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. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: Gastroenterology. 2009 Apr 8;137(1):221–230. doi: 10.1053/j.gastro.2009.03.060

Heparin-binding EGF-like Growth Factor Increases Intestinal Microvascular Blood Flow in Necrotizing Enterocolitis

Xiaoyi Yu 1, Andrei Radulescu 1, Nicholas Zorko 1, Gail E Besner 1
PMCID: PMC2704259  NIHMSID: NIHMS108721  PMID: 19361505

Abstract

Background & Aims

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates. Although the exact etiology remains unknown, decreased intestinal blood flow is thought to play a critical role. We have shown that heparin-binding EGF-like growth factor (HB-EGF) protects the intestines from injury in a rodent model of NEC. Our current goal was to assess the effect of HB-EGF on intestinal microvascular blood flow and intestinal injury in rat pups subjected to experimental NEC.

Methods

Newborn rat pups were subjected to stress by exposure to hypoxia, hypothermia, hypertonic feedings and lipopolysaccharide, with some pups receiving HB-EGF (800 μg/kg/dose) added to the feeds. Control animals received breast milk. Intestinal injury was graded using a standard histologic injury scoring system. Microvascular blood flow was assessed by FITC-dextran angiography with fluorescent images subjected to quantification, and by scanning electron microscopy.

Results

Intestinal microvascular blood flow (defined as the extent of vascular filling with FITC-dextran) was significantly decreased in pups subjected to stress compared to breast fed pups. Stressed pups treated with HB-EGF had significantly increased microvascular blood flow. The changes in villous microvasculature correlated with histologic injury scores, with stressed pups treated with HB-EGF showing decreased histologic injury.

Conclusions

HB-EGF significantly preserved intestinal microvascular blood flow in pups subjected to experimental NEC, indicating that HB-EGF may play a critical role in the therapy of various diseases manifested by decreased intestinal blood flow, including NEC.

Introduction

Necrotizing enterocolitis (NEC) is an often catastrophic disease that occurs predominantly in premature neonates. The most important risk factor for the development of NEC is prematurity. Although the exact etiology remains unknown, decreased intestinal blood flow is thought to play a critical role. It is thought that NEC commences within the intestinal intramural microcirculation, particularly the small arteries that penetrate the intestinal wall and the submucosal arteriolar plexus, representing the principal resistance regulators in the intestine.1-4 The histologic characteristics of NEC, including inflammatory cell infiltration, mucosal edema and ulceration, and coagulative necrosis, suggest that intestinal ischemia may be a pivotal contributing factor. However, the role of ischemia as a primary provocative factor or as a secondary outcome in the development of NEC has not been clearly defined.5, 6 A predominant feature of the newborn intestinal microcirculation is its low resting vascular resistance and relatively high blood flow rate compared with adult animals1, 7, 8 Regulation of vascular resistance within newborn intestine is principally determined by a balance between the endothelial production of the vasoconstrictor peptide endothelin-1 (ET-1) and the vasodilatory free radical nitric oxide (NO). 9, 10

HB-EGF was initially identified as a secreted product of cultured human macrophages11 and found to be a member of the EGF family.12 HB-EGF is a 22-kDa glycoprotein which is produced as a membrane-anchored precursor (pro-HBEGF) that is processed to a soluble, secreted, mature form (sHB-EGF). HB-EGF exerts its biological effects by binding to cell-surface EGF receptors (EGFR)13, 14 and to the HB-EGF-specific receptor Nardilysin (Nrdc).15, 16 Many cell types including epithelial cells produce HB-EGF which acts as an autocrine growth factor for these cells.17

We have shown that in vitro exposure of intestinal epithelial cells (IEC) to exogenous HB-EGF protects the cells from necrosis, rebuilds intracellular ATP stores and preserves cytoskeletal integrity upon exposure to hypoxia.18 We have also shown that HB-EGF decreases apoptosis in IEC exposed to pro-inflammatory cytokines in vitro18, 19 and in injured intestine in vivo.20 HB-EGF decreases oxygen free radical production both in vitro and in vivo.21 Using various models of intestinal injury invivo, we have shown that HB-EGF protects against injury due to intestinal ischemia/reperfusion (I/R)22, hemorrhagic shock and resuscitation (HS/R)23, and experimental NEC.24 We have shown that HB-EGF preserves mesenteric microvascular blood flow in adult rats subjected to HS/R.23 However, the effect of HB-EGF on intestinal microcirculatory blood flow in experimental NEC has not been examined. In addition, we recently showed that HB-EGF acts as a vasodilator of isolated terminal mesenteric arterioles examined ex vivo (unpublished observations). Based on these observations, we sought to determine whether the ability of HB-EGF to protect the intestines from experimental NEC could be due, in part, to an ability to preserve intestinal microvascular blood flow. In the current study, rat pups were subjected to a combination of hypoxia, hypothermia, hypertonic feedings and enteral administration of lipopolysaccharide. This particular combination of insults, as described by us previously (24, 25), successfully creates gross and histologic evidence of significant NEC in our hands.

Materials and Methods

Rat pup model of experimental NEC

All experimental procedures were carried out according to guidelines for the ethical treatment of experimental animals and approved by the Institutional Animal Care and Use Committee of Nationwide Children's Hospital (Protocol #04203AR). Experimental NEC was induced using a modification of the neonatal rat model of NEC initially described by Barlow et al.26 Timed pregnant rats (Harlan Sprague-Dawley, Indianapolis, IN) underwent Cesarean section under CO2 anesthesia, with pups delivered on day 21 of gestation. Newborn pups were placed in an incubator at 37°C and gavaged with hypertonic formula containing 15 g Similac 60/40 (Ross Pediatrics, Columbus, OH) in 75 ml Esbilac (Pet-Ag, New Hampshire, IL), a diet that provides 836.8 kJ/kg on a daily basis. Feeds were started at 0.1 ml every 4h and advanced to a maximum of 0.4 ml per feed. Pups were stressed by exposure to hypoxia with 100% nitrogen for 1 minute, followed by exposure to hypothermia at 4°C for 10 minutes twice daily for either 1, 2 or 3 days, with administration of intragastric lipopolysaccharide (LPS) (2 mg/kg) 8h after birth. The addition of LPS increases the incidence of NEC in our model, and has been used by others as well.27, 28 Exposure of pups to hypoxia, hypothermia, hypertonic feeds and LPS will subsequently be referred to as “stress”. Pups were euthanized by cervical dislocation upon the development of any clinical signs of NEC, or at the end of the experiment at 3 days after birth. Additional pups were stressed for 3 days, but were treated with HB-EGF (800 μg/kg/dose) added to each feed. Control pups were breast fed for 3 days using surrogate mothers (since their natural mothers were sacrificed after C-section) and were not stressed. Previous studies from our laboratory utilized recombinant mature HB-EGF produced in E. coli as previously described.29 The recombinant human HB-EGF used in the current experiments, corresponding to amino acids 74-148 of the mature HB-EGF precursor, was produced using a Pichia pastoris expression system under Good Manufacturing Practice (Trillium Therapeutics, Inc, Toronto, Canada).

Histologic injury grading

Upon sacrifice, intestines were removed fixed in 10% formalin for 24h. Four pieces each of duodenum, jejunum, ileum, and colon were harvested, paraffin-embedded, sectioned at 5 μm thickness, stained with hematoxylin and eosin, and viewed using phase contrast microscopy for the presence and degree of NEC using a standard histological scoring system.30 In the interest of space, results from the ileum are shown throughout this paper. Histologic changes in the intestines were graded as: grade 0, no damage; grade 1, epithelial cell lifting or separation; grade 2, necrosis to the mid villous level; grade 3, necrosis of the entire villus; and grade 4, transmural necrosis. All sections were graded blindly by two independent observers. Tissue damage with histologic injury scores of grade 2 or greater were considered positive for NEC.

FITC-dextran angiography

Pups were anesthetized with isoflurane inhalation and placed under a dissecting microscope on a heating pad. A midline incision was made in the chest, and the pectoralis muscles reflected, allowing visualization of the heart though the intercostal muscles. For each animal, 50 μl of a 20 mg/ml solution of high-molecular-weight (2,000 kDa) fluorescein isothiocyanate (FITC)-labeled dextran (FD2000; Sigma, St. Louis, MO) was injected over 10 seconds into the left ventricle using a closed-chest technique and a 30-gauge needle attached to a 1 ml syringe, with animals sacrificed 5 min later.

Analysis of villous microvascular blood flow in whole mount preparations

Confocal microscopy

Villous microvascular blood flow was examined by confocal microscopy using a modification of the methodology of Stappenbeck et al.31 To best visualize the newborn rat pup villous microvasculature and to quantify blood flow, whole-mount slides was used in this part of the study. Segments of small intestine were excised, washed with PBS and opened longitudinally. Specimens were placed in fixation solution containing 1% paraformaldehyde, 15% picric acid, and 0.1 M sodium phosphate buffer (pH 7.0) and shaken gently at 4°C overnight. Specimens were then rinsed in ice-cold PBS (x 3), followed by incubation in 10% sucrose/PBS for 3h at 4°C, and an overnight incubation in 20% sucrose/10% glycerol/PBS at 4°C. Tissue segments were stained with propidium iodide (0.5μg/ml) (Sigma, St. Louis, MO) for 15 min at RT, followed by PBS washes × 3. Whole-mount preparations were placed on glass slides villous side up, mounted with Gelvatol, coverslipped, and evaluated using an LSM 510 confocal microscope (Zeiss, Thornwood, NY) with scanning at 2 μm thickness intervals. Scans were projected in three dimensions by taking 30-60 serial images, aligning them at 6° intervals, and compiling/rotating them about the y axis using LSM 510 software (Zeiss) to get three dimensional (3D) movies for viewing the microvascular network from different perspectives. Fluorescent images were subjected to quantification using Zeiss LSM 5 software. The vascular villous network was quantified by calculating the ratio of green FITC-dextran signal intensity (representing the vascular network) to red propidium iodide signal intensity (representing nuclei) per window, as visualized from a fixed perspective of the three-dimensional reconstruction.

Fluorescent microscopy

To display villous structure together with villous vasculature in pups, whole mount tissue samples and slides were prepared as described above and examined using a Zeiss Axioskop fluorescent microscope (Carl Zeiss, Inc., New York, NY). Fluorescent images were obtained using equal exposure times for all slides, with fluorescent intensity of individual villous vascular images defined as those signals above background.

Analysis of intestinal submucosal blood flow in cryosection slides

Confocal microscopy

Cryosection slides were utilized to maximally display the details of the submucosa. Fixed intestinal segments were embedded in OCT. Eighty μm thick sections were air-dried in the dark for 2h at RT, rehydrated in ice cold PBS for 1 min and incubated in 3% deoxycholic acid (Sigma, St. Louis, MO) for 1h at RT. Sections were rinsed in water × 2 and PBS × 1 to remove residual deoxycholic acid, and then stained with propidium iodide (0.5 μg/ml; Sigma, St. Louis, MO) for 7 min at RT, followed by PBS washes × 3. Finally, sections were mounted with Gelvatol, and examined with an LSM 510 confocal microscope (Zeiss, Thornwood, NY). The scanning procedures and data quantification were the same as detailed above. The vascular network of the submucosal area was defined by a zone between the base of the crypt and the muscularis mucosa (Figure 1). The submucosal vascular network was quantified by calculating the ratio of green FITC-dextran signal intensity (representing the vascular network) to red propidium iodide signal intensity (representing nuclei) per window as visualized from a fixed perspective of the three-dimensional reconstruction.

Figure 1.

Figure 1

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A) Illustration of intestinal submucosal and villous blood flow. The major sites of resistance to flow (flow regulation) are in the small mesenteric (piercing) arteries and in the submucosal 1A and 2A arterioles arising from the piercing arteries. The 3A arterioles arising from the 2A arterioles proceed to the villi. Modified with permission from Elsevier Science [Seminars in Pediatric Surgery, 2005; 14:152-8, Figure 1 A]; B) The submucosal vessels (mainly 1A and 2A arterioles) were defined as vessels in the zone between the base of the crypt and the muscularis mucosa (enclosed by white rectangle).

Scanning electron microscopy (SEM) of vascular corrosion casts (VCC)

To better define the intestinal microvasculature, a modified technique for vascular casting32, 33 was utilized, with perfusion through the descending aorta.34 Warm normal saline (45°C; 3 ml) was infused followed by ice cold normal saline (1 ml), with the system vented through the vena cava. The vascular bed was infused by continuous injection of Mercox II resin (2 ml; Ladd Research, Williston, VT) at a constant rate of 0.1 ml/min. The inferior vena cava was ligated to maintain stable intravascular pressure while the resin polymerized. Small intestines were excised and placed in a water bath overnight at 60°C to achieve optimal polymerization. Intestines were incubated in 15% NaOH solution for 12h to remove soft tissue and rinsed with distilled water. Vascular casts were frozen in distilled water and ice-embedded casts were freeze-dried. Casts were glued onto specimen stubs, sputtered with gold, and viewed using a scanning electron microscope (FEI Nova 400 NanoSEM, Hillsboro, Oregon).

Statistical analyses

Microvascular blood flow in the villi and submucosa was compared using Student's t test to determine statistical significance. Data are presented as mean ± SE. The incidence of NEC was evaluated between groups using chi-square analysis. Differences were considered to be statistically significant if p < 0.05.

Results

HB-EGF decreases intestinal histological damage in experimental NEC

Histologic injury was compared between pups subjected to stress and pups subjected to stress with HB-EGF added to the feeds (Figures 2 and 3). Pups were subjected to stress for up to 72h, at which time all surviving animals were sacrificed. Sixty percent of pups subjected to stress had histologic injury consistent with NEC (grade 2 or greater injury), and of those animals that developed NEC, 77% had grade 2 injury and 23% had grade 3 injury. On the other hand, only 23% of pups subjected to stress but treated with HB-EGF developed NEC, and of those animals that developed NEC, 93% had grade 2 injury and only 7% had grade 3 injury. Thus, stressed pups treated with HB-EGF that did go on to develop NEC had much lower histologic injury scores, indicating that HB-EGF significantly decreases histological damage associated with NEC.

Figure 2.

Figure 2

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HB-EGF decreases intestinal histological injury in rat pups subjected to experimental NEC. Shown are representative H&E stained sections demonstrating: A) grade 0, normal intestine from pup fed with breast milk for 3 days; B) grade 1, epithelial cell lifting or separation from pup subjected to stress but treated with HB-EGF for 3 days; C) grade 2, sloughing of epithelial cells to mid villous level in pup subjected to stress for 3 days; D) grade 3, necrosis of entire villus in pup subjected to stress for 3 days. Magnification 100x.

Figure 3.

Figure 3

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Incidence of experimental NEC. Shown are the grades of injury for individual pups subjected to stress with or without HB-EGF added to the feeds. In this experiment, pups were subjected to stress for up to 72h, at which time all surviving animals were sacrificed. Each point represents an individual animal. The starting number of animals in the Stress group was 80 and in the Stress + HB-EGF group was 73. Animals were excluded from analysis if they expired a significant amount of time prior to harvesting of intestines, since this led to tissue autolysis. Points above the dashed line indicate histologic injury consistent with NEC (grade 2 or higher injury). *p<0.001 compared to stress alone.

HB-EGF increases intestinal villous microvascular blood flow in experimental NEC

To begin to examine the effect of HB-EGF on intestinal microvascular blood flow, we utilized FITC-dextran angiography with fluorescent microscopic imaging of whole mount intestinal specimens. Pups subjected to stress had significantly decreased villous microvascular blood flow compared to breast fed pups (Figure 4). Stressed pups treated with HB-EGF had significant preservation of villous microvascular blood flow compared to stressed pups that did not receive HB-EGF.

Figure 4.

Figure 4

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Effect of HB-EGF on villous microcirculatory blood flow in whole mount specimens visualized by fluorescent microscopy. Shown are representative examples of fluorescent microscopic images of the villous microvasculature as observed in a single image of whole mount slides obtained from the ileum (10x). The villous microvascular blood flow is demonstrated by FITC-labeled high-molecular-weight dextran (green) staining. Shown are images from: A) a pup fed with breast milk for 3 days, showing intact villous microvascular blood flow; B) a pup subjected to stress for 3 days, showing decreased villous microvascular blood flow; and C) a rat pup subjected to stress for 3 days but with HB-EGF added to the feeds, showing significant preservation of villous microvascular blood flow.

Since confocal microscopy makes three dimensional (3D) characterization of the villous microvasculature possible, we next utilized confocal microscopy to quantify intestinal villous microvascular blood flow and to create 3D movies. We found that pups stressed for 1 or 2 days had no significant change in villous microvascular blood flow, whereas pups stressed for 3 days had significantly decreased villous microvascular blood flow compared to breast fed, non-stressed pups (p<0.001) (Figure 5 and 3D movie-1). Pups subjected to stress for only 1 or 2 days with HB-EGF added to the feeds had no significant change in villous microvascular blood flow, whereas pups stressed for 3 days with HB-EGF added to the feeds had preservation of villous microvascular blood flow compared to pups stressed for 3 days that did not receive HB-EGF (p<0.001). The finding that pups subjected to stress for only 1 or 2 days had no change in villous microvascular blood flow was consistent with our findings that it usually takes about 3 days for pups to develop NEC in our model (Figure 6). Thus, the changes we observed in villous microcirculatory blood flow correlated with the changes we observed in histologic injury scores.

Figure 5.

Figure 5

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Effect of HB-EGF on villous microcirculatory blood as demonstrated by confocal microscopy. Shown are representative images of villous microcirculatory blood flow as observed using FITC-dextran angiography in a 3D confocal projection of whole mount slides obtained from the ileum (40x). The red staining represents nuclear staining with propidium iodide, and the green staining indicates the microvasculature. The representative panels show intestinal samples from: A) control pups fed with breast milk for 3 d; B) pups subjected to stress for 3 d; and C) pups subjected to stress for 3 d but with HB-EGF added to the feeds. Panel D represents quantification of villous microcirculatory blood flow. The number of animals represented by each bar is 13, with the exception of bars 5 and 6 which represent 4 animals each. For each animal studied, two measurements were subjected to quantification, with green and red channels separated and signal intensity calculated using Zeiss LSM 5 software. In each scanned file, the ratio of green channel signal intensity (representing villous microcirculatory blood flow) to red channel signal intensity (representing nuclear staining) was obtained. *p<0.001 compared to Control; **p<0.001 compared to Stress-Day-3. Also, refer to 3D movie-1. Panel A of this figure corresponds to movie BMF; panel B corresponds to movie Stress, and panel C corresponds to movie Stress+HB-EGF.

Figure 6.

Figure 6

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Time course for the development of experimental NEC. Shown are the grades of injury for individual pups subjected to stress for 1, 2, 3 or 4 days. Each point represents an individual animal. Points lying above the dashed line indicate histologic injury consistent with NEC (grade 2 or higher injury).

HB-EGF preserves intestinal villous central arterioles, venules and capillaries in experimental NEC

We next examined the arterioles, venules and capillaries of newborn rat villi using intestinal vascular corrosion casting and SEM (Figure 7). We found that breast fed pups had normal villous central arterioles, venules and capillaries, even though the villous capillary network in newborn pups was simple compared to older animals, with relatively few capillaries bridging the arterioles and venules. Pups stressed for 3 d had distorted, injured, broken villous central arterioles, venules and capillaries, suggesting a complete lack of perfusion of the microvasculature. When HB-EGF was added to the feeds of stressed pups, the intestinal villous microvasculature was greatly preserved, with villous microvascular structure comparable to that of breast fed pups. This further demonstrated that HB-EGF preserves intestinal villous microvascular structure in animals subjected to experimental NEC.

Figure 7.

Figure 7

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Effect of HB-EGF on intestinal villous microvascular structure as visualized by SEM. Shown are intestinal vascular corrosion casts and SEM images (upper panels 200x and lower panels 350x) obtained from the ileum of: A, D) a breast milk fed pup with normal villous central arterioles, capillaries and venules; B, E) a pup stressed for 3 d, showing injured villous arterioles, capillaries and venules; and C, F) a pup stressed for 3 days but treated with HB-EGF added to the feeds, showing comparable villous microvasculature to that of the breast fed control, with significant preservation of villous blood flow and microvascular architecture.

HB-EGF increases intestinal submucosal microvascular blood flow in experimental NEC

Others have shown that a major source of resistance (flow regulation) in the intestine are the 1A and 2A arterioles within the submucosa and the small piercing mesenteric arteries that give rise to the 1A and 2A arterioles.1, 3, 4 Based on this, we next investigated the effect of HB-EGF on submucosal blood flow. This also allows for an accurate quantification of blood flow in a manner that is independent of structural damage to the intestine, since the submucosal layer itself is never structurally damaged in our model of experimental NEC.

We found that pups stressed for 3 d had a significant decrease in submucosal blood flow compared to non-stressed, breast fed pups (p < 0.001) (Figure 8, 3D movie-2). Pups stressed for 3 d but treated with HB-EGF had significantly increased submucosal microvascular blood flow compared to pups stressed for 3 d without HB-EGF. In pups stressed for only 1 or 2 d (Figure 8, panels B, C, F), submucosal blood flow was unchanged, again correlating with the fact that experimental NEC takes approximately 3 days to develop in our model, with little significant histologic damage occurring after only 1 or 2 days. Preservation submucosal blood flow resulting from HB-EGF treatment likely results in increased villous microvascular blood flow leading to preservation of villous structure.

Figure 8.

Figure 8

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Effect of HB-EGF on intestinal submucosal blood flow. Shown are representative images of submucosal microcirculatory blood flow as observed in a 3D confocal projection of cryosection slides obtained from the ileum (40x). The vascular network, including 1A, 2A and 3A arteries/arterioles, is shown using FITC-dextran angiography. The red staining represents nuclear staining with propidium iodide, and the green staining shows the microvasculature. The representative panels show intestinal samples from: A) control pups fed with breast milk for 3 d; B) pups subjected to stress for 1 d; C) pups subjected to stress for 2 d; D) pups subjected to stress for 3 d; and E) pups subjected to stress for 3 d but with HB-EGF added to the feeds. Panel F represents quantification of sumucosal microcirculatory blood flow. The number of animals represented by each bar is 10, with the exception of bars 5 and 6 which represent 6 and 4 animals respectively. For each animal studied, two measurements were subjected to quantification, with green and red channels separated and signal intensity calculated using Zeiss LSM 5 software. In each scanned file, the ratio of the green channel signal intensity (representing submucosal microcirculatory blood flow) to the red channel signal intensity (representing nuclear staining) was obtained. *p<0.001 compared to Control; **p<0.001 compared to Stress-Day-3. Also, refer to the 3D movie-2. Panel A corresponds to movie BMF; panel D corresponds to movie Stress, and panel E corresponds to movie Stress+HB-EGF.

Discussion

Our findings provide evidence that enteral HB-EGF significantly increases intestinal villous and submucosal microvascular blood flow, and decreases the incidence and severity of experimental NEC. Preservation of the intestinal submucosal vasculature has been shown to lead to preservation of blood flow to the villi.1 In the intestinal hemorrhagic shock animal model, a substantial reduction of the entire microvasculature and the villous plexus has been reported.23, 32 We have previously shown that HB-EGF preserves villous microcirculatory blood flow and protects against intestinal damage in adult rats subjected to HS/R.23 We now show that HB-EGF has similar effects on intestinal microvascular blood flow in experimental NEC. Please note that our studies assessed microvascular blood flow to the intestine, but did not measure actual blood flow rates. Nevertheless, it appears that the ability of HB-EGF to protect the intestines from NEC is due, in part, to its ability to preserve the intestinal submucosal and villous microvasculature.

We acknowledge that a definitive cause and effect relationship proving that decreased blood flow leads to worsened histologic injury is difficult to prove in our animal model. The current studies have examined the microvasculature after sacrifice. To support a cause and effect relationship between blood flow and histologic injury, we plan in the future to examine the intestinal microvasculature in vivo utilizing a high resolution micro-ultrasound system (Vevo 770, VisualSonics, Toronto, Canada). This may provide spatial resolution down to 30 microns, allowing evaluation of submucosal and villous microvasular blood flow. Correlation of our vascular findings with the temporal development of histologic injury may help us to determine whether decreased blood flow preceeds histologic mucosal injury in our model of experimental NEC.

In addition, we also plan to study endothelial progenitor cells (EPC), in light of the fact that tissue ischemia is a potent stimulus for the recruitment of EPC. Vascular trauma, especially ischemia, releases VEGF and platelet-derived SDF-1 into the circulation system, thus promoting mobilization of EPC. In addition, VEGF and platelet-derived SDF-1 are elevated in ischemic foci, contributing to EPC recruitment from the circulation to the injury site.35, 36 We will evaluate EPC mobilization in animals stressed for 1, 2, or 3 days with and without HB-EGF treatment, and will correlate this with the development of mucosal histologic injury.

HB-EGF is a member of the EGF family that functions thorough activation of the tyrosine kinase EGF receptors (EGFR). EGF is known to have specific vasodilatory effects in the mucosa of the alimentary tract in sheep, and leads to direct relaxation of isolated rabbit mesenteric arteries through activation of EGFR.37, 38 Using a rat ethanol/toxicity model, EGF protected gastric mucosa from ethanol-induced mucosal injury via an increase in gastric blood flow.39 EGF was reported to exert its gastroprotective effects through capsaicin-sensitive afferent neurons, including the release of calcitonin gene-related peptide (CGRP).40, 41 Although the exact mechanisms by which HB-EGF increases intestinal microcirculatory blood flow in vivo remain to be determined, we have examined the effects of HB-EGF in isolated rat pup terminal mesenteric arterioles (TMA) ex vivo. We found that HB-EGF acts as a direct vasodilator of newborn rat TMA by increasing NO production and by increasing endothelin B (ETB) vasodilatory receptor expression in these vessels (unpublished data).

Pro-inflammatory mediators including platelet activating factor (PAF) and pro-inflammatory cytokines were shown to be generated within the damaged villi of human infants with NEC and are capable of inducing vascular dysfunction, vasoconstriction and ischemia.42, 43 Exposure of the intestinal mucosa to high local PAF concentrations leads to mucosal permeability, intestinal inflammation, activation of the inflammatory response, apoptosis and bowel necrosis.44 Interestingly, PAF has been shown to stimulate transcription of HB-EGF in monocytes through increased NF kappa B binding activity.45 Further studies are needed to determine the exact mechanisms by which HB-EGF regulates intestinal blood flow in experimental NEC.

The villous vasculature of adult rats consists of well-developed arterioles, capillary networks and venules. The capillary network, which descends from the apical to the basal portions of the villi, connects to the capillary network of adjacent villi by crossing the cryptal plexus.46 In rat pups, we see a very simple villous vasculature composed of immature arterioles, capillaries and venules. We observed shrunken and damaged villous arterioles, capillaries and venules in stressed pups, but found that HB-EGF preserved these microvascular structures in stressed pups treated with HB-EGF. In a rat model of hemorrhagic shock, others have shown injury to the subepithelial capillary plexus; with the central arteries appearing as rigid abnormal vessels without capillaries arising from them,32 similar to our findings.

The structural simplicity of the newborn intestinal microvasculature may lead to susceptibility of the newborn intestine to NEC. The ability of HB-EGF to preserve these simple, delicate vessels further supports its use in the prevention of this disease. We conclude that HB-EGF significantly preserves intestinal microcirculatory blood flow in rat pups subjected to experimental NEC, indicating that HB-EGF may play a critical role in the therapy of various diseases manifested by decreased intestinal blood flow, including NEC.

Acknowledgements

The authors thank Cynthia McAllister (Nationwide Children's Hospital) for her assistance with confocal microscopy, Brian Kemmenoe and Richard Montione (Campus Microscopy and Imaging Facility, The Ohio State University) for their assistance with SEM, and Xiaoli Zhang (Center for Biostatistics, The Ohio State University) for assistance with data analysis.

This work was supported by a grant from the National Institutes of Health R01 DK074611 (GEB), the Children's Hospital Firefighter's Endowment (XY) and by Trillium Therapeutics, Inc.

Abbreviations

NEC

necrotizing enterocolitis

HB-EGF

heparin-binding EGF-like growth factor

EGF

epidermal growth factor

LPS

lipopolysaccharide

Footnotes

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