ABSTRACT
BACKGROUND: We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) protects the intestines from injury in several different experimental animal models. In the current study, we investigated whether the ability of HB-EGF to protect the intestines from ischemia/reperfusion (I/R) injury was related to its effects on Wnt/ß-catenin signaling in intestinal stem cells (ISC). METHODS: Lucien-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-enhanced green fluorescent protein (EGFP) transgenic (TG) mice with fluorescently labeled ISC, as well as the same mice treated with intraluminal HB-EGF or genetically engineered to overexpress HB-EGF, were exposed to segmental mesenteric artery occlusion (sMAO) to the terminal ilium. Wnt/ß-catenin signaling was evaluated using immunofluorescent staining and Western blotting. RESULTS: LGR5 expression and Wnt/ß-catenin signaling in the ISC of the terminal ilium of LGR5-EGFP TG mice was significantly reduced 24 hours after sMAO. Intraluminal administration of HB-EGF or HB-EGF overexpression in these mice led to preservation of LGR5 expression and Wnt/ß-catenin signaling. CONCLUSION: These data show that HB-EGF preserves Wnt/ß-catenin signaling in ISC after I/R injury.
Subject(s)
Ileum/drug effects , Intercellular Signaling Peptides and Proteins/pharmacology , Protective Agents/pharmacology , Reperfusion Injury/drug therapy , Stem Cells/drug effects , Wnt Signaling Pathway/drug effects , Animals , Blotting, Western , Fluorescent Antibody Technique , Heparin-binding EGF-like Growth Factor , Ileum/blood supply , Ileum/metabolism , Intercellular Signaling Peptides and Proteins/therapeutic use , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protective Agents/therapeutic use , Random Allocation , Receptors, G-Protein-Coupled/metabolism , Reperfusion Injury/metabolism , Stem Cells/metabolismABSTRACT
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.
