Xiaoyi Yu

Heparin-binding EGF-like Growth Factor Protects Intestinal Stem Cells from Injury in a Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.

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

BACKGROUND & AIMSnNecrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates. Although the exact etiology remains unknown, decreased intestinal blood flow is believed to play a critical role. We have shown that heparin-binding epidermal growth factor-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.nnnMETHODSnNewborn rat pups were subjected to stress by exposure to hypoxia, hypothermia, hypertonic feedings, and lipopolysaccharide, with some pups receiving HB-EGF (800 microg x kg(-1) x dose(-1)) 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 fluorescein isothiocyanate/dextran angiography, with fluorescent images subjected to quantification, and by scanning electron microscopy.nnnRESULTSnIntestinal microvascular blood flow (defined as the extent of vascular filling with fluorescein isothiocyanate/dextran) was significantly decreased in pups subjected to stress compared with 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.nnnCONCLUSIONSnHB-EGF significantly preserved intestinal microvascular blood flow in pups subjected to experimental NEC, indicating that HB-EGF may play a critical role in the treatment of various diseases manifested by decreased intestinal blood flow, including NEC.

Heparin-binding epidermal growth factor–like growth factor overexpression in transgenic mice increases resistance to necrotizing enterocolitis

BACKGROUNDnNecrotizing enterocolitis (NEC) is the most common gastrointestinal emergency and the leading surgical cause of death in premature infants. We have shown that administration of exogenous heparin-binding epidermal growth factor-like growth factor (HB-EGF) in mice protects the intestines from experimental NEC. The aim of the current study was to evaluate the effect of gain-of-function of endogenous HB-EGF on susceptibility to NEC.nnnMETHODSnNeonatal HB-EGF transgenic (TG) mice and their wild-type (WT) counterparts were exposed to experimental NEC. An additional group of HB-EGF TG pups were also exposed to NEC, but received the HB-EGF antagonist cross-reacting material 197 (CRM197) injected subcutaneously immediately after birth. To examine gut barrier function, HB-EGF TG and WT pups received intragastric fluorescein isothiocyanate-labeled dextran under basal and stressed conditions, and serum fluorescein isothiocyanate-labeled dextran levels were measured.nnnRESULTSnWild-type mice had an incidence of NEC of 54.2%, whereas HB-EGF TG mice had a significantly decreased incidence of NEC of 22.7% (P = .03). Importantly, administration of CRM197 to HB-EGF TG pups significantly increased the incidence of NEC to 65% (P = .004). HB-EGF TG mice had significantly decreased intestinal permeability compared to WT mice both under basal and stressed conditions.nnnCONCLUSIONSnOur results provide evidence that overexpression of the HB-EGF gene decreases susceptibility to NEC and that administration of the HB-EGF antagonist CRM197 reverses this protective effect.

Deletion of the heparin-binding epidermal growth factor-like growth factor gene increases susceptibility to necrotizing enterocolitis

BACKGROUNDnNecrotizing enterocolitis (NEC) is the leading surgical cause of death in premature infants. We have accumulated evidence supporting a role for heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) in protection of the intestines from NEC. The aim of the current study was to evaluate the effect of loss-of-function of endogenous HB-EGF on susceptibility to NEC.nnnMETHODSnNeonatal HB-EGF((-/-)) knockout (KO) mice and their HB-EGF((+/+)) wild-type (WT) counterparts were exposed to experimental NEC. An additional group of HB-EGF KO pups were also exposed to NEC but had HB-EGF added to their formula. To examine gut barrier function, HB-EGF KO and WT pups received intragastric fluorescein isothiocyanate-labeled dextran (FITC dextran) under basal and stressed conditions, and serum FITC dextran levels were measured.nnnRESULTSnThe WT mice had an incidence of NEC of 53%, whereas HB-EGF KO mice had a significantly increased incidence of NEC of 80% (P = .04). Importantly, administration of exogenous HB-EGF to HB-EGF KO pups significantly reduced the incidence of NEC to 45% (P = .04). Heparin-binding EGF KO mice had significantly increased intestinal permeability compared to WT mice under basal and stressed conditions.nnnCONCLUSIONSnOur results provide evidence that loss of the HB-EGF gene increases susceptibility to NEC and that administration of exogenous HB-EGF reverses this susceptibility.

Preclinical Neonatal Rat Studies of Heparin-Binding EGF-Like Growth Factor in Protection of the Intestines From Necrotizing Enterocolitis

We have previously demonstrated that enterally administered heparin-binding EGF-like growth factor (HB-EGF) produced in Escherichia coli decreases the incidence and severity of intestinal injury in a neonatal rat model of necrotizing enterocolitis (NEC). In preparation for upcoming human clinical trials, large-scale production of HB-EGF according to Good Manufacturing Practice (GMP) has been successfully accomplished using a Pichia pastoris yeast system. The current studies used a neonatal rat model of NEC to elucidate several important preclinical characteristics of HB-EGF therapy. We found that enteral administration of HB-EGF (800 μg/kg/dose) four times a day effectively reduced the incidence and severity of NEC, that Pichia-derived HB-EGF was not significantly different from E. coli-derived HB-EGF in preventing NEC, that EGF was not superior to HB-EGF in preventing NEC, and that prophylactic administration of HB-EGF added to formula starting with the first feed or 12 h later significantly reduced the incidence of NEC, with no change in the incidence of NEC noted if HB-EGF was added to the formula starting 24, 48, or 72 h after birth. Thus, large-scale production of GMP-grade HB-EGF in Pichia pastoris yeast produces a biologically active molecule suitable for human clinical trials.

Original articleDeletion of the heparin-binding epidermal growth factor-like growth factor gene increases susceptibility to necrotizing enterocolitis☆

BACKGROUNDnNecrotizing enterocolitis (NEC) is the leading surgical cause of death in premature infants. We have accumulated evidence supporting a role for heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) in protection of the intestines from NEC. The aim of the current study was to evaluate the effect of loss-of-function of endogenous HB-EGF on susceptibility to NEC.nnnMETHODSnNeonatal HB-EGF((-/-)) knockout (KO) mice and their HB-EGF((+/+)) wild-type (WT) counterparts were exposed to experimental NEC. An additional group of HB-EGF KO pups were also exposed to NEC but had HB-EGF added to their formula. To examine gut barrier function, HB-EGF KO and WT pups received intragastric fluorescein isothiocyanate-labeled dextran (FITC dextran) under basal and stressed conditions, and serum FITC dextran levels were measured.nnnRESULTSnThe WT mice had an incidence of NEC of 53%, whereas HB-EGF KO mice had a significantly increased incidence of NEC of 80% (P = .04). Importantly, administration of exogenous HB-EGF to HB-EGF KO pups significantly reduced the incidence of NEC to 45% (P = .04). Heparin-binding EGF KO mice had significantly increased intestinal permeability compared to WT mice under basal and stressed conditions.nnnCONCLUSIONSnOur results provide evidence that loss of the HB-EGF gene increases susceptibility to NEC and that administration of exogenous HB-EGF reverses this susceptibility.

HEPARIN-BINDING EGF-LIKE GROWTH FACTOR PROMOTES INTESTINAL ANASTOMOTIC HEALING

BACKGROUNDnWe 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.nnnMATERIALS AND METHODSnHB-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.nnnRESULTSnHB-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.nnnCONCLUSIONnOur results demonstrate that HB-EGF is an important factor involved in the healing of intestinal anastomoses.

Heparin-binding EGF-like growth factor protects pericytes from injury

BACKGROUNDnWe have previously shown that heparin-binding EGF-like growth factor (HB-EGF) promotes angiogenesis and preserves mesenteric microvascular blood flow in several models of intestinal injury. The current study was designed to evaluate the effect of HB-EGF on pericytes, since these cells function to regulate capillary blood flow and new capillary growth.nnnMATERIALS AND METHODSnC3H/10T1/2 mouse mesenchymal cells were differentiated into pericyte-like cells in vitro using transforming growth factor-β1 (TGF-β1). In addition, primary pericyte cultures were established from rat brain. The effect of HB-EGF on pericyte proliferation was assessed. In addition, cells were stressed by exposure to anoxia, and apoptosis determined. In vivo, we examined the effect of HB-EGF on pericytes in a model of intestinal I/R injury based on superior mesenteric artery occlusion (SMAO) in mice.nnnRESULTSnDifferentiated C3H/10T1/2 cells (pericyte-like cells) demonstrated morphologic characteristics of pericytes, and expressed pericyte specific markers. Addition of HB-EGF led to significant cell proliferation in differentiated pericyte-like cells, even under conditions of anoxic stress. Addition of the EGF receptor inhibitor AG 1478 led to complete inhibition of the proliferative effects of HB-EGF on pericyte-like cells. In addition, HB-EGF protected pericyte-like cells from anoxia-induced apoptosis. In addition, HB-EGF promoted cell proliferation in primary pericyte cultures. In vivo, administration of HB-EGF to mice subjected to intestinal I/R injury led to protection of pericytes from injury.nnnCONCLUSIONSnThese results suggest that HB-EGF may function as a microcirculatory blood flow regulator, at least in part, via its effects on pericytes.