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Original Articles: Gastroenterology

Comparison of Epidermal Growth Factor and Heparin-binding Epidermal Growth Factor–like Growth Factor for Prevention of Experimental Necrotizing Enterocolitis

Dvorak, Bohuslav*; Khailova, Ludmila*; Clark, Jessica A; Hosseini, Dania Molla*; Arganbright, Kelly M*; Reynolds, Charity A*; Halpern, Melissa D*

Author Information
Journal of Pediatric Gastroenterology and Nutrition: July 2008 - Volume 47 - Issue 1 - p 11–18
doi: 10.1097/MPG.0b013e3181788618
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Abstract

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease predominantly of prematurely born infants. The pathophysiology of this disease remains poorly understood; however, prematurity, enteral feeding, intestinal hypoxia-ischemia, and bacterial colonization are considered major risk factors (1,2). No predictive diagnostic tests, prevention, or effective treatments are available as far as we are aware.

Epidermal growth factor (EGF) is a peptide that has trophic, maturational, and healing effects on the intestinal mucosa (3). Maternal colostrum and milk are the major sources of EGF for the developing neonate (4). It has been suggested that EGF insufficiency may play an important role in the pathogenesis of NEC (5). In an experimental rat model of NEC, we have previously shown that supplementation of EGF into formula reduces the incidence of NEC (6), downregulates the overproduction of proinflammatory cytokines (7,8), and maintains bile acid homeostasis (9). The molecular mechanisms underlying EGF-mediated protection against NEC include the reduction of intestinal apoptosis (10) and an improvement of intestinal barrier function (11).

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the family of EGF-related peptides. The presence of HB-EGF has been reported in human amniotic fluid and human milk, suggesting a role for HB-EGF in the early stages of the development of the gastrointestinal tract (12). An initial report from our laboratory showed that HB-EGF reduces the incidence of experimental NEC (13). Recently, a similar protective effect of HB-EGF against NEC-like injury has been reported (14).

Although EGF shows promise as a preventive of and/or treatment for NEC, it is possible that other EGF-related growth factors found in breast milk may have additional protective effects against NEC injury. Whereas EGF activates predominantly EGF-R receptor complexes (15), HB-EGF is capable of binding to and activating 2 tyrosine kinase receptors, EGF-R and ErbB-4 (16). Therefore, it has been suggested that the cytoprotective effects of HB-EGF against intestinal injury may be mediated via a mechanism different from that of EGF.

The aim of this study was to clarify the effect of enteral administration of HB-EGF, EGF, or a combination of both (E+HB) on the development of NEC in a neonatal rat model. First, we established the most efficient dosage of HB-EGF to decrease the incidence of experimental NEC. Second, we compared the efficacy of HB-EGF treatment versus EGF treatment and tested a possible synergistic effect of HB-EGF and EGF against the development of NEC. Finally, to clarify possible molecular mechanisms underlying HB-EGF and EGF-mediated protection against NEC, intestinal expression of EGF-R and ErbB-4 receptors, goblet cell density, and apoptotic proteins were evaluated in this study.

MATERIALS AND METHODS

Experimental NEC Model

This protocol was approved by the Animal Care and Use Committee of the University of Arizona (A-324801-95081). Neonatal Sprague-Dawley rats (Charles River Labs, Pontage, MI) were collected by caesarian section 1 day before scheduled birth. Animals were hand fed for 96 hours with rat milk substitute formula based on cow's milk and free of growth factor (6). Experimental NEC was induced by asphyxia (breathing 100% nitrogen gas for 60 seconds) and cold stress (4°C for 10 minutes) twice daily (9).

Experimental Design

Dosage Efficiency Experiments

To examine the effect of enteral administration of HB-EGF on the development of NEC and to identify the most efficient dosage of HB-EGF, 3 separate studies were performed. Pups were hand fed with either rat milk substitute formula (NEC, n = 24) or formula supplemented with 5 (HB-5, n = 16), 50 (HB-50, n = 16), 500 (HB-500, n = 16), or 1000 (HB-1000, n = 16) ng/mL human HB-EGF (EMD Biosciences, San Diego, CA). Dam-fed littermates were used for comparison (DF, n = 15).

HB-EGF and EGF Experiments

To compare the effect of HB-EGF, EGF (rat EGF from Harlan Bioproducts, Indianapolis, IN), or both on the development of NEC, a series of additional experiments were performed. We have shown that the most efficient dosage of EGF to reduce experimental NEC is 500 ng/mL (6,10). The same dosage of HB-EGF was also the most effective. Rats were hand fed with either rat milk substitute formula (NEC; n = 36) or formula supplemented with 500 ng/mL HB-EGF (HB, n = 26), or 500 ng/mL rat EGF (EGF, n = 24) or 500 ng/mL of each growth factor (E+HB, n = 25). Dam-fed littermates served as a control group (DF, n = 22). Animals in all of the above-described experimental groups were exposed to asphyxia and were cold stressed to induce experimental NEC. After 96 hours, all of the surviving animals were killed by decapitation.

NEC Evaluation

Pathological changes in intestinal architecture were evaluated according to our published NEC scoring system (6,9): 0 = no damage; 1 = slight submucosal and/or lamina propria separation; 2 = moderate separation of submucosa and/or lamina propria, and/or edema in submucosal and muscular layers; 3 = severe separation of submucosa and/or lamina propria, and/or severe edema in submucosa and muscular layers, region villous sloughing; 4 = loss of villi and necrosis. Intermediate scores of 0.5, 1.5, 2.5, and 3.5 were also used to more accurately assess levels of ileal damage when necessary (6,9). To determine the incidence of NEC, animals with histological scores <2 did not have NEC; animals with histological scores of ≥2 did have NEC (6,9).

RNA Preparation and Real-time Polymerase Chain Reaction

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 (10,11). Real-time polymerase chain reaction (PCR) assays were performed to quantify steady-state mRNA levels of MUC2 (11). cDNA was synthesized from 0.5 μg Dnase-treated total RNA. Real-time PCR amplification (17) was performed by Primer Express software (Applied Biosystems). The following MUC2 sequences were used (GenBank BC036170): sense primer 5′-actgggaatgtgactgctactg-3′; anti-sense primer 5′-accctggtaactgtagtaaagtccat-3′; and probe 5′-acaaagtgtgggtcccc-3′.

Reporter dye emission was detected by an automated sequence detector combined with ABI Prism 7700 Sequence Detection System software (Applied Biosystems). Real-time PCR quantification was then performed by use of TaqMan 18S controls (10,11).

Immunohistology of MUC2 and Enumeration of Goblet Cells

A 2-cm section of distal ileum was collected from each animal and fixed overnight in 70% ethanol, paraffin embedded, and sectioned at 4 to 6 μm. Serial sections were stained for MUC2. After deparaffinization and rehydration, sections were blocked with 1.5% goat serum (Vector Laboratories, Burlingame, CA) in phosphate-buffered saline for 30 min, then incubated with 4.0 μg/mL rabbit anti-MUC2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min, washed with phosphate-buffered saline 3 times, and incubated with a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) for 30 min. Sections were counterstained with hematoxylin, dehydrated, and mounted with coverslips. MUC2-positive goblet cells were enumerated from 12 animals per group. The total number of epithelial cells per crypt-villus unit was also enumerated.

Western Blot

Individual frozen ileum samples were homogenized with a hand-held homogenizer (Pellet Pestle, Kimble/Kontes, Vineland, NJ) in a 5× volume of ice-cold homogenization buffer (50 mmol/L Tris HCl, pH 7.4; 150 mmol/L NaCl; 1 mmol/L EDTA; 0.1% SDS; 1% Na-deoxycholic acid; 1% Triton X-100; 50 mmol/L DTT; 50 μg/mL aprotinin; 50 μg/mL leupeptin; 5 mmol/L PMSF). The homogenates were centrifuged at 10,000 rpm for 5 minutes at 4°C, and the supernatant was collected. Total protein concentration was quantified by use of the Bradford protein assay (18). For protein analysis, 40 μg of protein was added to an equal volume of 2× Laemmli sample buffer and boiled for 5 minutes. The samples were run on a gradient of 8% or 15% polyacrylamide gel (Bio-Rad, Hercules, CA) at 95 V for 1 hour. Protein was transferred to Immuno-Blot PVDF membranes (Bio-Rad) at 35 to 50 V for 1 hour. Membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (Sigma, St Louis, MO) for 1 hour at room temperature and then incubated with a rabbit polyclonal anti-EGF receptor antibody (Cell Signaling, Beverly, MA), a rabbit polyclonal ErbB-4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit polyclonal Bax antibody (BD Pharmigen, San Jose, CA), or a mouse polyclonal Bcl-2 antibody (Santa Cruz Biotechnology) overnight at 4°C. After extensive washing, the membranes were incubated for 1 hour at room temperature with horseradish peroxidase–conjugated donkey antimouse or antirabbit IgG (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.

Statistics

Statistical analyses between the DF, NEC, HB, EGF, and E+HB groups were performed by analysis of variance, followed by Fisher PLSD test. The χ2 test was used to analyze differences in the incidence of disease. All of the numerical data are expressed as mean ± SE.

RESULTS

Dose Response of HB-EGF on the Incidence of NEC

Four different dosages of HB-EGF were tested. The most efficient dosage of orally administered HB-EGF for protection against NEC was 500 ng/mL (Table 1). The incidence of NEC was decreased by 40%, and ileal damage was markedly reduced in comparison with the nonsupplemented NEC group. Supplementation of low-dosage (5 ng/mL) and high-dosage (1000 ng/mL) HB-EGF did not provide significant protective effects against NEC.

TABLE 1
TABLE 1:
Effect of enteral HB-EGF on incidence and severity of NEC

Comparison of the Protective Effects of HB, EGF, and E+HB Against NEC

To compare the efficacy of HB-EGF and EGF treatment and to evaluate a possible synergistic effect of EGF and HB-EGF against the development of NEC, a separate set of 5 experiments was performed. Supplementation of EGF alone into milk formula was the most efficient preventive treatment against the development of NEC, reducing the incidence of disease in the NEC group from 62% to 14% in the EGF group (Fig. 1). HB-EGF alone or in combination with EGF (E+HB, 500 ng/mL of each growth factor) also significantly reduced the incidence of NEC (by 35% or 43%, respectively). Supplementation of formula with a lower dosage of both EGF and HB-EGF together (250 ng/mL of each) had markedly lower efficiency in the prevention of NEC (data not shown).

FIG. 1
FIG. 1:
Effect of enteral administration of HB-EGF, EGF, or E+HB on incidence of NEC. All animals (DF, n = 22; NEC, n = 36; HB-EGF, n = 26; EGF, n = 24, E + HB, n = 25) with NEC scores ≥2 are considered NEC positive; animals with ileal damage <2 do not have NEC.*P ≤ 0.01 vs NEC, #P ≤ 0.001 vs NEC, χ2 analysis.

EGF-R and ErbB-4 Expression

Levels of EGF-R and ErbB-4 mRNA were similar in all of the experimental groups (data not shown). However, the mean EGF-R mRNA levels in the terminal ileum were approximately 650 to 750 times higher than the ErbB-4 mRNA levels in the same tissue. To quantify changes in protein expression, EGF-R and ErbB-4 levels were evaluated in the terminal ileum of pups by use of Western blot analysis. EGF-R protein levels were markedly increased in the NEC and HB groups compared with DF pups (Fig. 2). Treatment with EGF or with E+HB markedly reduced ileal EGF-R protein expression to levels similar to that seen in DF pups. Protein expression of the ErbB-4 receptor was not detected in any sample even when more sensitive detection techniques were used.

FIG. 2
FIG. 2:
A, Representative 170-kDa protein bands for EGF-R in ileal tissue from DF (n = 6), NEC (n = 8), HB-EGF (n = 8), EGF (n = 8), and E + HB (n = 8) groups. B, Mean relative optical density of EGF-R protein in the ileum.*P ≤ 0.05 vs DF, #P ≤ 0.05 vs NEC.

MUC2 Production in the Ileum

The mucus layer is an essential part of intestinal barrier function. MUC2 is the major secretory mucin produced by intestinal goblet cells in rats. Gene expression of MUC2 was evaluated in the ileum by use of real-time PCR. Supplementation of HB, EGF, or E+HB resulted in a significant increase in MUC2 mRNA levels compared with either the DF or the NEC group. Enumeration of ileal MUC2-positive goblet cells indicated a significant decrease in the MUC2-positive cells in the NEC group compared with DF control pups. Supplementation with HB, EGF, or E+HB markedly increased the number of MUC2-secreting goblet cells compared with either DF or NEC animals (Table 2).

TABLE 2
TABLE 2:
Enumeration of MUC2 cells and gene expression of MUC2 in the ileum

Levels of Ileal Proapoptotic and Antiapoptotic Proteins

Regulation of the balance between proapoptotic and antiapoptotic proteins in the site of injury is a possible mechanism by which intestinal integrity can be maintained. The shift in the balance of apoptotic proteins that favors either cell survival or cell death is often expressed as the ratio of Bax to Bcl-2 proteins. In our studies, ileal protein levels of proapoptotic Bax and antiapoptotic Bcl-2 were evaluated by Western blot. The Bax-to-Bcl-2 ratio was markedly increased in the NEC and HB groups compared with DF animals (Fig. 3). Supplementation of EGF or E+HB into formula significantly decreased this ratio, suggesting a shift in the balance of apoptotic proteins to favor cell survival (Fig. 3).

FIG. 3
FIG. 3:
A, Representative 21-kDa protein bands for Bax and 26-kDa protein bands for Bcl-2 in ileal tissue from DF (n = 6), NEC (n = 8), HB-EGF (n = 8), EGF (n = 8), and E + HB (n = 8) groups. B, Mean ratio of Bax to Bcl-2 protein in the ileum.*P ≤ 0.001 vs DF, #P ≤ 0.05 vs NEC.

DISCUSSION

Oral administration of EGF or HB-EGF has been suggested as a potential novel treatment for neonatal NEC, but the comparative efficiency of these treatments has not been evaluated. The present study demonstrates that supplementation of either EGF or HB-EGF into milk formula reduces the incidence and severity of this disease in a rat NEC model. Simultaneous administration of both growth factors does not result in any additional protective effects against NEC. Our results also show that EGF provides protection against the development of NEC in physiologically relevant doses compared with HB-EGF. A shift in the balance of apoptotic proteins in the ileum in favor of cell survival may be a mechanism responsible for the higher efficiency of EGF protection against NEC.

Neonatal NEC primarily affects prematurely born infants, and decreasing gestational age of newborns clearly correlates with increasing incidence of disease (19,20). After birth, maternal milk is usually the major source of nutrients and growth-promoting substances (21). A multicenter study from the United Kingdom has shown that the incidence of NEC among premature babies fed with human milk is 6 to 10 times lower than the incidence in formula-fed babies (22). Although the component(s) of breast milk associated with these protective effects were not identified, the results from rodent studies suggest EGF and HB-EGF as possible candidates (13,14,23,24). Human milk EGF levels are the highest in the first days after parturition (approximately 100 ng/mL) and then gradually decrease during the first month of lactation (4,25). Interestingly, EGF levels in the milk of mothers with extremely preterm neonates are 50% to 80% higher than those in the milk of mothers with full-term infants (4). Although the physiological relevance of this observation is still not fully understood, elevated levels of EGF in human milk can be potentially responsible for the protective effect of maternal milk against neonatal diseases, such as NEC. Indeed, the results from current and previous work from our laboratory have shown that supplementation of EGF into formula reduced the incidence of experimental NEC in comparison with nonsupplemented formula-fed littermates (6).

In comparison with EGF, concentrations of HB-EGF in human milk are approximately 1000 to 10,000 times lower (20–230 pg/mL (12)), raising a question about the role of HB-EGF in the developing intestine. The results from the present study indicate that HB-EGF can have protective effects against experimental NEC. However, a high pharmacological dosage of HB-EGF (500 ng/mL) is necessary to reduce the incidence and severity of NEC. Lower dosages of HB-EGF were less efficient or not efficient at all. These results are in agreement with a recently published study wherein intestinal injury was developed in neonatal rats by challenging them with hypoxia, hypothermia, hypertonic feedings, and intragastric administration of lipopolysaccharide; only high pharmacological amounts of HB-EGF (600 or 800 μg/kg) were able to reduce intestinal injury (26). However, these dosages correspond to concentrations of 1500 to 6500 ng/mL HB-EGF in milk formula, which is 4 to 5 orders higher than the normal physiological levels of HB-EGF in maternal milk. We speculate that discrepancies between the dosages of HB-EGF treatment in the present study and the study by Feng and Besner (26) are likely due to the differences in experimental protocol, the severity of intestinal injury in these models, and the type of HB-EGF peptide used in these studies.

It is not clear why the highest dosage of HB-EGF (HB-1000) did not have any protective effect against NEC. The structure of HB-EGF is characterized by the presence of both EGF-like and heparin-binding domains. In vitro studies have shown that HB-EGF, but not EGF, requires cell surface heparin sulfate proteoglycans for binding and activation of its receptor (27–29). In addition, it has been shown that heparin regulates binding of HB-EGF to the EGFR in a dose-dependent manner (27). A recent report suggests that the heparin-binding domain of HB-EGF suppresses the activity of the EGF-like domain of HB-EGF peptide and thus regulates the biological activity of this peptide. The authors conclude that the heparin-binding domain of HB-EGF may serve as a negative regulator of this growth factor (30). We speculate that in our NEC model, similar regulatory mechanisms of HB-EGF action may apply.

Furthermore, the ability of HB-EGF to bind to and activate both EGF-R and ErbB-4 receptors leads to the assumption that HB-EGF could be more efficient for the treatment of intestinal injury than EGF, which binds only to the EGF-R (26). However, in our NEC model the supplementation of either HB-EGF or the combination of EGF and HB-EGF into formula did not exceed the protective effect observed with EGF alone. These data raise the question about the relevance of ErbB-4 signaling during neonatal intestinal injury. Gene and protein levels of both EGF-R and ErbB-4 receptors were evaluated and compared in the site of NEC injury: the terminal ileum. Gene expression of EGF-R and ErbB-4 was not significantly different among the experimental groups. However, mean EGF-R mRNA levels were about 650 to 750 times higher than the ErbB-4 mRNA levels in the same tissue. We have shown increased immunohistochemical staining for EGF-R in the epithelial cells of the ileum of NEC rats, whereas oral administration of EGF diminished these levels to levels observed in healthy control animals (6). In the present study, protein expression of EGF-R in the ileum was quantified and compared between all of the treatment groups. EGF-R expression was markedly increased in the NEC group but also in HB-EGF-treated animals. By contrast, treatment with EGF or E+HB normalized EGF-R protein expression to the levels seen in the control animals. As expected from the gene expression data, protein levels of ErbB-4 receptor in the ileum of neonatal rats were low, and we could not detect any ErbB-4 signal using standard Western blot technique. Because immunoprecipitation followed by Western blot is a less accurate method for protein quantification and requires a larger amount of tissue for analysis, we could not use this approach for ErbB-4 detection. Thus, we speculate that the role of ErbB-4 signaling in the injured intestine of neonatal rats is perhaps limited.

Perturbation of the intestinal barrier has been implicated in the pathogenesis of NEC (31–34), and a recent study from our laboratory has shown that intestinal permeability, ileal goblet cell density, and mucin production are altered in rats with NEC (11). Because HB-EGF and EGF have been associated with improved intestinal barrier function (35,36), we evaluated structural and functional changes of the intestinal barrier in rats treated with HB-EGF, EGF, or E+HB. Ileal goblet cell density and MUC2 production were markedly increased in all of the groups treated with growth factor(s), but there were no differences between the efficiency of individual treatments.

A possible molecular mechanism for EGF or HB-EGF protection against bowel injury is via regulation of intestinal epithelial cell apoptosis (37,38). Although severe NEC is associated with extensive bowel necrosis, apoptosis has been shown to play an important role in the initial stages of experimental NEC (10,39). The Bcl-2 family of proteins is a key regulator of enterocyte apoptosis (40), and proapoptotic Bax and antiapoptotic Bcl-2 play an important role in the regulation of apoptosis in the developing intestinal epithelium. Furthermore, the tissue Bax-to-Bcl-2 ratio has been used as an indicator of proapoptotic and antiapoptotic stimuli. In animals with NEC, the molecular ratio of Bax to Bcl-2 proteins in the site of injury is shifted toward cell death (10). We have recently shown that reduction of experimental NEC with anti-TNF-α therapy is associated with decreased protein expression of proapoptotic Bax in the site of injury (41).

Supplementation of formula with either EGF alone or EGF plus HB-EGF leads to a shift in the balance between Bax to Bcl-2 proteins in favor of cell survival. By contrast, supplementation of HB-EGF into formula had no effect on the Bax-to-Bcl-2 ratio. We conclude that the inability of HB-EGF to reduce ileal epithelial cell apoptosis is at least partially responsible for its lower efficacy in prevention of NEC compared with EGF.

In summary, this study shows for the first time, to our knowledge, the comparative efficacy of EGF and/or HB-EGF to prevent the development of NEC. Oral administration of EGF provides protection against disease development in more physiological dosages compared with HB-EGF. Although the majority of possible molecular mechanisms responsible for EGF or HB-EGF protective effects on intestinal injury are similar, the higher efficiency of EGF-mediated reduction of epithelial cell apoptosis is likely an important factor in mucosal healing processes.

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Keywords:

Epidermal growth factor; Heparin-binding epidermal growth factor-like growth factor; Intestinal injury; Rat

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