Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis

Jessica A. Clark,1 Robert H. Lane,2 Nicole K. MacLennan,2 Hana Holubec,3 Katerina Dvorakova,3 Melissa D. Halpern,1 Catherine S. Williams,1 Claire M. Payne,3 and Bohuslav Dvorak1,3

1Departments of Pediatrics and Steele Memorial Children's Research Center and 3Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona; and 2Department of Pediatrics, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California

Submitted 19 April 2004 ; accepted in final form 3 November 2004


    ABSTRACT
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 METHODS
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Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC+EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury.

epithelial injury; ileum; rat; Bcl-2; Bax


NECROTIZING ENTEROCOLITIS (NEC) is the most common gastrointestinal disease of premature infants (50). Recent reports suggest increasing occurrence of NEC with up to 9,000 cases in the United States every year, with death occurring in 20–40% of affected individuals (41, 42). Although the etiology of NEC is unknown, the major risk factors for development of the disease are prematurity, enteral feeding, intestinal hypoxia-ischemia, and bacterial colonization (6). Despite significant morbidity and mortality, there is currently no effective preventive treatment (58).

The incidence of NEC is significantly decreased in breast milk-fed compared with formula-fed infants (5, 17, 45, 59, 67). Furthermore, maternal rat milk reduces the severity of experimental NEC in the neonatal rat model of NEC (14). The components of maternal milk that are responsible for protection against NEC remain unknown, but epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis (12). EGF is a peptide that exhibits trophic, maturational, and healing effects on intestinal mucosa (11, 16, 53). The major sources of EGF in the developing neonate are maternal colostrum, milk, and saliva (7, 9, 13, 47, 51, 57). Diminished serum and saliva EGF levels were reported in neonates suffering NEC compared with healthy controls (28, 62). In the experimental rat model of NEC, we have previously shown that supplementation of EGF into cow's milk-based formula reduces the incidence of NEC by 50% (15). However, the mechanisms underlying EGF-mediated reduction of NEC are still not understood.

The Bcl-2 family of cytoplasmic proteins is an important class of molecules that regulates enterocyte apoptosis (37). Bcl-2 is an anti-apoptotic protein that attenuates the effects of cytochrome c release from the mitochondria and counters the effects of the pro-apoptosis protein Bax (35, 38). Bcl-2 and Bax contribute to the signaling pathways that modulate caspase-3 activity, which is necessary for the chromatin condensation and DNA fragmentation that characterize apoptosis (31, 54). An increase in the expression of pro-apoptotic Bax relative to a reduced expression of anti-apoptotic Bcl-2 may create an environment that favors apoptosis (63).

Although end-stage NEC is characterized histopathologically as extensive necrosis, recent reports suggest that apoptosis, or programmed cell death, accounts for the initial loss of cells in the apical villi before full development of the disease (19, 33, 48). It has been suggested that activation of the EGF-receptor signaling pathway blunts apoptosis (10), thereby preserving the villus architecture. The aim of this study was to investigate if EGF-mediated reduction of NEC is connected with the alteration of the expression of pro- and anti-apoptotic genes and proteins, causing a shift in the ratio that promotes cell survival. To achieve this aim, we induced NEC in neonatal rats using formula feeding coupled with exposure to asphyxia/cold stress. We investigated the effects of enteral administration of EGF on changes in intestinal morphology and expression of the apoptotic genes Bcl-2, Bcl-xL, Bcl-w, Bax, and Bad, as well as protein expression of Bax and Bcl-2, in the terminal ileum (site of NEC injury). Furthermore, histological localization of cleaved caspase-3, a specific marker for apoptotic cells, was evaluated in the terminal ileum. To evaluate if EGF treatment induced epithelial cell proliferation in the terminal ileum, expression of proliferating cell nuclear antigen (PCNA) was measured.


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Animal model and diets. The protocol was approved by the Animal Care and Use Committee of the University of Arizona (A-324801–95081). Neonatal Sprague-Dawley rats (Charles River Laboratories, Pontage, MI) originating from 14 separate litters were used in 5 different experiments. Newborn rats were collected immediately after birth to prevent suckling of maternal milk. Animals were assigned to one of the following three experimental groups: NEC, pups artificially fed with growth factor-free rat milk substitute (RMS); NEC+EGF, pups artificially fed with RMS supplemented with 500 ng/ml rat EGF (Harlan Bioproducts, Indianapolis, IN), or dam fed (DF). Experimental NEC was induced by asphyxia and cold stress, as previously described (15, 24, 25). After 96 h, all surviving animals were killed via decapitation. Animals that died before 96 h were excluded from the study because postmortem tissue is not suitable for evaluations.

Corticosterone measurement. Blood was collected from the trunk and centrifuged for 5 min at 10,000 rpm. Plasma samples were then frozen until the assay was performed. Corticosterone was assayed in duplicate by using a double-antibody 125I RIA kit (ICN Pharmaceuticals, Costa Mesa, CA). Plasma samples were diluted 1:200 in the assay buffer before RIA.

RNA preparation. Total RNA was isolated from ileal tissue using the RNeasy Mini Kit (Qiagen, Santa Clarita, CA) as described in the manufacturer's protocol and our previous studies (15, 24). All samples were incubated with RNase-free DNase (20 U/reaction) for 10 min at 37°C to eliminate DNA contamination. RNA concentration was quantified by ultraviolet spectrophotometry at 260 nm, and the purity was determined by the ratio of absorbance at 260 nm to that at 280 nm (SPECTRAmax PLUS; Molecular Devices, Sunnyvale, CA). The integrity of RNA was verified by electrophoresis on a 1.2% agarose gel containing formaldehyde (2.2 mol/l) and ethidium bromide in 1x 3-(N-morpholino)propanesulfonic acid (MOPS) buffer [in mmol/l: 40 MOPS (pH 7.0), 10 sodium acetate, and 1 EDTA (pH 8.0); see Refs. 15 and 24].

RT and real-time PCR. RT real-time PCR assays were performed to specifically quantify rat Bcl-2, Bcl-xL, Bcl-w, Bax, and Bad steady-state mRNA levels. cDNA was synthesized from 0.5 µg DNase-treated total RNA. Target (Bcl-2, Bcl-xL, Bax, and Bad) primers and probes were designed using Primer Express Software (Applied Biosystems, Foster, CA; Table 1); target probes were labeled with fluorescent reporter dye FAM (52). Predeveloped TaqMan primer and probe were used for detection of Bcl-w (Applied Biosystems). Before initiation of real-time PCR, all primer pairs were tested with serial Mg2+ and primer concentrations to determine the optimal reaction conditions and to demonstrate the specificity of each primer pair. Reporter dye emission is detected by an automated sequence detector combined with ABI Prism 7700 Sequence Detection System software (Applied Biosystems). An algorithm normalizes the reporter signal (Rn) to a passive reference and multiplies the SD of the background Rn in the first cycle by a default factor of 10 to determine the threshold cycle (CT). CThas a linear relation with the logarithm of the initial template copy number (29). Real-time PCR quantification is then performed using TaqMan glyceraldehyde-3-phosphate dehydrogenase (GAPDH) controls. Before the use of GAPDH as a control, serial dilutions of cDNA are quantified to prove the validity of using GAPDH as an internal control. Relative quantification of PCR products are then based on the value differences between the target and GAPDH control using the comparative CT method (46). Cycle parameters were 55°C for 5 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 58°C for 60 s. For every sample, each PCR reaction was performed on three separate occasions; in each set of reactions, every sample was present in triplicate.


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Table 1. Sequences of PCR primers and probes

 
Western blot. Individual frozen ileum samples were homogenized with a hand-held homogenizer (Pellet Pestle; Kimble/Kontes, Vineland, NJ) in a 5x volume of ice-cold homogenization buffer (50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% sodium deoxycholic acid, 1% Triton X-100, 50 mM DTT, 50 µg/ml aprotinin, 50 µg/ml leupeptin, and 5 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was collected. Total protein concentration was quantified using the Bradford (4) protein assay. For protein analysis, 50 µg protein were added to an equal volume of 2x Laemmli sample buffer and boiled for 5 min. The samples were run on a 10–20% gradient polyacrylamide gel (Bio-Rad, Hercules, CA) at 95 volts for 1 h. Protein was transferred to Immuno-Blot polyvinylidene difluoride membranes (Bio-Rad) at 15 volts for 1 h. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (Sigma, St. Louis, MO) for 1 h at room temperature and then incubated with a rabbit polyclonal anti-Bax antibody (Pharmingen, San Diego, CA) or mouse monoclonal anti-Bcl-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. After being washed, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (for Bax) or goat anti-mouse IgG (for Bcl-2; Santa Cruz Biotechnology). Proteins were visualized with a chemiluminescent system (Pierce, Rockford, IL) and exposed to X-ray film. Densitometry was performed to compare protein expression between groups with Bio-Rad QuantityOne software.

Ileal morphometry. From each animal, a 2-cm section of distal ileum next to the ileocecal valve was fixed overnight in 70% ethanol, paraffin-embedded, sectioned at 4–6 µm, and stained with hematoxylin and eosin for morphometric measurements; 8–10 villi were measured for each animal, and 10–13 animals were evaluated per experimental group. Sections from animals with a NEC score of four were not included in analyses because of the lack of intact tissue to evaluate. Villi were measured from the tip to the crypt base, and the number of epithelial cells in this crypt-villus axis was enumerated. These measurements were performed using an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). Analysis of all morphological data was performed in a blind manner to prevent observer bias.

Immunohistochemistry. Expression of cleaved caspase-3 protein and PCNA were evaluated. Serial sections from ileal samples were processed as previously described (15, 24). After deparaffinization, rehydration, and incubation in hydrogen peroxide, sections were blocked with 1.5% appropriate serum (Vector Laboratories, Burlingame, CA) and then incubated with the following antibodies: 2.0 µg/ml rabbit polyclonal anti-human cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) and 2.0 µg/ml goat polyclonal anti-rat PCNA (Santa Cruz Biotechnology), followed by biotinylated secondary antibody (Vector Laboratories), Vectastain Elite ABC reagent (Vector Laboratories), diaminobenzidine activated by hydrogen peroxide. Sections were then counterstained with hematoxylin. Control sections were treated with the same procedure except they were incubated with 2.0 µg/ml rabbit Ig (for caspase-3) or 2.0 µg/ml goat Ig (for PCNA; Sigma). No immunostaining was observed in the controls. Sections from both experimental groups were immunostained for a specific antigen at the same time so that comparisons between groups could be assessed. For caspase-3 measurements, stained slides were evaluated by a blind observer, and enumeration of positively stained cells was accomplished by counting 8–10 villi/animal and evaluating 5 rats/experimental group (100x magnification).

Statistics. Statistical analyses between NEC, NEC+EGF, and DF groups were performed using ANOVA followed by Fishers protected least-significant difference test. For histological NEC scores, analyses were performed using the Kruskal-Wallis test followed by pairwise comparisons using the Mann-Whitney U-test. For protein ratios and caspase-3 measurements, analyses were performed using the paired t-test. All statistical analyses were conducted using the statistical program Statview for Macintosh computers (Abacus Concepts, Berkely, CA). All numerical data are expressed as means ± SE.


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Morphometric measurements. Intestinal morphology was examined by light microscopy. The NEC group had significantly shorter villi compared with DF animals (P ≤ 0.05). Villi in the NEC+EGF group were significantly longer than those not given EGF (Fig. 1A; P ≤ 0.003). To determine whether the increase in villus height in the EGF group was a result of hypertrophy or hyperplasia, the total number of epithelial cells in each measured villus was counted, and results from these measurements were expressed as the number of epithelial cells per micrometer of villus. There were no significant differences seen between the groups, indicating that EGF caused hyperplasia of the epithelial cells and not hypertrophy (Fig. 1B).



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Fig. 1. Morphologic measurements of villus length (A) and no. of villi epithelial cells (B) in the ileum of neonatal rats that were dam fed (DF), fed growth factor-free rat milk substitute [necrotizing enterocolitis (NEC)], and fed rat milk substitute supplemented with 500 ng/ml rat epidermal growth factor (NEC+EGF); n = 10–13 rats/experimental group. *P ≤ 0.05 for NEC vs. DF and P ≤ 0.003 for NEC vs. NEC+EGF.

 
Cell proliferation induced by EGF. To determine if EGF induces mitogenic effects on epithelial cells, the cell proliferation marker PCNA was evaluated by immunohistochemistry (Fig. 2). There were no significant differences observed between NEC and NEC+EGF groups, indicating that supplementation of EGF into formula did not induce epithelial cell proliferation in the terminal ileum.



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Fig. 2. Histological localization of proliferating cell nuclear antigen (PCNA) in the ileum. Representative sections from DF, NEC, and NEC+EGF animals stained for PCNA are shown. There were no significant differences between groups; n = 6 rats/experimental group. No staining was observed in the controls (Neg.).

 
Plasma corticosterone levels. The exposure to stress can result in increased epithelial apoptosis of the gastrointestinal tract (20, 33). To characterize animal response to repeated asphyxia/cold stress during the study, plasma corticosterone levels were measured using RIA. To assure that serum corticosterone levels were not the result of immediate asphyxia/cold stress treatment, animals were not stressed for 16 h before harvest. Rat pups artificially fed with formula (NEC) or with formula supplemented with 500 ng EGF (NEC+EGF) had significantly elevated levels of corticosterone compared with DF animals (Fig. 3; P ≤ 0.0001 and P ≤ 0.001, respectively). However, there was no statistically significant difference among NEC and NEC+EGF groups. This indicates that, although artificially fed animals are more stressed than DF animals, supplementation of EGF into formula did not reduce this effect.



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Fig. 3. Corticosterone (ng/ml) in plasma of neonatal DF, NEC, NEC+EGF rats; n = 15 rats/experimental group. *P ≤ 0.0001 for DF vs. NEC and P ≤ 0.001 for DF vs. NEC+EGF.

 
Ileal mRNA levels of pro- and anti-apoptotic genes. Gene expression of pro-apoptotic Bax and Bad and anti-apoptotic Bcl-2, Bcl-w, and Bcl-xL was evaluated in the ileum using RT Real-time PCR. Pro-apoptotic Bax mRNA levels were significantly increased in the NEC group compared with DF animals (Fig. 4A; P < 0.01). EGF supplementation markedly decreased Bax mRNA levels compared with animals fed formula alone (Fig. 4A; P < 0.05). Conversely, anti-apoptotic Bcl-2 mRNA levels were significantly decreased in the NEC group compared with DF animals (Fig. 4B; P < 0.01). Supplementation of formula with EGF significantly increased Bcl-2 mRNA levels compared with NEC (P < 0.05) but still remained markedly lower than DF animals (Fig. 4B; P < 0.01). There were no statistically significant differences in Bad, Bcl-xL, and Bcl-w expression between NEC and NEC+EGF groups (results not shown). To determine if there is a shift in the balance of apoptotic genes that favor cell survival in EGF-supplemented animals, the ratio of Bax to Bcl-2 levels was evaluated (Table 2). The Bax-to-Bcl-2 ratio was markedly increased in the NEC group compared with DF animals. Supplementation with EGF decreased the ratio compared with animals given formula alone.



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Fig. 4. Ileal mRNA levels of Bax (A) and Bcl-2 (B) were evaluated using real-time RT-PCR. The mean steady-state mRNA level for the DF group was assigned a value of 1.0, and the mean mRNA from the NEC and NEC+EGF groups was determined relative to this number; n = 10 rats/experimental group. *Statistically significant vs. DF, P ≤ 0.01. {ddagger}Statistically significant vs. NEC, P ≤ 0.05.

 

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Table 2. Ratio of Bax to Bcl-2 mRNA expression in the ileum

 
Ileal levels of pro- and anti-apoptotic proteins. Ileal expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins was evaluated using Western blot. Pro-apoptotic Bax protein levels were markedly increased in NEC animals compared with DF animals. Supplementation with EGF decreased expression of Bax compared with animals fed formula alone. Anti-apoptotic Bcl-2 protein levels were increased in animals supplemented with EGF compared with NEC animals (Fig. 5). The Bax-to-Bcl-2 ratio for protein expression was also evaluated and was significantly decreased in NEC+EGF animals compared with the NEC group (n = 10/experimental group, P ≤ 0.0001). This indicates that EGF treatment shifts the balance of apoptotic proteins in favor of cell survival.



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Fig. 5. Representative 21-kDa protein bands for Bax and 26 kDa protein bands for Bcl-2 by Western blot are shown for DF, NEC, and NEC+EGF groups. Expression of Bax is increased in NEC animals and decreased with supplementation of EGF in formula compared with the NEC group. Expression of Bcl-2 is increased in NEC+EGF animals compared with NEC animals. DF levels are shown as age-matched controls.

 
Apoptosis. Cleaved caspase-3, a specific marker for apoptotic cells, was used to detect and localize apoptotic changes in the terminal ileum of neonatal rats in all experimental groups (Fig. 6A). In the NEC group, there was a significant increase in the number of caspase-3-positive epithelial cells at the tip of the villus (Fig. 6B; P ≤ 0.001). EGF treatment significantly decreased cleaved caspase-3 staining compared with the NEC group, with only occasional positive apoptotic cells observed in the EGF-treated rats. No cleaved caspase-3-positive cells were detected in DF controls.



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Fig. 6. Cleaved caspase-3 localization in the terminal ileum of neonatal rats. A: representative slides for DF, NEC, and NEC+EGF groups. In the NEC group, many epithelial cells at the tip of the villi exhibited positive signal for cleaved caspase-3 (arrows). Light micrographs x100 oil immersion. B: caspase-3-positive epithelial cells are significantly increased in animals with NEC. Positive cells were counted from 8 to 10 villi/animal (x100 magnification), and results are expressed as percentage of caspase-3-positive cells per villi; n = 5 rats/experimental group. *P ≤ 0.001, NEC vs. NEC+EGF.

 

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Alterations in apoptosis may predispose the intestine to development of NEC (33). We are the first to show that EGF treatment of NEC elicits changes in expression of apoptotic genes and proteins in the terminal ileum, shifting the balance between pro- and anti-apoptotic proteins in favor of cell survival. We have previously shown that supplementation of EGF into milk formula significantly reduces the incidence of NEC in a neonatal rat model (15). These data suggest a possible mechanism of EGF-mediated reduction of the experimental NEC.

Several studies have demonstrated an important role of EGF insufficiency in the pathogenesis of NEC. Diminished serum and saliva EGF levels were reported in neonates suffering NEC compared with healthy controls (28, 62). Furthermore, a critically ill infant, diagnosed with NEC-like symptoms, was successfully treated by continuous intravenous infusion of EGF (65). In a rat model of NEC, EGF supplementation in formula reduced the incidence of experimental NEC (15), and EGF-mediated reduction of NEC was associated with downregulation of proinflammatory IL-18 and increased production of anti-inflammatory IL-10 in the site of injury (23). The biological actions of EGF are mediated through binding to EGF receptor (EGF-R); the presence of EGF-R has been reported in intestinal epithelium of infants diagnosed with NEC (18) and in a rat NEC model (15). Previous studies have shown that activation of the EGF-R may blunt apoptosis, thereby protecting intestinal structure during NEC (10).

In the normal small intestine, epithelial homeostasis is maintained by balancing the rate of cell proliferation and cell loss. It has been shown that apoptosis, and not simple exfoliation of enterocytes from the tip of villus, accounts for the majority of cell loss in the gut lumen (22, 55, 56, 61). Berseth (3) has shown that supplementation of EGF in milk formula enhances neonatal intestinal growth and intestinal cellular proliferation in suckling rats. In our NEC model, a significant reduction of villus length was measured in the intact portions of the terminal ileum of the NEC group compared with DF controls. Supplementation of formula with EGF resulted in normalization of villus size. Because the number of epithelial cells per micrometer of villus length was not different between the NEC and NEC+EGF groups, we conclude that hyperplasia rather than hypertrophy is occurring in the EGF-treated group. This conclusion is further supported by our previously published observation that ileal protein-to-DNA ratios are not different among these experimental groups (15). In addition to its mitogenic effects, EGF has multiple nonmitogenic actions within the gastrointestinal tract (see review in Ref. 66). In rat and mouse models of small bowel resection (SBR), increased intestinal morphological parameters, such as villus height or crypt depth, with exogenous EGF have been reported (8, 27, 43). The results from the present study with a NEC model indicate no changes in intestinal epithelial proliferation as a result of enteral administration of EGF. Thus the data suggest that enhanced hyperplasia of the intestinal mucosa in the NEC+EGF group results from inhibition of apoptosis of epithelial cells rather than from increased mitogenic effects of EGF.

Apoptosis is a physiological mode of cell death, distinct from necrosis, that plays an important role in many physiological and pathological processes (36, 60, 68). The intestinal epithelium has a high turnover rate that necessitates the apoptotic removal of cells at the end of their normal life cycle (2). In neonatal rats, the exposure to stress results in increased apoptosis in the gastric (20) and the intestinal epithelium (33). In vitro studies using the rat intestinal epithelial cell line IEC-6 demonstrated a dose-dependent effect of increasing corticosteroid levels on the induction of apoptosis (34). In our studies, formula feeding combined with repeated asphyxia/cold stress resulted in a significant increase of plasma levels of corticosterone in both the NEC and NEC+EGF groups (4- to 5-fold) compared with the DF group. Therefore, apoptotic changes measured between NEC and NEC+EGF groups cannot be attributed to differences in corticosterone levels. Expression of pro-apoptotic genes and proteins were significantly higher in the NEC group compared with the NEC+EGF group, and EGF treatment resulted in a shift in the balance of pro- and anti-apoptotic markers in favor cell survival. Thus we speculate that EGF has a direct effect on blunting apoptosis at the site of NEC injury.

Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis, and the balance of their pro- and anti-apoptotic members is a critical factor for cell survival (1). The mechanism by which EGF protects intestinal cells against apoptosis may include the reduction of Bax expression (21), the increase of Bcl-2 (44) and Bcl-xL (49, 64) expression, and the inhibition of caspases (40). Studies using the murine model of SBR indicate that enterocyte apoptosis during intestinal adaptation is attenuated by EGF (26) and exaggerated when the EGF-R is defective (27). Similarly, Stern et al. (63) and Knott et al. (37) have shown that exogenous EGF retards rates of enterocyte apoptosis and modifies the expression of critical Bcl-2 family members. By decreasing pro-apoptotic Bax and increasing anti-apoptotic Bcl-w expression, the balance between pro- and anti-survival genes shifts in favor of cell survival. Inhibition of EGF-R signaling in the SBR model accelerates the rate of apoptosis and modifies the expression of Bcl-2-related peptides in favor of apoptosis (37). Our results using the neonatal rat model of NEC are in agreement with findings from the adult murine SBR model.

Bcl-2 and Bax are critical factors in apoptosis regulation because the molecular ratio of Bax to Bcl-2 acts as a cellular "rheostat" determining cellular flux toward or away from apoptosis (38, 52). Tissue Bax-to-Bcl-2 ratio is often used as an indicator of sensitivity to pro- or anti-apoptotic stimuli (30, 39). In this study, the development of experimental NEC in neonatal rats was associated with a huge increase in the Bax-to-Bcl-2 mRNA ratio (250-fold) in injured ileum compared with healthy DF littermates. Bax protein expression was also significantly increased in animals with NEC compared with DF animals. These findings suggest that massive apoptosis occurred before full necrosis of ileal mucosa developed. Jilling et al. (33) recently showed that caspase-3 activity, a specific marker for apoptotic cells, is significantly increased in intestinal lysates of rats with NEC. However, it was not specified in which part of the small intestine and in which intestinal cells the increase in caspase-3 activity is occurring. In this study, the number of cleaved caspase-3-positive cells was significantly increased in the epithelial cells of the terminal ileum, the site of intestinal injury. Thus results from our study support previous finding and further clarify this observation. In conclusion, inhibition of the EGF-R signaling pathway or knockout models of NEC are necessary to further clarify the exact molecular mechanism of EGF-mediated protection. However, a recent study using waved-2 mice (defective EGF-R signaling) crossbred with Bax-null mice in a SBR model clearly indicated that impairment of EGF-R signaling augments intestinal epithelial cell apoptosis (32).

Supplementation of EGF in formula markedly decreased the Bax-to-Bcl-2 mRNA and protein ratios and dramatically reduced apoptosis. These results indicate that EGF treatment of experimental NEC alters apoptotic gene expression, thereby shifting the balance of pro- and anti-apoptotic genes toward cell survival. We speculate that EGF-mediated reduction of epithelial cell apoptosis is an important factor by which EGF reduces mucosal injury in the neonatal rat model of NEC. Better understanding of molecular processes underlying EGF-mediated reduction of experimental NEC might provide the basis for the future therapeutic strategies for the treatment of human NEC.


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This work was supported by National Institute of Child Health and Human Development Grant HD-39657 (to B. Dvorak).


    ACKNOWLEDGMENTS
 
We thank Dr. Jon Wispé for editing the manuscript.

Current address for R. H. Lane: University of Utah, Department of Pediatrics, 30 N. 1900 East, Salt Lake City, UT, 84132.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. Dvorak, Dept. of Pediatrics, Univ. of Arizona, 1501 N. Campbell Ave, P.O. Box 245073, Tucson, AZ 85724-5073 (E-mail: dvorakb{at}peds.arizona.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Adams JM and Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 281: 1322–1326, 1998.[Abstract/Free Full Text]
  2. Benedetti A, Mancini R, Marucci L, Paolucci F, Jezequel AM, and Orlandi F. Quantitative study of apoptosis in normal rat gastroduodenal mucosa. J Gastroenterol Hepatol 5: 369–374, 1990.[ISI][Medline]
  3. Berseth CL. Enhancement of intestinal growth in neonatal rats by epidermal growth factor in milk. Am J Physiol Gastrointest Liver Physiol 253: G662–G665, 1987.[Abstract/Free Full Text]
  4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  5. Buescher ES. Host defense mechanisms of human milk and their relations to enteric infections and necrotizing enterocolitis. Clin Perinatol 21: 247–262, 1994.[ISI][Medline]
  6. Caplan MS and MacKendrick W. Necrotizing enterocolitis: a review of pathogenetic mechanisms and implications for prevention. Pediatr Pathol 13: 357–369, 1993.[ISI][Medline]
  7. Carpenter G. Epidermal growth factor is a major growth-promoting agent in human milk. Science 210: 198–199, 1980.[ISI][Medline]
  8. Chaet MS, Arya G, Ziegler MM, and Warner BW. Epidermal growth factor enhances intestinal adaptation after massive small bowel resection. J Pediatr Surg 29: 1035–1039, 1994.[CrossRef][ISI][Medline]
  9. Connolly JM and Rose DP. Epidermal growth factor-like proteins in breast fluid and human milk. Life Sci 42: 1751–1756, 1988.[CrossRef][ISI][Medline]
  10. Danielsen AJ and Maihle NJ. The EGF/ErbB receptor family and apoptosis. Growth Factors 20: 1–15, 2002.[CrossRef][ISI][Medline]
  11. Duh G, Mouri N, Warburton D, and Thomas DW. EGF regulates early embryonic mouse gut development in chemically defined organ culture. Pediatr Res 48: 794–802, 2000.[Abstract/Free Full Text]
  12. Dvorak B. Epidermal growth factor and necrotizing enterocolitis. Clin Perinatol 31: 183–192, 2004.[ISI][Medline]
  13. Dvorak B, Fituch CC, Williams CS, Hurst NM, and Schanler RJ. Increased epidermal growth factor levels in human milk of mothers with extremely premature infants. Pediatr Res 54: 15–19, 2003.[Abstract/Free Full Text]
  14. Dvorak B, Halpern MD, Holubec H, Dvorakova K, Dominguez JA, Williams CS, Meza YG, Kozakova H, and McCuskey RS. Maternal milk reduces severity of necrotizing enterocolitis and increases intestinal IL-10 in a neonatal rat model. Pediatr Res 53: 426–433, 2003.[Abstract/Free Full Text]
  15. Dvorak B, Halpern MD, Holubec H, Williams CS, McWilliam DL, Dominguez JA, Stepankova R, Payne CM, and McCuskey RS. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model. Am J Physiol Gastrointest Liver Physiol 282: G156–G164, 2002.[Abstract/Free Full Text]
  16. Dvorak B, Philipps AF, and Koldovsky O. Milk-Borne Growth Factors and Gut Development. In: Nutrition of the Very Low Birthweight Infant, edited by Zeigler EE, Lucas A, and Moro GE. Philadelphia, PA: Williams & Wilkins, 1999, p. 245–255.
  17. Eibl MM, Wolf HM, Furnkranz H, and Rosenkranz A. Prevention of necrotizing enterocolitis in low-birth-weight infants by IgA-IgG feeding. N Engl J Med 319: 1–7, 1988.[Abstract]
  18. Fagbemi AO, Wright N, Lakhoo K, and Edwards AD. Immunoreactive epidermal growth factor receptors are present in gastrointestinal epithelial cells of preterm infants with necrotising enterocolitis. Early Hum Dev 65: 1–9, 2001.[CrossRef][ISI][Medline]
  19. Ford H, Watkins S, Reblock K, and Rowe M. The role of inflammatory cytokines and nitric oxide in the pathogenesis of necrotizing enterocolitis. J Pediatr Surg 32: 275–282, 1997.[CrossRef][ISI][Medline]
  20. Gama P, Goldfeder EM, de Moraes JC, and Alvares EP. Cell proliferation and death in the gastric epithelium of developing rats after glucocorticoid treatments. Anat Rec 260: 213–221, 2000.[CrossRef][ISI][Medline]
  21. Gibson S, Tu S, Oyer R, Anderson SM, and Johnson GL. Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation. J Biol Chem 274: 17612–17618, 1999.[Abstract/Free Full Text]
  22. Hall PA, Coates PJ, Ansari B, and Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107: 3569–3577, 1994.[Abstract/Free Full Text]
  23. Halpern MD, Dominguez JA, Dvorakova K, Holubec H, Williams CS, Meza YG, Ruth MC, and Dvorak B. Ileal cytokine dysregulation in experimental necrotizing enterocolitis is reduced by epidermal growth factor. J Pediatr Gastroenterol Nutr 36: 126–133, 2003.[CrossRef][ISI][Medline]
  24. Halpern MD, Holubec H, Dominguez JA, Meza YG, Williams CS, Ruth MC, McCuskey RS, and Dvorak B. Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 284: G695–G702, 2003.[Abstract/Free Full Text]
  25. Halpern MD, Holubec H, Dominguez JA, Williams CS, Meza YG, McWilliam DL, Payne CM, McCuskey RS, Besselsen DG, and Dvorak B. Up-regulation of IL-18 and IL-12 in the ileum of neonatal rats with necrotizing enterocolitis. Pediatr Res 51: 733–739, 2002.[Abstract/Free Full Text]
  26. Helmrath MA, Erwin CR, and Warner BW. A defective EGF-receptor in waved-2 mice attenuates intestinal adaptation. J Surg Res 69: 76–80, 1997.[CrossRef][ISI][Medline]
  27. Helmrath MA, Shin CE, Erwin CR, and Warner BW. The EGF:EGF-receptor axis modulates enterocyte apoptosis during intestinal adaptation. J Surg Res 77: 17–22, 1998.[CrossRef][ISI][Medline]
  28. Helmrath MA, Shin CE, Fox JW, Erwin CR, and Warner BW. Epidermal growth factor in saliva and serum of infants with necrotising enterocolitis. Lancet 351: 266–267, 1998.[ISI][Medline]
  29. Higuchi R, Fockler C, Dollinger G, and Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11: 1026–1030, 1993.[CrossRef][ISI][Medline]
  30. Ina K, Itoh J, Fukushima K, Kusugami K, Yamaguchi T, Kyokane K, Imada A, Binion DG, Musso A, West GA, Dobrea GM, McCormick TS, Lapetina EG, Levine AD, Ottaway CA, and Fiocchi C. Resistance of Crohn's disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance. J Immunol 163: 1081–1090, 1999.[Abstract/Free Full Text]
  31. Janicke RU, Sprengart ML, Wati MR, and Porter AG. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273: 9357–9360, 1998.[Abstract/Free Full Text]
  32. Jarboe MD, Juno RJ, Bernal NP, Knott AW, Zhang Y, Erwin CR, and Warner BW. Bax deficiency rescues resection-induced enterocyte apoptosis in mice with perturbed EGF receptor function. Surgery 136: 121–126, 2004.[CrossRef][ISI][Medline]
  33. Jilling T, Lu J, Jackson M, and Caplan MS. Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental model of neonatal necrotizing enterocolitis. Pediatr Res 55: 622–629, 2004.[Abstract/Free Full Text]
  34. Jung S, Fehr S, Harder-d'Heureuse J, Wiedenmann B, and Dignass AU. Corticosteroids impair intestinal epithelial wound repair mechanisms in vitro. Scand J Gastroenterol 36: 963–970, 2001.[CrossRef][ISI][Medline]
  35. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, and Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95: 4997–5002, 1998.[Abstract/Free Full Text]
  36. Kerr JF, Wyllie AH, and Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257, 1972.[ISI][Medline]
  37. Knott AW, Juno RJ, Jarboe MD, Zhang Y, Profitt SA, Thoerner JC, Erwin CR, and Warner BW. EGF receptor signaling affects bcl-2 family gene expression and apoptosis after massive small bowel resection. J Pediatr Surg 38: 875–880, 2003.[CrossRef][ISI][Medline]
  38. Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE, and Oltvai ZN. Bcl-2/Bax: a rheostat that regulates an anti-oxidant pathway and cell death. Semin Cancer Biol 4: 327–332, 1993.[ISI][Medline]
  39. Krajewski S, Krajewska M, Shabaik A, Miyashita T, Wang HG, and Reed JC. Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am J Pathol 145: 1323–1336, 1994.[Abstract]
  40. Lan L and Wong NS. Phosphatidylinositol 3-kinase and protein kinase C are required for the inhibition of caspase activity by epidermal growth factor. FEBS Lett 444: 90–96, 1999.[CrossRef][ISI][Medline]
  41. Lee JS and Polin RA. Treatment and prevention of necrotizing enterocolitis. Semin Neonatol 8: 449–459, 2003.[CrossRef][Medline]
  42. Lemons JA, Bauer CR, Oh W, Korones SB, Papile LA, Stoll BJ, Verter J, Temprosa M, Wright LL, Ehrenkranz RA, Fanaroff AA, Stark A, Carlo W, Tyson JE, Donovan EF, Shankaran S, and Stevenson DK. Very low birth weight outcomes of the National Institute of Child health and human development neonatal research network, January 1995 through December 1996. NICHD Neonatal Research Network Pediatrics 107: E1, 2001.
  43. Liu CD, Rongione AJ, Shin MS, Ashley SW, and McFadden DW. Epidermal growth factor improves intestinal adaptation during somatostatin administration in vivo. J Surg Res 63: 163–168, 1996.[CrossRef][ISI][Medline]
  44. Loo DT, Bradford S, Helmrich A, and Barnes DW. Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation. Cell Biol Toxicol 14: 375–382, 1998.[CrossRef][ISI][Medline]
  45. Lucas A and Cole TJ. Breast milk and neonatal necrotising enterocolitis. Lancet 336: 1519–1523, 1990.[CrossRef][ISI][Medline]
  46. Menon RK, Shaufl A, Yu JH, Stephan DA, and Friday RP. Identification and characterization of a novel transcript of the murine growth hormone receptor gene exhibiting development- and tissue-specific expression. Mol Cell Endocrinol 172: 135–146, 2001.[CrossRef][ISI][Medline]
  47. Moran JR, Courtney ME, Orth DN, Vaughan R, Coy S, Mount CD, Sherrell BJ, and Greene HL. Epidermal growth factor in human milk: daily production and diurnal variation during early lactation in mothers delivering at term and at premature gestation. J Pediatr 103: 402–405, 1983.[ISI][Medline]
  48. Nadler EP, Dickinson E, Knisely A, Zhang XR, Boyle P, Beer-Stolz D, Watkins SC, and Ford HR. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis. J Surg Res 92: 71–77, 2000.[CrossRef][ISI][Medline]
  49. Nass SJ and Dickson RB. Epidermal growth factor-dependent cell cycle progression is altered in mammary epithelial cells that overexpress c-myc. Clin Cancer Res 4: 1813–1822, 1998.[Abstract]
  50. Neu J. Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention. Pediatr Clin North Am 43: 409–432, 1996.[ISI][Medline]
  51. Okada M, Ohmura E, Kamiya Y, Murakami H, Onoda N, Iwashita M, Wakai K, Tsushima T, and Shizume K. Transforming growth factor (TGF)-alpha in human milk. Life Sci 48: 1151–1156, 1991.[CrossRef][ISI][Medline]
  52. Pham TD, MacLennan NK, Chiu CT, Laksana GS, Hsu JL, and Lane RH. Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney. Am J Physiol Regul Integr Comp Physiol 285: R962–R970, 2003.[Abstract/Free Full Text]
  53. Pollack PF, Goda T, Colony PC, Edmond J, Thornburg W, Korc M, and Koldovsky O. Effects of enterally fed epidermal growth factor on the small and large intestine of the suckling rat. Regul Pept 17: 121–132, 1987.[CrossRef][ISI][Medline]
  54. Porter AG and Janicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6: 99–104, 1999.[CrossRef][ISI][Medline]
  55. Potten CS. Epithelial cell growth and differentiation. II. Intestinal apoptosis. Am J Physiol Gastrointest Liver Physiol 273: G253–G257, 1997.[Free Full Text]
  56. Potten CS, Wilson JW, and Booth C. Regulation and significance of apoptosis in the stem cells of the gastrointestinal epithelium. Stem Cells 15: 82–93, 1997.[Abstract/Free Full Text]
  57. Read LC, Upton FM, Francis GL, Wallace JC, Dahlenberg GW, and Ballard FJ. Changes in the growth-promoting activity of human milk during lactation. Pediatr Res 18: 133–139, 1984.[Abstract]
  58. Schanler RJ. Overview: the clinical perspective. J Nutr 130: 417S–419S, 2000.[ISI][Medline]
  59. Schanler RJ. The use of human milk for premature infants. Pediatr Clin North Am 48: 207–219, 2001.[ISI][Medline]
  60. Searle J, Kerr JF, and Bishop CJ. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol Annu 17: 229–259, 1982.[ISI][Medline]
  61. Shin CE, Falcone RA Jr, Kemp CJ, Erwin CR, Litvak DA, Evers BM, and Warner BW. Intestinal adaptation and enterocyte apoptosis following small bowel resection is p53 independent. Am J Physiol Gastrointest Liver Physiol 277: G717–G724, 1999.[Abstract/Free Full Text]
  62. Shin CE, Falcone RA Jr, Stuart L, Erwin CR and Warner BW. Diminished epidermal growth factor levels in infants with necrotizing enterocolitis. J Pediatr Surg 35: 173–176, 2000.[CrossRef][ISI][Medline]
  63. Stern LE, Falcone RA Jr, Huang F, Kemp CJ, Erwin CR and Warner BW. Epidermal growth factor alters the bax:bcl-w ratio following massive small bowel resection. J Surg Res 91: 38–42, 2000.[CrossRef][ISI][Medline]
  64. Stoll SW, Benedict M, Mitra R, Hiniker A, Elder JT, and Nunez G. EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL. Oncogene 16: 1493–1499, 1998.[CrossRef][ISI][Medline]
  65. Sullivan PB, Brueton MJ, Tabara ZB, Goodlad RA, Lee CY, and Wright NA. Epidermal growth factor in necrotising enteritis. Lancet 338: 53–54, 1991.[ISI][Medline]
  66. Uribe JM and Barrett KE. Nonmitogenic actions of growth factors: an integrated view of their role in intestinal physiology and pathophysiology. Gastroenterology 112: 255–268, 1997.[ISI][Medline]
  67. Walker WA. Breast milk and the prevention of neonatal and preterm gastrointestinal disease states: a new perspective. Chung-Hua Min Kuo Hsiao Erh Ko i Hsueh Hui Tsa Chih 38: 321–331, 1997.
  68. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284: 555–556, 1980.[CrossRef][ISI][Medline]