Gastrointestinal
Heparin-Binding EGF-Like Growth Factor Promotes Intestinal Anastomotic Healing

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Background

We have accumulated multiple lines of evidence supporting the ability of HB-EGF to protect the intestines from injury and to augment the healing of partial-thickness scald burns of the skin. The aim of the current study was to investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in intestinal anastomotic wound healing.

Materials and Methods

HB-EGF (–/–) knockout (KO) mice (n = 42) and their HB-EGF (+/+) wild type (WT) counterparts (n = 33), as well as HB-EGF transgenic (TG) mice (n = 26) and their (WT) counterparts (n = 27), underwent division and reanastomosis of the terminal ileum. In addition, WT mice (n = 21) that received enteral HB-EGF (800 μg/kg) underwent the same operative procedure. Anastomotic bursting pressure was measured at 3 and 6 d postoperatively. Tissue sections were stained with hematoxylin and eosin to assess anastomotic healing, and Picrosirus red to assess collagen deposition. Immunohistochemistry using anti-von Willebrand factor antibodies was performed to assess angiogenesis. Complications and mortality were also recorded.

Results

HB-EGF KO mice had significantly lower bursting pressures, lower healing scores, higher mortality, and higher complication rates postoperatively compared with WT mice. Collagen deposition and angiogenesis were significantly decreased in KO mice compared with WT mice. Conversely, HB-EGF TG mice had increased anastomotic bursting pressure, higher healing scores, lower mortality, lower complication rates, increased collagen deposition, and increased angiogenesis postoperatively compared with WT mice. WT mice that received HB-EGF had increased bursting pressures compared with non-HB-EGF treated mice.

Conclusion

Our results demonstrate that HB-EGF is an important factor involved in the healing of intestinal anastomoses.

Introduction

Rapid and effective wound-healing is of paramount importance to the surgeon. Failure of wound-healing generally leads to potentially life-threatening complications, additional surgical procedures, increased length of hospital stay, increased cost, and long-term disability [1]. Inadequate healing and consequent leakage from bowel anastomosis are a significant cause of postoperative morbidity and mortality [2].

During the past few years, a series of candidate key players in the wound-healing scenario have been identified. These include not only a variety of different growth factors and cytokines, but also molecules that are involved in cell–cell and cell–matrix interactions, and proteins responsible for cell stability and cell migration [3]. The effects of growth hormone and different growth factors on the healing of bowel anastomoses are currently being evaluated in an attempt to identify agents that may promote and improve the anastomotic healing process. This is especially important in seriously ill patients with altered metabolism and impaired tissue healing [2].

Heparin-binding EGF-like growth factor (HB-EGF) was initially identified in the conditioned medium of cultured human macrophages [4] and later found to be a member of the epidermal growth factor (EGF) family [5]. Like other family members, HB-EGF binds to the EGF receptor (EGFR; ErbB-1), inducing its phosphorylation. Unlike most EGF family members, HB-EGF has the ability to bind strongly to heparin. Cell-surface heparan-sulfate proteoglycans (HSPG) can act as highly abundant, low affinity receptors for HB-EGF. HB-EGF mRNA expression has been demonstrated in multiple tissues including skin, lung, heart, intestine, kidney, skeletal muscle, brain, male reproductive system, lymph nodes, thymus, and spleen 6, 7. HB-EGF is produced by many different cell types including epithelial cells, and it is mitogenic and chemotactic for smooth muscle cells, keratinocytes, hepatocytes, and fibroblasts. HB-EGF exerts its mitogenic effects by binding to and activation of EGF receptor subtypes ErbB-1 and ErbB-4 [8]. In addition, HB-EGF exerts chemotactic effects when binding to the HB-EGF specific receptor N-arginine dibasic convertase (NrdC) [9]. Importantly, endogenous HB-EGF is protective in various pathologic conditions and plays a pivotal role in mediating the earliest cellular responses to proliferative stimuli and cellular injury [10].

We have previously demonstrated the presence of HB-EGF protein in the basal layer of normal human dermis [11]. In addition, we have identified HB-EGF in human burn wound fluid following thermal injury, and have localized it immunohistochemically to epithelial cells of the marginal epidermis and dermal appendages of excised human burn tissue [12]. Furthermore, after thermal injury of the skin there is increased expression of endogenous HB-EGF in marginal surface keratinocytes [13]. We have also shown that topical application of HB-EGF accelerates partial thickness burn wound healing by increasing keratinocyte proliferative activity, in part via enhanced production of endogenous transforming growth factor α mRNA [14].

We have accumulated multiple lines of evidence supporting the role of HB-EF in protection of the intestines from a variety of insults including intestinal ischemia/reperfusion (I/R) injury [15], hemorrhagic shock and resuscitation(HS/R) [16], and necrotizing enterocolitis (NEC) 17, 18. Several investigators have shown that significant metabolic differences exist between wound healing in the skin and in the gastrointestinal tract [19]. Our findings of the ability of HB-EGF to promote cutaneous burn wound healing and to protect the intestines from various types of injuries prompted us to investigate the ability of HB-EGF to affect intestinal anastomotic wound healing.

Section snippets

Animals

To investigate the effects of HB-EGF loss-of-function on intestinal anastomotic wound healing, HB-EGF(–/–) knockout (KO) mice (n = 42) and their HB-EGF(+/+) wild type (WT) counterparts (n = 33) were randomly selected to undergo terminal ileal division and reanastomosis. HB-EGF KO mice on a C57BL/6J x 129 background and their HB-EGF WT C57BL/6J x 129 counterparts were kindly provided by Dr. David Lee (Chapel Hill, NC) [20].

In HB-EGF KO mice, HB-EGF exons 1 and 2 were replaced with PGK-Neo, thus

Anastomotic Bursting Pressure

Anastomotic bursting pressure measurements on POD 3 revealed that WT mice had slightly higher bursting pressures compared to HB-EGF KO mice (38.34 ± 14.47 mmHg versus 33.18 ± 15.63 mmHg; P = 0.36) (Fig. 1A). However, on POD 6, WT mice had significantly higher bursting pressures than KO mice (110.05 ± 22.93 mmHg versus 55.95 ± 19.82 mmHg; P < 0.01).

We next compared anastomotic bursting pressure in HB-EGF TG mice and their WT counterparts. We found that HB-EGF TG mice had slightly increased

Discussion

HB-EGF is an immediate early gene that plays a pivotal role in mediating the earliest cellular responses to proliferative stimuli and cellular injury [26]. Previous studies from our laboratory and from others have shown that expression of endogenous HB-EGF is significantly increased in response to tissue damage 13, 27, hypoxia [28], oxidative stress [29], and during wound healing and regeneration [12]. Endogenous HB-EGF has been shown to play a role in the healing of various tissues, as

Acknowledgments

The authors acknowledge the following financial support for this work: NIH R01 DK074611 and R01 GM061193 (GEB), The Samuel J. Roessler Memorial Fellowship (JKO) and The Firefighters Endowment Fund of Nationwide Children’s Hospital (XY). The authors thank Dr. David Lee (Chapel Hill, NC) for supplying HB-EGF KO and WT mice, Dr. Deborah Gumucio (Ann Arbor, MI) for providing them with the pBSII-12.4 kb Vill plasmid containing the 12.4 kb promoter fragment from the villin gene, Laurie Goodchild, DVM

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    These authors contributed equally to this work.

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