villous tips, Grade 2 ⫽ midvillous damage, Grade 3 ⫽ villi absent but crypts still detectable, and Grade 4 ⫽ complete absence of epithelial structures and transmural necrosis (Fig. 1). Intestinal cross sections with changes higher than or equal to Grade 2 were classified as NEC. Histology and tissue staining. Paraffin-embedded tissues were sectioned to a 4- to 7-m thickness and stained with hematoxylin and eosin as published (15). Immunohistochemistry. Immunohistochemistry was performed using the Standard avidin biotin complex (ABC) method with a Vectastain ABC kit following the manufacturer’s protocols. Tissues were fixed in 4% formaldehyde and paraffin-embedded using standard protocols. Sections were deparaffinized and hydrated in graded ethanol. Specific staining was enhanced by using a heat-induced epitope retrieval method. Sections were then exposed to 3% H2O2 in PBS for 30 min followed by blocking with normal serum blocker buffer. The sections were then incubated overnight with the primary antibody. The following day, sections were incubated with biotinylated second-
ary antibody for 60 min. Sections were incubated in ABC solution for 1 h at room temperature. Finally, the sections were incubated in peroxidase substrate solution (3,3=-diaminobenzidine) and viewed under a microscope. Incubation without primary antibody served as the negative control for all incubations. The following primary antibodies were used in the immunostaining experiments: ASL monoclonal antibody (M01) (no. H00000435-M01, Abnova, Walnut, CA), neutrophil antibody Ly-6B.2 Alloantigen Antibody (AbD Serotec, Raleigh, NC). Western blot. The intestinal tissues were homogenized and lysed in protein lysis buffer (50 mM Tris·HCl, pH 7.5; 150 mM NaCl; 1% Triton-100) with a complete protease inhibitor cocktail (Roche) and centrifuged at 14,000 rpm for 10 min. Laemmli loading buffer and 5% -mercaptoethanol were added to the supernatants and boiled for 10 min at 95°C. Equal amounts of protein from these samples were separated on 10% SDS polyacrylamide gels and then blotted onto polyvinylidene difluoride membranes which were incubated with the primary antibodies overnight at 4°C. The following day, these membranes were incubated with the corresponding secondary antibodies. Protein quantification was performed using a Licor Odyssey quantitative fluorescence imaging system according to the manufacturer’s instructions. The following primary antibodies were used in the Western blot experiments: ASL monoclonal antibody (M01) (no. H00000435-M01, Abnova, BAX (Bcl2 associated X protein) antibody (no. 2772, Cell Signaling, Danvers, MA), AIF (apoptosis-inducing factor) antibody (no. 4642, Cell Signaling), caspase-3 (no. 9662, Cell Signaling), anti-GAPDH (no. AM4300, Ambion, Grand Island, NY), monoclonal anti-␣-tubulin antibody (T5168, Sigma-Aldrich, St. Louis, MO).
A Day 1
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Exclusive Formula feed by gavage Every 3 hours for 72 hours Twice daily X 3 days Hypoxia (1 min 99% N2)+ Hypothermia (4C 10 minutes) LPS in milk (2 mg/Kg once on day 1) C section birth E 18.5
Tissue Analysis
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Fig. 1. Schematic representation of the protocol used for mouse model of necrotizing enterocolitis (NEC). Images of hematoxylin and eosin sections of terminal ileum of mouse pups representative of histological changes suggestive of Grades 1– 4 of NEC are shown. LPS, lipopolysaccharide; C section, caesarean section; E18.5, 18.5 days of gestation. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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Real-time quantitative PCR. RNA was extracted from either whole intestinal homogenates or enterocytes using TRIzol (Invitrogen, Carlsbad, CA), and first-strand cDNA synthesis was performed with the SuperScript III reverse transcriptase kit (Invitrogen, Grand Island, NY). Real-time quantitative PCR (qPCR) was performed on the LightCycler version 1.1 instruments with Roche Applied Science (Indianapolis, IN) reagents, according to the manufacturer’s recommendations. cDNA template concentrations were determined using Ribogreen (Invitrogen), and equivalent concentrations were used for each qPCR. Fluorescence was captured at the end of each extension cycle, over a total of 40 cycles. Crossing points were determined by a second derivative algorithm intrinsic to the LightCycler software and normalized to a constitutively expressed gene (2-microglobulin or GAPDH). RNA fold-change was calculated to show the difference in transcript expression between samples normalized to an unregulated reference gene of equal abundance in the specimens tested. Enterocyte isolation by the mechanical dissociation method. Intestinal segments from neonatal pups were everted onto a glass rod attached to a vibrating source (electric toothbrush). The everted intestinal segments were then shaken in HBSS media, without calcium and magnesium, with 40 mM EDTA for 20 min on ice. Following this, the media with the dissociated enterocytes were centrifuged at 700 g to collect the enterocyte pellet. Apoptosis assays. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining was performed by using the Apoptag in situ apoptosis detection kit (Chemicon International, Billerica, MA) according to the manufacturer’s instructions. TUNEL-positive enterocytes were counted in 10 randomly selected villi in five ⫻200 high-power fields per each intestinal cross section. Preparation of intestinal villi samples by laser capture microscopy. The intestines were flushed with PBS, opened along their cephalo-
A
caudal axis, and frozen in optimal cutting temperature compound. Frozen sections of 10 m were generated on polyethylene napthalate membrane slides. Enterocytes from the villi and the crypts excluding the core of the villi were captured under a laser capture microscope (Pix Cell II; Arcturus, Mountain View, CA) using HS Capsure LCM caps by Applied Biosciences Arcturus Systems. RNA was purified using a Picopure RNA isolation kit according to the manufacturer’s directions. Independent RNA preparations were extracted from three control mice and three CKO mice. Microarray analysis. Microarrays were performed with mouse WG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA). Data were processed by using the lumi package within the R statistical package. Variance-stabilizing transformation was performed, followed by quantile normalization of the resulting expression values. Differential expression was calculated using the limma package within R. A heat map was generated using normalized fold change. The resulting lists were then annotated and reviewed for candidates. Cell culture and transduction. The rat intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (Manassas, VA). The cells were grown in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose, and supplemented with 0.1 U/ml bovine insulin and 10% FBS. Lentiviral vectors (based on pLKO.1 plasmid) were obtained from Cell Based Assay Screening Services (Baylor College of Medicine, Houston, TX). The short hairpin RNA (shRNA) against Asl corresponds to clones V3LMM_425167, V3LMM_425163, V3LMM_425168, and V3LMM_425165. The nonsilencing scrambled shRNA corresponds to clone V2LXX 00001. Lentiviruses were produced as described using packaging plasmids pMD2.G and psPAX2 in 293T cells (14). Transduction of IEC-6 cells required a “spin-infection” of 5 min at 500 g.
C
D ASL Tubulin Control
Relative ASL/tubulin density
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Fig. 2. A: cross sections of terminal ileum from double-heterozygote mice containing -galactosidase gene lacZ inserted into Rosa 26 locus and Villin-cre showing selective staining of the enterocytes, thereby confirming tissue-specific expression of cre recombinase. B: quantitative PCR from the enterocytes demonstrating significant decrease in the relative Asl mRNA expression in CKO. *P ⬍ 0.05, n ⫽ 4 in each group. C: representative Western blot of the enterocytes demonstrating significant loss of argininosuccinate lyase (ASL) in CKO mice (top) and graphical representation of the quantification (bottom). *P ⬍ 0.05, n ⫽ 3 in each group. D: representative images comparing immunostaining of the terminal ileum cross sections for ASL between control (top) and CKO (bottom) mice. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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Cell culture and migration assays. IEC-6 cells were lifted with 0.05% trypsin plus 0.53 mM EDTA in HBSS without calcium and magnesium. They were counted in a hemocytometer and plated at 0.3 ⫻ 106 cells/cm2 in six-well plates in DMEM with 5% FBS and 4.5 g/l glucose and supplemented with 0.1 U/ml bovine insulin and puromycin at 1 g/ml. IEC-6 cells grown to confluence over 24 – 48 h were starved by using starvation media for 12 h (media as described
A
NEC severity score
% of animals with NEC
Histological Score
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Incidence of NEC
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* 63%
60 37%
40 20
RESULTS
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n = 88
n = 88
n = 46
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earlier but without FBS). The confluent cells were scratched with a 20-l pipette tip to cause wounding. Images were taken of all cultures immediately following wounding, and the location was marked. Following this, media containing 10% FBS were replaced. Images of the referenced locations were captured at intervals of 6, 12, 18, and 24 h. The wound gap was measured, and the percentage of wound repair was determined for each time point with Image J 1.46r computer software (National Institutes of Health, Bethesda, MD). All treatments were assessed in triplicate. LPS, when used, was added 12 h prior to the wound assays at a concentration of 50 g/ml. NO donor DEA NONOate (diethylenetriamine NONOate) (Cayman Chemical, Ann Arbor, MI), when used, was at a concentration of 10 mM in combination with regular media as described earlier. Media for migration experiments were replaced every 12 h. Statistical analysis. Statistical analysis was performed with SigmaPlot version 11.0 (Systat Software, San Jose, CA). Student’s t-test, Mann-Whitney U-test, and ANOVA were used as appropriate when comparisons were made between two or more experimental groups. The 2-test was used to compare proportions. P ⬍ 0.05 was considered statistically significant, and all analyses were two sided.
CKO
n = 46
Enterocyte deletion of Asl. We generated enterocyte-specific knockout mice of Asl by intercrossing Aslflox/flox mice with transgenic mice expressing Cre recombinase under the control of the villin promoter (Aslflox/flox; VillinCretg/⫹ or CKO), as described in Methods. Double-heterozygote mice containing the -galactosidase gene lacZ inserted into the Rosa 26 locus and Villin-cre alleles demonstrated enterocyte-specific expression (Fig. 2A) of -galactosidase, confirming the tissue-specific expression of Cre recombinase. The CKO mice were grossly indistinguishable from their littermate Aslflox/flox controls and exhibited similar growth curves and life span. Enterocytes isolated from CKO mice demonstrated an 80% reduction in mRNA expression of Asl (Fig. 2B). Western blots performed on the enterocytes revealed significantly decreased ASL protein in CKO mice (Fig. 2C). Whereas immunostaining of cross sections of terminal ileum from neonatal mice showed robust expression of ASL, especially near the tips of the villi, near-absence of ASL was observed in the CKO (Fig. 2D). Similar to our past experience, loss of Asl did not induce significant changes in the expression of any of the NOS isoforms (data not shown) (6). Loss of enterocyte ASL results in increased incidence of NEC. CKO and control littermate pups were subjected to the NEC protocol (Fig. 1A). The efficacy of NEC induction from this protocol was demonstrable by gross pathological and radiographic examinations (Figs. 1B and 3, C–F). CKO mice demonstrated a significantly higher histological injury score compared with their control littermates. The median score of
Fig. 3. A: comparison of the severity of tissue injury between controls and CKO mice as assessed by hematoxylin and eosin staining of the terminal ileum. Median histological score in controls ⫽ 1; CKO ⫽ 2; *P ⬍ 0.05; n ⫽ 88 controls, 46 CKO; Mann-Whitney U-test. B: comparison of the incidence of NEC between controls and CKO. *P ⬍ 0.05; n ⫽ 88 controls, 46 CKO. C and D: representative images of the gastrointestinal tract of a mouse pup; normal appearance in control pup (C); bluish-black discoloration of the terminal ileum and colon suggestive of NEC in a CKO pup (D). E and F: representative radiographs of mouse pups; control pup with normal X-ray (E); CKO pup with abdominal distension, showing dilated bowel loops (F). G and H: representative histological images of cross sections of ileum depicting severity of injury in control pups (G) and CKO (H).
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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Table 1. List of transcription factors predicted to be up- or downregulated based on ingenuity analysis Upstream Regulator
Activation z-Score
P Value of Overlap
FOS
⫺2.646
4.39E-02
NFE2L2
⫺2.29
7.92E-04
ERG HNF4A
⫺1.633 ⫺1
1.76E-02 3.51E-03
STAT4 TP53
0.186 1.915
2.21E-02 6.15E-02
Target Molecules in Dataset
AGPS,ANXA4,BGLAP,CLIP1,CMTM4,GIT2,HSPA5, KRT8,LETMD1,PCYT2,PLD1,PSMA7,PSMB3,RALB, SELENBP1,UTRN,VAMP7 BGLAP,COPS5,FTL,GNA11,HM13,KLK3,NELF,NQO2,PDIA4,PSMA4,PSMA7,PSMB1,PSMB3, PSMB6,PSMD1,PSMD14,PSMD7,PTPN1,RARS CLIP1,MYO10,PXN,RAB7A,TRIOBP,UTRN ANXA5,ASUN,AXIN2,BCKDHA,BLVRB,BRAP,C11orf73,C19orf66,C20orf43,CEACAM1,COPS7A,CPT2, DAG1DCAF13,DSCR3,DUSP11,EIF5,FYCO1,G3BP2,HNF4A,HSPA5,IL11RA,IPO13,KIAA0141,KRT8, LMAN2L,MAP7,MRPL22 (includes EG:100142645),MRPL4 (includes EG:363023),MRPS23,MTMR2,NAPA,NDUFA1,NDUFA5,NDUFS3,NDUFS4,NSA2,OGFR,PAFAH2, PJA2,PPP1R11,PRCP,PSMB1,PSMB7,PSMD1,PSMD7,RAB7A,RAP2C,RPS6KA1,RXRB, SLIRP,SNW1,SRSF11,TCIRG1,TEF,TERF2IP,TFPT,THOC3,TRAF6,TSN,WARS2,ZNF317 DNAJB6,FCER1G,Gdap10,HES6,HLA-DQB1,INTS12,RGS16,SELENBP1,SH2B1,SRPK1 ACTB,ADD3,ANXA4,AP1B1,BAG1,BBC3,COX5B (includes EG:100002384),CYB5A,ECH1,FAT1,GTF3C2,H2AFY,HRAS,KLK3,KRT8,MAP2K4,MCM6,MTDH, NLRC4,OAS1,PPM1B,PPP1R13B,PQLC3,PSMD1,PTPN1,RFWD2,RGS16,RPS6KA1, SMARCB1,STRN3,TANK,UBA1,UIMC1,UPP1,XPNPEP1
A positive z-score indicates activation, and a negative z-score indicates inhibition in the CKO.
histological injury in the controls was one, compared with a median score of two in CKO (P ⬍ 0.05; Fig. 3, A and C–H). The CKO mice also demonstrated an overall 63% incidence of NEC compared with a 37% incidence of NEC in the control littermates (relative risk 1.7, 95% CI ⫽ 1.2–2.4, P ⫽ 0.006; Fig. 3B). The 70% increase in incidence of NEC in the CKO
2
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Nu. of neutrophils / HPF Control
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1.5
12
6
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3
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IL-10
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9
TGF-β
IL-4
TNF-α
IL-6
on t
Relative mRNA expression
A
mice and increased histological scores demonstrate that the CKO mice were not only more susceptible to develop NEC but also developed a more severe form of the disease. Upregulation of apoptotic and inflammatory pathways in CKO mice. Because the CKO mice demonstrated increases in both tissue injury and the incidence of NEC, we performed an
Fig. 4. A: quantitative PCR comparing the levels of various cytokines in the enterocytes between controls and CKO mice showing significant elevation in the relative mRNA expression of IL-6. IL, interleukin; TNF-␣, tumor necrosis factor-␣; TGF-, transforming growth factor-; ns, not significant; *P ⬍ 0.05, n ⫽ 4 in each group. B: representative images showing the cross sections of terminal ileum with immunostaining for neutrophils. Arrows, neutrophils in the lamina propria. C: graphical representation of comparison of neutrophil counts in the lamina propria between controls and CKO. HPF, high-power field; *P ⬍ 0.05. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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unbiased assay of pathways affected by loss of ASL that could explain the mechanisms behind this phenotype. We performed transcriptional profiling on the enterocytes obtained by laser capture microscopy in control vs. CKO neonatal mice (Supplemental Table 1).1 Pathway and network analyses were completed using Ingenuity pathway analysis (Ingenuity Systems, Redwood City, CA, www.ingenuity.com). We found that apoptotic and T-cell signaling pathways demonstrated differences between CKO and controls (Supplemental Table 2). Specifically, there was increased activation of TP53 and STAT4 pathways in the CKO mice (Table 1). Thus, immuneand apoptosis-related studies were pursued. Increased interleukin-6 expression and augmented neutrophilic infiltration in CKO mice. Because transcriptional profiling of the enterocytes from CKO showed upregulation of inflammatory pathways, we compared the markers of inflammation both in the plasma and at the cellular level within the enterocytes. We compared relative mRNA expression of the various cytokines and chemokines including interleukins (ILs)-4, -6, and -10, transforming growth factor-,
The online version of this article contains supplemental data.
A
CKO
Control
B
DAPI
TUNEL
* 50 40 30. 20 10 0 Control
D
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BAX
* 1.4
AIF
1.2
Caspase
1
GAPDH
0.8 0.6
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0.4 0.2 0
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Relative BAX mRNA expression
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MERGED
Nu. of TUNEL positive cells/ HPF
1
interferon-␥, and tumor necrosis factor-␣. The CKO mice demonstrated increased IL-6 mRNA expression in the enterocytes, but no significant differences were found in the expression of the other cytokines and chemokines (Fig. 4A). Because IL-6 is a primary chemoattractant, we performed immunostaining for neutrophils on the ileal cross sections of the mouse pups subjected to the NEC protocol. The CKO pups demonstrated a significantly higher number of neutrophils in the lamina propria compared with the controls (P ⬍ 0.05), supporting the presence of an exaggerated inflammatory state and corroborating the increased histological severity in the CKO (Fig. 4, B and C). Loss of enterocyte ASL results in increased apoptosis in the context of NEC. Apart from the inflammatory pathways, apoptotic pathways were significantly upregulated in the transcriptional profiling on the enterocytes from the CKO. To quantify the degree of apoptosis as a contributing factor in the susceptibility of NEC, terminal ileal cross sections from the animals that underwent NEC protocol were subjected to TUNEL staining. Compared with controls, the histological sections from the CKO demonstrated significantly increased levels of apoptosis (Fig. 5, A and B). Intestinal homogenates
Fig. 5. A: images representing intestinal cross sections stained with the Apoptag in situ apoptosis detection kit and DAPI (4=,6-diamidino-2-phenylindole) (blue) and viewed under a microscope for apoptotic cells. The numbers of apoptotic cells per high-power field (HPF) were measured in 10 villi in 5– 6 HPFs on each slide. B: graphical representation of the quantification of cells positive for apoptosis using the method stated in A. *P ⬍ 0.05, n ⫽ 3 in each group. C: quantitative PCR comparing relative mRNA expression of the BAX gene between controls and CKO. *P ⬍ 0.05, n ⫽ 4 in each group. D: representative Western blot of intestinal homogenates comparing levels of proapoptotic proteins BAX, AIF, and caspase-3 between CKO and controls, along with loading control GAPDH. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
ENTEROCYTE-DERIVED ASL MODULATES INCIDENCE OF NEC
from the animals that underwent the NEC protocol were analyzed for markers of apoptosis. qPCR analysis revealed significantly increased expression of the proapoptotic gene Bax in the CKO compared with the controls (Fig. 5C). Western blot analyses performed on intestinal homogenates from pups that were subjected to stresses of NEC protocol showed elevated levels of apoptotic proteins BAX and AIF (Fig. 5D). Loss of Asl impairs migration of IEC-6 cells in the presence of LPS. Because CKO mice demonstrated elevated apoptosis similar to findings observed with human infants with NEC, we explored the effect of loss of ASL on the ability of enterocytes to migrate. The ability of the enterocytes to migrate is an integral part in the process of repair following intestinal injury. We knocked down Asl in IEC-6 cells using lentivirus encoding shRNA targeting Asl. The movement of IEC-6 cells across a wounded area of the cell monolayer is representative of the ability of the cells to migrate (17). As depicted (Fig. 6, A and B), the ability of cells to migrate was similar in the Asl knockdown and in control cells with nonsilencing scrambled shRNA under basal conditions. On addition of LPS, the rate of migration in control cells increased considerably, while Asl knockdown cells failed to show a similar response, suggesting that the loss of ASL impairs the migration of IEC-6 cells during conditions of stress.
infants with NEC (4, 21) and supplementation with arginine has been shown to be beneficial in some studies (1, 19). The mechanisms by which arginine therapy might be protective are not clear, and hence conclusive proof for the protection of arginine is still elusive. Arginine is a substrate for synthesis of many biologically important molecules including NO, agmatine, polyamines, and creatine. These metabolites have roles in energy metabolism, gene expression, apoptosis, and cell proliferation and differentiation, which are crucial in intestinal homeostasis (7). Perturbations of these critical cellular functions can lead to an increase in apoptosis and inflammation and to a decreased capacity to repair, some of which are hallmarks of NEC (2, 18). Our report highlights the protective action of enterocytederived ASL in the pathogenesis of NEC. The increased histological injury score in the CKO was associated with an exaggerated inflammatory state with elevated levels of IL-6 and increased neutrophilic infiltration in the gut. In addition, activation of proapoptotic pathways and the resultant enterocyte loss were significantly higher in the CKO. Whereas there were no differences in the histology of intestine in the CKO during unstressed conditions compared with wild-type littermates, induction of NEC led to increased inflammation, and enterocyte death, with supportive in vitro evidence for decreased migration with loss of ASL. These findings are consistent with the exaggerated inflammation and apoptosis observed in the intestines from human infants with NEC (2). We acknowledge the limitations in our study. NEC was induced in the animals by exposing newborn pups to the risk factors thought to play an important role in human NEC, namely prematurity, absence of breast-feeding, and hypoxia. It is widely recognized that, although this model mimics the intestinal injury of NEC at the histological level, it is perhaps not most representative of human NEC. However, in spite of its limitations, the mouse model of NEC with hypoxia-hypothermia-formula feeding is one of the best available tools to study NEC in the laboratory. This is mainly due to the availability of mutant mouse models and tractability of mouse genetics. The
DISCUSSION
*
*
100 80 60
Fig. 6. A: images representing IEC-6 cells culture grown at a monolayer with a horizontal scratch at 0 h (top) and 12 h (bottom). B: graphical representation of quantification of the area of migration in 12 h at basal conditions and on addition of LPS. *P ⬍ 0.05.
40 20
LP S
S A
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+
LP Sh
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ro l+
Sh
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on tr
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B
C
0 hours
Percent area of wound healing in 12 hours
We successfully generated a mouse model to study the role of enterocyte-derived ASL in the pathogenesis of NEC and to demonstrate that loss of ASL results in increased incidence and severity of NEC. Mechanistically, we show that loss of enterocyte-derived ASL is associated with increased apoptosis and inflammation and with reduced migration capacity of mutant enterocytes. Because enterocytes are the main sites of ASL expression in perinatal mammals, they are also the principal sites of arginine synthesis. ASL expression in the enterocytes is at the maximum by 10 –14 days of age in mice and 2–3 yr in humans (13). Studies have demonstrated that lower plasma arginine levels in
A
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maximum ASL expression in the enterocytes in mice is around 10 –14 days (13). Although it seemed appropriate to test the role of ASL in NEC at this age in mice, pups of this age group are very resistant to induction of NEC, primarily due to the exposure to breast milk and its protective effects. Hence our studies with NEC were conducted in pups immediately after birth before they came in contact with mother’s milk. Whereas previous studies manipulated arginine at the whole body level, leading to inconclusive results, our approach of manipulating ASL metabolism specifically in enterocytes allowed us to specifically study the cell-autonomous contribution of arginine synthesis in this cell type to NEC susceptibility. Our report offers avenues to harness this protective role of ASL in devising methods of prevention or treatment in NEC. ACKNOWLEDGMENTS We acknowledge the contributions of Yuqing Chen and Elda Munivez for technical assistance in this project. GRANTS This work was supported in part by Public Health Service Grant DK-56338 to M. H. Premkumar and National Institutes of Health Grant GM-90310 to B. Lee. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS M.H.P., P.M.C., A.E., and B.L. conception and design of research; M.H.P., G.S., S.C.N., S.C., A.N., M.Z.R., T.B., J.Z., D.S., and N.S.B. performed experiments; M.H.P., G.S., M.J., B.C.D., and N.S.B. analyzed data; M.H.P., M.Z.R., and D.S. interpreted results of experiments; M.H.P. and P.M.C. prepared figures; M.H.P. drafted manuscript; M.H.P., S.C.N., P.M.C., A.E., and B.L. edited and revised manuscript; M.H.P., S.C.N., P.M.C., A.E., and B.L. approved final version of manuscript. REFERENCES 1. Amin HJ, Zamora SA, McMillan DD, Fick GH, Butzner JD, Parsons HG, Scott RB. Arginine supplementation prevents necrotizing enterocolitis in the premature infant. J Pediatr 140: 425–431, 2002. 2. Ballance WA, Dahms BB, Shenker N, Kliegman RM. Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 117 (1 Pt 2): S6 –S13, 1990. 3. Barlow B, Santulli TV. Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model. Surgery 77: 687–690, 1975. 4. Becker RM, Wu G, Galanko JA, Chen W, Maynor AR, Bose CL, Rhoads JM. Reduced serum amino acid concentrations in infants with necrotizing enterocolitis. J Pediatr 137: 785–793, 2000. 5. Caplan MS, Hedlund E, Adler L, Hsueh W. Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis. Pediatr Pathol 14: 1017–1028, 1994.
6. Erez A, Nagamani SC, Shchelochkov OA, Premkumar MH, Campeau PM, Chen Y, Garg HK, Li L, Mian A, Bertin TK, Black JO, Zeng H, Tang Y, Reddy AK, Summar M, O’Brien WE, Harrison DG, Mitch WE, Marini JC, Aschner JL, Bryan NS, Lee B. Requirement of argininosuccinate lyase for systemic nitric oxide production. Nat Med 17: 1619 –1626, 2011. 7. Flynn NE, Meininger CJ, Haynes TE, Wu G. The metabolic basis of arginine nutrition and pharmacotherapy. Biomed Pharmacother 56: 427– 438, 2002. 8. Guthrie SO, Gordon PV, Thomas V, Thorp JA, Peabody J, Clark RH. Necrotizing enterocolitis among neonates in the United States. J Perinatol 23: 278 –285, 2003. 9. Holman RC, Stoll BJ, Clarke MJ, Glass RI. The epidemiology of necrotizing enterocolitis infant mortality in the United States. Am J Public Health 87: 2026 –2031, 1997. 10. Holman RC, Stoll BJ, Curns AT, Yorita KL, Steiner CA, Schonberger LB. Necrotising enterocolitis hospitalisations among neonates in the United States. Paediatr Perinat Epidemiol 20: 498 –506, 2006. 11. Horbar JD, Carpenter JH, Badger GJ, Kenny MJ, Soll RF, Morrow KA, Buzas JS. Mortality and neonatal morbidity among infants 501 to 1500 grams from 2000 to 2009. Pediatrics 129: 1019 –1026, 2012. 12. Kafetzis DA, Skevaki C, Costalos C. Neonatal necrotizing enterocolitis: an overview. Curr Opin Infect Dis 16: 349 –355, 2003. 13. Kohler ES, Sankaranarayanan S, van Ginneken CJ, van Dijk P, Vermeulen JL, Ruijter JM, Lamers WH, Bruder E. The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes. BMC Dev Biol 8: 107, 2008. 14. Kutner RH, Zhang XY, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4: 495–505, 2009. 15. Morello R, Bertin TK, Chen Y, Hicks J, Tonachini L, Monticone M, Castagnola P, Rauch F, Glorieux FH, Vranka J, Bächinger HP, Pace JM, Schwarze U, Byers PH, Weis M, Fernandes RJ, Eyre DR, Yao Z, Boyce BF, Lee B. CRTAP is required for prolyl 3-hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell 127: 291–304, 2006. 16. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915–918, 1994. 17. Nusrat A, Delp C, Madara JL. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest 89: 1501–1511, 1992. 18. Pender SL, Braegger C, Gunther U, Monteleone G, Meuli M, Schuppan D, Macdonald TT. Matrix metalloproteinases in necrotising enterocolitis. Pediatr Res 54: 160 –164, 2003. 19. Polycarpou E, Zachaki S, Tsolia M, Papaevangelou V, Polycarpou N, Briana DD, Gavrili S, Kostalos C, Kafetzis D. Enteral L-arginine supplementation for prevention of necrotizing enterocolitis in very low birth weight neonates: a double-blind randomized pilot study of efficacy and safety. JPEN J Parenter Enteral Nutr 37: 617–622, 2013. 20. Radulescu A, Zhang HY, Yu X, Olson JK, Darbyshire AK, Chen Y, Besner GE. Heparin-binding epidermal growth factor-like growth factor overexpression in transgenic mice increases resistance to necrotizing enterocolitis. J Pediatr Surg 45: 1933–1939, 2010. 21. Zamora SA, Amin HJ, McMillan DD, Kubes P, Fick GH, Bützner JD, Parsons HG, Scott RB. Plasma L-arginine concentrations in premature infants with necrotizing enterocolitis. J Pediatr 131: 226 –232, 1997.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
Argininosuccinate lyase in enterocytes protects from development of necrotizing enterocolitis M. H. Premkumar,1 G. Sule,2 S. C. Nagamani,2 S. Chakkalakal,2 A. Nordin,2 M. Jain,2 M. Z. Ruan,2 T. Bertin,2 B. Dawson,2 J. Zhang,2 D. Schady,3 N. S. Bryan,4 P. M. Campeau,2 A. Erez,5 and B. Lee2,6 1
Division of Neonatology, Texas Children’s Hospital, Baylor College of Medicine; 2Department of Molecular and Human Genetics, Baylor College of Medicine; 3Department of Pathology, Texas Children’s Hospital, Houston; 4University of Texas Health Science Center at Houston, Texas; 5Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel; and 6Howard Hughes Medical Institute, Houston, Texas Submitted 21 November 2013; accepted in final form 3 June 2014
Premkumar MH, Sule G, Nagamani SC, Chakkalakal S, Nordin A, Jain M, Ruan MZ, Bertin T, Dawson B, Zhang J, Schady D, Bryan NS, Campeau PM, Erez A, Lee B. Argininosuccinate lyase in enterocytes protects from development of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 307: G347–G354, 2014. First published June 5, 2014; doi:10.1152/ajpgi.00403.2013.— Necrotizing enterocolitis (NEC), the most common neonatal gastrointestinal emergency, results in significant mortality and morbidity, yet its pathogenesis remains unclear. Argininosuccinate lyase (ASL) is the only enzyme in mammals that is capable of synthesizing arginine. Arginine has several homeostatic roles in the gut and its deficiency has been associated with NEC. Because enterocytes are the primary sites of arginine synthesis in neonatal mammals, we evaluated the consequences of disruption of arginine synthesis in the enterocytes on the pathogenesis of NEC. We devised a novel approach to study the role of enterocyte-derived ASL in NEC by generating and characterizing a mouse model with enterocyte-specific deletion of Asl (Aslflox/flox; VillinCretg/⫹, or CKO). We hypothesized that the presence of ASL in a cell-specific manner in the enterocytes is protective in the pathogenesis of NEC. Loss of ASL in enterocytes resulted in an increased incidence of NEC that was associated with a proinflammatory state and increased enterocyte apoptosis. Knockdown of ASL in intestinal epithelial cell lines resulted in decreased migration in response to lipopolysaccharide. Our results show that enterocyte-derived ASL has a protective role in NEC. NEC; ASL; arginine
is a critical step in conversion of waste nitrogen into urea, whereas, in most other tissues, this is the sole pathway for synthesis of endogenous arginine. We recently showed that ASL is an essential regulator in the synthesis of nitric oxide (NO) (6). We demonstrated that ASL is required to maintain a NO synthesis complex containing NO synthase (NOS), argininosuccinate synthase, HSP90, and the arginine transporter SLC7A1. In the absence of ASL, this complex is assembled less efficiently, despite unaltered levels of the remaining components. Loss of ASL not only leads to loss of arginine recycling from citrulline, that is, intracellular de novo arginine synthesis, but also to loss of channeling of extracellular arginine to NOS for NO production. Hence, loss of ASL leads to deficiency of NOS-dependent NO production at the cellular level. Moreover, we have shown that this structural requirement for ASL in NO production is conserved, because humans with structural and catalytic deficiency of ASL are also NO deficient. To study the cell-autonomous role of ASL in NEC, we generated a novel mouse model with enterocyte-specific deletion of Asl. We show that the loss of enterocyte-derived ASL results in increased incidence of NEC, and hence manipulating ASL expression or activity could be of potential benefit for treatment of NEC. METHODS
(NEC), the most common gastrointestinal emergency in extremely low birth weight newborn infants, is caused by complex pathogenic mechanisms (12, 16). The incidence of NEC in North American neonatal intensive care units is reported to be 3–11% (8 –10). A recent study from over 600 hospitals in the United States demonstrated a significant increase in the incidence of NEC between 1999 and 2009, while all the other neonatal morbidities demonstrated a stable or declining trend (8, 10, 11). Observational studies have shown decreased levels of plasma arginine in the immediate weeks preceding the onset of NEC (4, 21), and arginine supplementation has been shown to have some beneficial effects in the prevention of NEC (1, 19). Enterocytes are the main sites of arginine synthesis in neonatal mammals. The synthesis of arginine is dependent on argininosuccinate lyase (ASL), which catalyzes the conversion of argininosuccinate to arginine and fumarate. In the liver, this
NECROTIZING ENTEROCOLITIS
Address for reprint requests and other correspondence: B. Lee, Dept. of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, R814, MS225 Houston, TX 77030 (e-mail: [email protected]). http://www.ajpgi.org
The experimental procedures were approved by the Animal Care and Use Committee of Baylor College of Medicine, Houston, TX. Generation of enterocyte-specific conditional ASL knockout mice. Aslflox[FRT-neo-FRT]/⫹ mice carrying the neomycin phosphotransferase (neo) gene were generated as previously described (6). The Aslflox[FRT-neo-FRT]/⫹ mice were crossed with transgenic mice expressing Flp recombinase to remove the neo cassette, leaving behind the loxP sites flanking exons 7–9 of Asl . The resulting mice with the Aslflox/⫹ allele were interbred to generate Aslflox/flox mice, which were further crossed with transgenic mice bearing cre-recombinase expressed under the control of the enterocyte-specific mouse villin 1 promoter [B6.SJL-Tg(Vil-cre)997 Gum/J; Jackson Laboratory, Bar Harbor, ME] to generate mice specifically lacking ASL within the enterocytes, Aslflox/flox, VillinCretg/⫹, or CKO. Induction of NEC. NEC was induced by techniques previously described by Barlow et al. (3) and Caplan et al. (5). Briefly, newborn mice pups were delivered at 18.5 days of gestation by cesarean section and were maintained in an incubator with constant temperature of 36°C and humidity of 55%. These pups were gavage fed infant formula (Similac PM60/40, Abbott Nutrition, Columbus, OH) every 3 h for 72 h using a 2F polystyrene catheter. Infant formula was prepared at a concentration of 1 kcal/ml and fed at a volume of 200 ml·kg⫺1·day⫺1. Lipopolysaccharide (LPS; L-1887; Sigma-Aldrich,
0193-1857/14 Copyright © 2014 the American Physiological Society
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St. Louis, MO) was introduced into the feed on day 1 at 2 mg/kg. Pups were exposed to hypoxia (99% N2) for 1 min in a hypoxic chamber, followed by hypothermia (4°C) for 10 min twice daily for 3 days (Fig. 1A) (20). The animals were killed when they appeared to be severely ill with signs such as abdominal distension, lethargy, cyanosis, or at the end of 72 h. Terminal ilea were obtained for histological and biochemical analysis. The diagnosis of NEC was made based on the assessment of two independent observers, including a pathologist, both of whom were blinded to the genotype. NEC was graded based on the severity of the damage from the mucosal to serosal surface as follows: Grade 0 ⫽ intact villi, Grade 1⫽ sloughing of cells on villous tips, Grade 2 ⫽ midvillous damage, Grade 3 ⫽ villi absent but crypts still detectable, and Grade 4 ⫽ complete absence of epithelial structures and transmural necrosis (Fig. 1). Intestinal cross sections with changes higher than or equal to Grade 2 were classified as NEC. Histology and tissue staining. Paraffin-embedded tissues were sectioned to a 4- to 7-m thickness and stained with hematoxylin and eosin as published (15). Immunohistochemistry. Immunohistochemistry was performed using the Standard avidin biotin complex (ABC) method with a Vectastain ABC kit following the manufacturer’s protocols. Tissues were fixed in 4% formaldehyde and paraffin-embedded using standard protocols. Sections were deparaffinized and hydrated in graded ethanol. Specific staining was enhanced by using a heat-induced epitope retrieval method. Sections were then exposed to 3% H2O2 in PBS for 30 min followed by blocking with normal serum blocker buffer. The sections were then incubated overnight with the primary antibody. The following day, sections were incubated with biotinylated second-
ary antibody for 60 min. Sections were incubated in ABC solution for 1 h at room temperature. Finally, the sections were incubated in peroxidase substrate solution (3,3=-diaminobenzidine) and viewed under a microscope. Incubation without primary antibody served as the negative control for all incubations. The following primary antibodies were used in the immunostaining experiments: ASL monoclonal antibody (M01) (no. H00000435-M01, Abnova, Walnut, CA), neutrophil antibody Ly-6B.2 Alloantigen Antibody (AbD Serotec, Raleigh, NC). Western blot. The intestinal tissues were homogenized and lysed in protein lysis buffer (50 mM Tris·HCl, pH 7.5; 150 mM NaCl; 1% Triton-100) with a complete protease inhibitor cocktail (Roche) and centrifuged at 14,000 rpm for 10 min. Laemmli loading buffer and 5% -mercaptoethanol were added to the supernatants and boiled for 10 min at 95°C. Equal amounts of protein from these samples were separated on 10% SDS polyacrylamide gels and then blotted onto polyvinylidene difluoride membranes which were incubated with the primary antibodies overnight at 4°C. The following day, these membranes were incubated with the corresponding secondary antibodies. Protein quantification was performed using a Licor Odyssey quantitative fluorescence imaging system according to the manufacturer’s instructions. The following primary antibodies were used in the Western blot experiments: ASL monoclonal antibody (M01) (no. H00000435-M01, Abnova, BAX (Bcl2 associated X protein) antibody (no. 2772, Cell Signaling, Danvers, MA), AIF (apoptosis-inducing factor) antibody (no. 4642, Cell Signaling), caspase-3 (no. 9662, Cell Signaling), anti-GAPDH (no. AM4300, Ambion, Grand Island, NY), monoclonal anti-␣-tubulin antibody (T5168, Sigma-Aldrich, St. Louis, MO).
A Day 1
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Exclusive Formula feed by gavage Every 3 hours for 72 hours Twice daily X 3 days Hypoxia (1 min 99% N2)+ Hypothermia (4C 10 minutes) LPS in milk (2 mg/Kg once on day 1) C section birth E 18.5
Tissue Analysis
B
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Fig. 1. Schematic representation of the protocol used for mouse model of necrotizing enterocolitis (NEC). Images of hematoxylin and eosin sections of terminal ileum of mouse pups representative of histological changes suggestive of Grades 1– 4 of NEC are shown. LPS, lipopolysaccharide; C section, caesarean section; E18.5, 18.5 days of gestation. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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Real-time quantitative PCR. RNA was extracted from either whole intestinal homogenates or enterocytes using TRIzol (Invitrogen, Carlsbad, CA), and first-strand cDNA synthesis was performed with the SuperScript III reverse transcriptase kit (Invitrogen, Grand Island, NY). Real-time quantitative PCR (qPCR) was performed on the LightCycler version 1.1 instruments with Roche Applied Science (Indianapolis, IN) reagents, according to the manufacturer’s recommendations. cDNA template concentrations were determined using Ribogreen (Invitrogen), and equivalent concentrations were used for each qPCR. Fluorescence was captured at the end of each extension cycle, over a total of 40 cycles. Crossing points were determined by a second derivative algorithm intrinsic to the LightCycler software and normalized to a constitutively expressed gene (2-microglobulin or GAPDH). RNA fold-change was calculated to show the difference in transcript expression between samples normalized to an unregulated reference gene of equal abundance in the specimens tested. Enterocyte isolation by the mechanical dissociation method. Intestinal segments from neonatal pups were everted onto a glass rod attached to a vibrating source (electric toothbrush). The everted intestinal segments were then shaken in HBSS media, without calcium and magnesium, with 40 mM EDTA for 20 min on ice. Following this, the media with the dissociated enterocytes were centrifuged at 700 g to collect the enterocyte pellet. Apoptosis assays. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining was performed by using the Apoptag in situ apoptosis detection kit (Chemicon International, Billerica, MA) according to the manufacturer’s instructions. TUNEL-positive enterocytes were counted in 10 randomly selected villi in five ⫻200 high-power fields per each intestinal cross section. Preparation of intestinal villi samples by laser capture microscopy. The intestines were flushed with PBS, opened along their cephalo-
A
caudal axis, and frozen in optimal cutting temperature compound. Frozen sections of 10 m were generated on polyethylene napthalate membrane slides. Enterocytes from the villi and the crypts excluding the core of the villi were captured under a laser capture microscope (Pix Cell II; Arcturus, Mountain View, CA) using HS Capsure LCM caps by Applied Biosciences Arcturus Systems. RNA was purified using a Picopure RNA isolation kit according to the manufacturer’s directions. Independent RNA preparations were extracted from three control mice and three CKO mice. Microarray analysis. Microarrays were performed with mouse WG-6 v2.0 Expression BeadChip (Illumina, San Diego, CA). Data were processed by using the lumi package within the R statistical package. Variance-stabilizing transformation was performed, followed by quantile normalization of the resulting expression values. Differential expression was calculated using the limma package within R. A heat map was generated using normalized fold change. The resulting lists were then annotated and reviewed for candidates. Cell culture and transduction. The rat intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (Manassas, VA). The cells were grown in DMEM with 4 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate and 4.5 g/l glucose, and supplemented with 0.1 U/ml bovine insulin and 10% FBS. Lentiviral vectors (based on pLKO.1 plasmid) were obtained from Cell Based Assay Screening Services (Baylor College of Medicine, Houston, TX). The short hairpin RNA (shRNA) against Asl corresponds to clones V3LMM_425167, V3LMM_425163, V3LMM_425168, and V3LMM_425165. The nonsilencing scrambled shRNA corresponds to clone V2LXX 00001. Lentiviruses were produced as described using packaging plasmids pMD2.G and psPAX2 in 293T cells (14). Transduction of IEC-6 cells required a “spin-infection” of 5 min at 500 g.
C
D ASL Tubulin Control
Relative ASL/tubulin density
* 1 0.8 0.6 0.4 0.2 0
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*
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O
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B
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Fig. 2. A: cross sections of terminal ileum from double-heterozygote mice containing -galactosidase gene lacZ inserted into Rosa 26 locus and Villin-cre showing selective staining of the enterocytes, thereby confirming tissue-specific expression of cre recombinase. B: quantitative PCR from the enterocytes demonstrating significant decrease in the relative Asl mRNA expression in CKO. *P ⬍ 0.05, n ⫽ 4 in each group. C: representative Western blot of the enterocytes demonstrating significant loss of argininosuccinate lyase (ASL) in CKO mice (top) and graphical representation of the quantification (bottom). *P ⬍ 0.05, n ⫽ 3 in each group. D: representative images comparing immunostaining of the terminal ileum cross sections for ASL between control (top) and CKO (bottom) mice. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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Cell culture and migration assays. IEC-6 cells were lifted with 0.05% trypsin plus 0.53 mM EDTA in HBSS without calcium and magnesium. They were counted in a hemocytometer and plated at 0.3 ⫻ 106 cells/cm2 in six-well plates in DMEM with 5% FBS and 4.5 g/l glucose and supplemented with 0.1 U/ml bovine insulin and puromycin at 1 g/ml. IEC-6 cells grown to confluence over 24 – 48 h were starved by using starvation media for 12 h (media as described
A
NEC severity score
% of animals with NEC
Histological Score
*
B
4 3 2 1 0
Incidence of NEC
100 80
* 63%
60 37%
40 20
RESULTS
O K
tr on C
C
C
C
ol
O K C
on
tr
ol
0
n = 88
n = 88
n = 46
Control
D
E
F
G
H
earlier but without FBS). The confluent cells were scratched with a 20-l pipette tip to cause wounding. Images were taken of all cultures immediately following wounding, and the location was marked. Following this, media containing 10% FBS were replaced. Images of the referenced locations were captured at intervals of 6, 12, 18, and 24 h. The wound gap was measured, and the percentage of wound repair was determined for each time point with Image J 1.46r computer software (National Institutes of Health, Bethesda, MD). All treatments were assessed in triplicate. LPS, when used, was added 12 h prior to the wound assays at a concentration of 50 g/ml. NO donor DEA NONOate (diethylenetriamine NONOate) (Cayman Chemical, Ann Arbor, MI), when used, was at a concentration of 10 mM in combination with regular media as described earlier. Media for migration experiments were replaced every 12 h. Statistical analysis. Statistical analysis was performed with SigmaPlot version 11.0 (Systat Software, San Jose, CA). Student’s t-test, Mann-Whitney U-test, and ANOVA were used as appropriate when comparisons were made between two or more experimental groups. The 2-test was used to compare proportions. P ⬍ 0.05 was considered statistically significant, and all analyses were two sided.
CKO
n = 46
Enterocyte deletion of Asl. We generated enterocyte-specific knockout mice of Asl by intercrossing Aslflox/flox mice with transgenic mice expressing Cre recombinase under the control of the villin promoter (Aslflox/flox; VillinCretg/⫹ or CKO), as described in Methods. Double-heterozygote mice containing the -galactosidase gene lacZ inserted into the Rosa 26 locus and Villin-cre alleles demonstrated enterocyte-specific expression (Fig. 2A) of -galactosidase, confirming the tissue-specific expression of Cre recombinase. The CKO mice were grossly indistinguishable from their littermate Aslflox/flox controls and exhibited similar growth curves and life span. Enterocytes isolated from CKO mice demonstrated an 80% reduction in mRNA expression of Asl (Fig. 2B). Western blots performed on the enterocytes revealed significantly decreased ASL protein in CKO mice (Fig. 2C). Whereas immunostaining of cross sections of terminal ileum from neonatal mice showed robust expression of ASL, especially near the tips of the villi, near-absence of ASL was observed in the CKO (Fig. 2D). Similar to our past experience, loss of Asl did not induce significant changes in the expression of any of the NOS isoforms (data not shown) (6). Loss of enterocyte ASL results in increased incidence of NEC. CKO and control littermate pups were subjected to the NEC protocol (Fig. 1A). The efficacy of NEC induction from this protocol was demonstrable by gross pathological and radiographic examinations (Figs. 1B and 3, C–F). CKO mice demonstrated a significantly higher histological injury score compared with their control littermates. The median score of
Fig. 3. A: comparison of the severity of tissue injury between controls and CKO mice as assessed by hematoxylin and eosin staining of the terminal ileum. Median histological score in controls ⫽ 1; CKO ⫽ 2; *P ⬍ 0.05; n ⫽ 88 controls, 46 CKO; Mann-Whitney U-test. B: comparison of the incidence of NEC between controls and CKO. *P ⬍ 0.05; n ⫽ 88 controls, 46 CKO. C and D: representative images of the gastrointestinal tract of a mouse pup; normal appearance in control pup (C); bluish-black discoloration of the terminal ileum and colon suggestive of NEC in a CKO pup (D). E and F: representative radiographs of mouse pups; control pup with normal X-ray (E); CKO pup with abdominal distension, showing dilated bowel loops (F). G and H: representative histological images of cross sections of ileum depicting severity of injury in control pups (G) and CKO (H).
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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Table 1. List of transcription factors predicted to be up- or downregulated based on ingenuity analysis Upstream Regulator
Activation z-Score
P Value of Overlap
FOS
⫺2.646
4.39E-02
NFE2L2
⫺2.29
7.92E-04
ERG HNF4A
⫺1.633 ⫺1
1.76E-02 3.51E-03
STAT4 TP53
0.186 1.915
2.21E-02 6.15E-02
Target Molecules in Dataset
AGPS,ANXA4,BGLAP,CLIP1,CMTM4,GIT2,HSPA5, KRT8,LETMD1,PCYT2,PLD1,PSMA7,PSMB3,RALB, SELENBP1,UTRN,VAMP7 BGLAP,COPS5,FTL,GNA11,HM13,KLK3,NELF,NQO2,PDIA4,PSMA4,PSMA7,PSMB1,PSMB3, PSMB6,PSMD1,PSMD14,PSMD7,PTPN1,RARS CLIP1,MYO10,PXN,RAB7A,TRIOBP,UTRN ANXA5,ASUN,AXIN2,BCKDHA,BLVRB,BRAP,C11orf73,C19orf66,C20orf43,CEACAM1,COPS7A,CPT2, DAG1DCAF13,DSCR3,DUSP11,EIF5,FYCO1,G3BP2,HNF4A,HSPA5,IL11RA,IPO13,KIAA0141,KRT8, LMAN2L,MAP7,MRPL22 (includes EG:100142645),MRPL4 (includes EG:363023),MRPS23,MTMR2,NAPA,NDUFA1,NDUFA5,NDUFS3,NDUFS4,NSA2,OGFR,PAFAH2, PJA2,PPP1R11,PRCP,PSMB1,PSMB7,PSMD1,PSMD7,RAB7A,RAP2C,RPS6KA1,RXRB, SLIRP,SNW1,SRSF11,TCIRG1,TEF,TERF2IP,TFPT,THOC3,TRAF6,TSN,WARS2,ZNF317 DNAJB6,FCER1G,Gdap10,HES6,HLA-DQB1,INTS12,RGS16,SELENBP1,SH2B1,SRPK1 ACTB,ADD3,ANXA4,AP1B1,BAG1,BBC3,COX5B (includes EG:100002384),CYB5A,ECH1,FAT1,GTF3C2,H2AFY,HRAS,KLK3,KRT8,MAP2K4,MCM6,MTDH, NLRC4,OAS1,PPM1B,PPP1R13B,PQLC3,PSMD1,PTPN1,RFWD2,RGS16,RPS6KA1, SMARCB1,STRN3,TANK,UBA1,UIMC1,UPP1,XPNPEP1
A positive z-score indicates activation, and a negative z-score indicates inhibition in the CKO.
histological injury in the controls was one, compared with a median score of two in CKO (P ⬍ 0.05; Fig. 3, A and C–H). The CKO mice also demonstrated an overall 63% incidence of NEC compared with a 37% incidence of NEC in the control littermates (relative risk 1.7, 95% CI ⫽ 1.2–2.4, P ⫽ 0.006; Fig. 3B). The 70% increase in incidence of NEC in the CKO
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mice and increased histological scores demonstrate that the CKO mice were not only more susceptible to develop NEC but also developed a more severe form of the disease. Upregulation of apoptotic and inflammatory pathways in CKO mice. Because the CKO mice demonstrated increases in both tissue injury and the incidence of NEC, we performed an
Fig. 4. A: quantitative PCR comparing the levels of various cytokines in the enterocytes between controls and CKO mice showing significant elevation in the relative mRNA expression of IL-6. IL, interleukin; TNF-␣, tumor necrosis factor-␣; TGF-, transforming growth factor-; ns, not significant; *P ⬍ 0.05, n ⫽ 4 in each group. B: representative images showing the cross sections of terminal ileum with immunostaining for neutrophils. Arrows, neutrophils in the lamina propria. C: graphical representation of comparison of neutrophil counts in the lamina propria between controls and CKO. HPF, high-power field; *P ⬍ 0.05. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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unbiased assay of pathways affected by loss of ASL that could explain the mechanisms behind this phenotype. We performed transcriptional profiling on the enterocytes obtained by laser capture microscopy in control vs. CKO neonatal mice (Supplemental Table 1).1 Pathway and network analyses were completed using Ingenuity pathway analysis (Ingenuity Systems, Redwood City, CA, www.ingenuity.com). We found that apoptotic and T-cell signaling pathways demonstrated differences between CKO and controls (Supplemental Table 2). Specifically, there was increased activation of TP53 and STAT4 pathways in the CKO mice (Table 1). Thus, immuneand apoptosis-related studies were pursued. Increased interleukin-6 expression and augmented neutrophilic infiltration in CKO mice. Because transcriptional profiling of the enterocytes from CKO showed upregulation of inflammatory pathways, we compared the markers of inflammation both in the plasma and at the cellular level within the enterocytes. We compared relative mRNA expression of the various cytokines and chemokines including interleukins (ILs)-4, -6, and -10, transforming growth factor-,
The online version of this article contains supplemental data.
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TUNEL
* 50 40 30. 20 10 0 Control
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AIF
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Caspase
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GAPDH
0.8 0.6
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Nu. of TUNEL positive cells/ HPF
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interferon-␥, and tumor necrosis factor-␣. The CKO mice demonstrated increased IL-6 mRNA expression in the enterocytes, but no significant differences were found in the expression of the other cytokines and chemokines (Fig. 4A). Because IL-6 is a primary chemoattractant, we performed immunostaining for neutrophils on the ileal cross sections of the mouse pups subjected to the NEC protocol. The CKO pups demonstrated a significantly higher number of neutrophils in the lamina propria compared with the controls (P ⬍ 0.05), supporting the presence of an exaggerated inflammatory state and corroborating the increased histological severity in the CKO (Fig. 4, B and C). Loss of enterocyte ASL results in increased apoptosis in the context of NEC. Apart from the inflammatory pathways, apoptotic pathways were significantly upregulated in the transcriptional profiling on the enterocytes from the CKO. To quantify the degree of apoptosis as a contributing factor in the susceptibility of NEC, terminal ileal cross sections from the animals that underwent NEC protocol were subjected to TUNEL staining. Compared with controls, the histological sections from the CKO demonstrated significantly increased levels of apoptosis (Fig. 5, A and B). Intestinal homogenates
Fig. 5. A: images representing intestinal cross sections stained with the Apoptag in situ apoptosis detection kit and DAPI (4=,6-diamidino-2-phenylindole) (blue) and viewed under a microscope for apoptotic cells. The numbers of apoptotic cells per high-power field (HPF) were measured in 10 villi in 5– 6 HPFs on each slide. B: graphical representation of the quantification of cells positive for apoptosis using the method stated in A. *P ⬍ 0.05, n ⫽ 3 in each group. C: quantitative PCR comparing relative mRNA expression of the BAX gene between controls and CKO. *P ⬍ 0.05, n ⫽ 4 in each group. D: representative Western blot of intestinal homogenates comparing levels of proapoptotic proteins BAX, AIF, and caspase-3 between CKO and controls, along with loading control GAPDH. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
ENTEROCYTE-DERIVED ASL MODULATES INCIDENCE OF NEC
from the animals that underwent the NEC protocol were analyzed for markers of apoptosis. qPCR analysis revealed significantly increased expression of the proapoptotic gene Bax in the CKO compared with the controls (Fig. 5C). Western blot analyses performed on intestinal homogenates from pups that were subjected to stresses of NEC protocol showed elevated levels of apoptotic proteins BAX and AIF (Fig. 5D). Loss of Asl impairs migration of IEC-6 cells in the presence of LPS. Because CKO mice demonstrated elevated apoptosis similar to findings observed with human infants with NEC, we explored the effect of loss of ASL on the ability of enterocytes to migrate. The ability of the enterocytes to migrate is an integral part in the process of repair following intestinal injury. We knocked down Asl in IEC-6 cells using lentivirus encoding shRNA targeting Asl. The movement of IEC-6 cells across a wounded area of the cell monolayer is representative of the ability of the cells to migrate (17). As depicted (Fig. 6, A and B), the ability of cells to migrate was similar in the Asl knockdown and in control cells with nonsilencing scrambled shRNA under basal conditions. On addition of LPS, the rate of migration in control cells increased considerably, while Asl knockdown cells failed to show a similar response, suggesting that the loss of ASL impairs the migration of IEC-6 cells during conditions of stress.
infants with NEC (4, 21) and supplementation with arginine has been shown to be beneficial in some studies (1, 19). The mechanisms by which arginine therapy might be protective are not clear, and hence conclusive proof for the protection of arginine is still elusive. Arginine is a substrate for synthesis of many biologically important molecules including NO, agmatine, polyamines, and creatine. These metabolites have roles in energy metabolism, gene expression, apoptosis, and cell proliferation and differentiation, which are crucial in intestinal homeostasis (7). Perturbations of these critical cellular functions can lead to an increase in apoptosis and inflammation and to a decreased capacity to repair, some of which are hallmarks of NEC (2, 18). Our report highlights the protective action of enterocytederived ASL in the pathogenesis of NEC. The increased histological injury score in the CKO was associated with an exaggerated inflammatory state with elevated levels of IL-6 and increased neutrophilic infiltration in the gut. In addition, activation of proapoptotic pathways and the resultant enterocyte loss were significantly higher in the CKO. Whereas there were no differences in the histology of intestine in the CKO during unstressed conditions compared with wild-type littermates, induction of NEC led to increased inflammation, and enterocyte death, with supportive in vitro evidence for decreased migration with loss of ASL. These findings are consistent with the exaggerated inflammation and apoptosis observed in the intestines from human infants with NEC (2). We acknowledge the limitations in our study. NEC was induced in the animals by exposing newborn pups to the risk factors thought to play an important role in human NEC, namely prematurity, absence of breast-feeding, and hypoxia. It is widely recognized that, although this model mimics the intestinal injury of NEC at the histological level, it is perhaps not most representative of human NEC. However, in spite of its limitations, the mouse model of NEC with hypoxia-hypothermia-formula feeding is one of the best available tools to study NEC in the laboratory. This is mainly due to the availability of mutant mouse models and tractability of mouse genetics. The
DISCUSSION
*
*
100 80 60
Fig. 6. A: images representing IEC-6 cells culture grown at a monolayer with a horizontal scratch at 0 h (top) and 12 h (bottom). B: graphical representation of quantification of the area of migration in 12 h at basal conditions and on addition of LPS. *P ⬍ 0.05.
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Percent area of wound healing in 12 hours
We successfully generated a mouse model to study the role of enterocyte-derived ASL in the pathogenesis of NEC and to demonstrate that loss of ASL results in increased incidence and severity of NEC. Mechanistically, we show that loss of enterocyte-derived ASL is associated with increased apoptosis and inflammation and with reduced migration capacity of mutant enterocytes. Because enterocytes are the main sites of ASL expression in perinatal mammals, they are also the principal sites of arginine synthesis. ASL expression in the enterocytes is at the maximum by 10 –14 days of age in mice and 2–3 yr in humans (13). Studies have demonstrated that lower plasma arginine levels in
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AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
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maximum ASL expression in the enterocytes in mice is around 10 –14 days (13). Although it seemed appropriate to test the role of ASL in NEC at this age in mice, pups of this age group are very resistant to induction of NEC, primarily due to the exposure to breast milk and its protective effects. Hence our studies with NEC were conducted in pups immediately after birth before they came in contact with mother’s milk. Whereas previous studies manipulated arginine at the whole body level, leading to inconclusive results, our approach of manipulating ASL metabolism specifically in enterocytes allowed us to specifically study the cell-autonomous contribution of arginine synthesis in this cell type to NEC susceptibility. Our report offers avenues to harness this protective role of ASL in devising methods of prevention or treatment in NEC. ACKNOWLEDGMENTS We acknowledge the contributions of Yuqing Chen and Elda Munivez for technical assistance in this project. GRANTS This work was supported in part by Public Health Service Grant DK-56338 to M. H. Premkumar and National Institutes of Health Grant GM-90310 to B. Lee. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS M.H.P., P.M.C., A.E., and B.L. conception and design of research; M.H.P., G.S., S.C.N., S.C., A.N., M.Z.R., T.B., J.Z., D.S., and N.S.B. performed experiments; M.H.P., G.S., M.J., B.C.D., and N.S.B. analyzed data; M.H.P., M.Z.R., and D.S. interpreted results of experiments; M.H.P. and P.M.C. prepared figures; M.H.P. drafted manuscript; M.H.P., S.C.N., P.M.C., A.E., and B.L. edited and revised manuscript; M.H.P., S.C.N., P.M.C., A.E., and B.L. approved final version of manuscript. REFERENCES 1. Amin HJ, Zamora SA, McMillan DD, Fick GH, Butzner JD, Parsons HG, Scott RB. Arginine supplementation prevents necrotizing enterocolitis in the premature infant. J Pediatr 140: 425–431, 2002. 2. Ballance WA, Dahms BB, Shenker N, Kliegman RM. Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 117 (1 Pt 2): S6 –S13, 1990. 3. Barlow B, Santulli TV. Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model. Surgery 77: 687–690, 1975. 4. Becker RM, Wu G, Galanko JA, Chen W, Maynor AR, Bose CL, Rhoads JM. Reduced serum amino acid concentrations in infants with necrotizing enterocolitis. J Pediatr 137: 785–793, 2000. 5. Caplan MS, Hedlund E, Adler L, Hsueh W. Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis. Pediatr Pathol 14: 1017–1028, 1994.
6. Erez A, Nagamani SC, Shchelochkov OA, Premkumar MH, Campeau PM, Chen Y, Garg HK, Li L, Mian A, Bertin TK, Black JO, Zeng H, Tang Y, Reddy AK, Summar M, O’Brien WE, Harrison DG, Mitch WE, Marini JC, Aschner JL, Bryan NS, Lee B. Requirement of argininosuccinate lyase for systemic nitric oxide production. Nat Med 17: 1619 –1626, 2011. 7. Flynn NE, Meininger CJ, Haynes TE, Wu G. The metabolic basis of arginine nutrition and pharmacotherapy. Biomed Pharmacother 56: 427– 438, 2002. 8. Guthrie SO, Gordon PV, Thomas V, Thorp JA, Peabody J, Clark RH. Necrotizing enterocolitis among neonates in the United States. J Perinatol 23: 278 –285, 2003. 9. Holman RC, Stoll BJ, Clarke MJ, Glass RI. The epidemiology of necrotizing enterocolitis infant mortality in the United States. Am J Public Health 87: 2026 –2031, 1997. 10. Holman RC, Stoll BJ, Curns AT, Yorita KL, Steiner CA, Schonberger LB. Necrotising enterocolitis hospitalisations among neonates in the United States. Paediatr Perinat Epidemiol 20: 498 –506, 2006. 11. Horbar JD, Carpenter JH, Badger GJ, Kenny MJ, Soll RF, Morrow KA, Buzas JS. Mortality and neonatal morbidity among infants 501 to 1500 grams from 2000 to 2009. Pediatrics 129: 1019 –1026, 2012. 12. Kafetzis DA, Skevaki C, Costalos C. Neonatal necrotizing enterocolitis: an overview. Curr Opin Infect Dis 16: 349 –355, 2003. 13. Kohler ES, Sankaranarayanan S, van Ginneken CJ, van Dijk P, Vermeulen JL, Ruijter JM, Lamers WH, Bruder E. The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes. BMC Dev Biol 8: 107, 2008. 14. Kutner RH, Zhang XY, Reiser J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat Protoc 4: 495–505, 2009. 15. Morello R, Bertin TK, Chen Y, Hicks J, Tonachini L, Monticone M, Castagnola P, Rauch F, Glorieux FH, Vranka J, Bächinger HP, Pace JM, Schwarze U, Byers PH, Weis M, Fernandes RJ, Eyre DR, Yao Z, Boyce BF, Lee B. CRTAP is required for prolyl 3-hydroxylation and mutations cause recessive osteogenesis imperfecta. Cell 127: 291–304, 2006. 16. Nathan C, Xie QW. Nitric oxide synthases: roles, tolls, and controls. Cell 78: 915–918, 1994. 17. Nusrat A, Delp C, Madara JL. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest 89: 1501–1511, 1992. 18. Pender SL, Braegger C, Gunther U, Monteleone G, Meuli M, Schuppan D, Macdonald TT. Matrix metalloproteinases in necrotising enterocolitis. Pediatr Res 54: 160 –164, 2003. 19. Polycarpou E, Zachaki S, Tsolia M, Papaevangelou V, Polycarpou N, Briana DD, Gavrili S, Kostalos C, Kafetzis D. Enteral L-arginine supplementation for prevention of necrotizing enterocolitis in very low birth weight neonates: a double-blind randomized pilot study of efficacy and safety. JPEN J Parenter Enteral Nutr 37: 617–622, 2013. 20. Radulescu A, Zhang HY, Yu X, Olson JK, Darbyshire AK, Chen Y, Besner GE. Heparin-binding epidermal growth factor-like growth factor overexpression in transgenic mice increases resistance to necrotizing enterocolitis. J Pediatr Surg 45: 1933–1939, 2010. 21. Zamora SA, Amin HJ, McMillan DD, Kubes P, Fick GH, Bützner JD, Parsons HG, Scott RB. Plasma L-arginine concentrations in premature infants with necrotizing enterocolitis. J Pediatr 131: 226 –232, 1997.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00403.2013 • www.ajpgi.org
