Jixin Yang

Heparin-Binding Epidermal Growth Factor-Like Growth Factor and Mesenchymal Stem Cells Act Synergistically to Prevent Experimental Necrotizing Enterocolitis

BACKGROUND We have shown that administration of heparin-binding EGF (epidermal growth factor)-like growth factor (HB-EGF) protects the intestines from experimental necrotizing enterocolitis (NEC). We have also demonstrated that systemically administered mesenchymal stem cells (MSC) can engraft into injured intestines. This study investigated the effects of HB-EGF on MSC in vitro, and whether MSC and HB-EGF can act synergistically to prevent NEC in vivo. STUDY DESIGN In vitro, the effect of HB-EGF on MSC proliferation, migration, and apoptosis was determined. In vivo, rat pups received MSC either intraperitoneally (IP) or intravenously (IV). Pups were assigned to 1 of 7 groups: Group 1, breast-fed; Group 2, experimental NEC; Group 3, NEC+HB-EGF; Group 4, NEC+MSC IP; Group 5, NEC+HB-EGF+MSC IP; Group 6, NEC+MSC IV; or Group 7, NEC+HB-EGF+MSC IV. Mesechymal stem cell engraftment, histologic injury, intestinal permeability, and mortality were determined. RESULTS Heparin-binding EGF-like growth factor promoted MSC proliferation and migration, and decreased MSC apoptosis in vitro. In vivo, MSC administered IV had increased engraftment into NEC-injured intestine compared with MSC administered IP (p < 0.05). Heparin binding EGF-like growth factor increased engraftment of IP-administered MSC (p < 0.01) and IV-administered MSC (p < 0.05). Pups in Groups 3 to 7 had a decreased incidence of NEC compared with nontreated pups (Group 2), with the lowest incidence in pups treated with HB-EGF+MSC IV (p < 0.01). Pups in Group 7 had a significantly decreased incidence of intestinal dilation and perforation, and had the lowest intestinal permeability, compared with other treatment groups (p < 0.01). Pups in all experimental groups had significantly improved survival compared with pups exposed to NEC, with the best survival in Group 7 (p < 0.05). CONCLUSIONS Heparin-binding EGF-like growth factor and MSC act synergistically to reduce injury and improve survival in experimental NEC.

Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy

IntroductionIntestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury.MethodsHuman intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility.ResultsThere was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups.ConclusionsSignificant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis.

HB-EGF promotes intestinal restitution by affecting integrin- extracellular matrix interactions and intercellular adhesions

Restitution is a critical form of intestinal epithelial cell (IEC) healing. We have previously shown that heparin-binding epidermal-like growth factor (HB-EGF) is necessary for IEC restitution; however, the mechanisms by which HB-EGF promotes restitution remain poorly understood. This study was designed to investigate whether HB-EGF promotes intestinal restitution by affecting integrin–extracellular matrix (ECM) interactions and intercellular adhesions. The effect of HB-EGF administration was examined in a murine necrotizing enterocolitis (NEC) model in vivo and an IEC line scrape-wound healing model in vitro. We evaluated the effect of HB-EGF on the expression of integrins, E-cadherin/β-catenin, and integrin α5β1-dependent cell–ECM interactions. We found that HB-EGF promoted intestinal restitution and the expression of integrin α5β1. HB-EGF promoted integrin α5β1-dependent cell adhesion and spreading. In addition, HB-EGF decreased the expression E-cadherin/β-catenin, via the activation of v-erb-b2 erythroblastic leukemia viral oncogene homolog (ErbB-1). We conclude that HB-EGF promotes intestinal restitution by affecting integrin–ECM interactions and intercellular adhesions.

HMGB1-Promoted and TLR2/4-Dependent NK Cell Maturation and Activation Take Part in Rotavirus-Induced Murine Biliary Atresia

Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.

Synergistic Effects of HB-EGF and Mesenchymal Stem Cells in a Murine Model of Intestinal Ischemia/Reperfusion Injury

BACKGROUND We have previously demonstrated that heparin-binding EGF-like growth factor (HB-EGF) administration protects the intestines from ischemia/reperfusion (I/R) injury in vivo. We have also shown that HB-EGF promotes mesenchymal stem cell (MSC) proliferation and migration in vitro. The goals of the current study were to examine the effects of HB-EGF and both bone marrow (BM)- and amniotic fluid (AF)-derived MSC on intestinal I/R injury in vivo. MATERIALS AND METHODS MSC were isolated from pan-EGFP mice, expanded, and purified. Pluripotency was confirmed by induced differentiation. Mice were subjected to terminal ileum I/R and received either: (1) no therapy; (2) HB-EGF; (3) BM-MSC; (4) HB-EGF+BM-MSC; (5) AF-MSC; or (6) HB-EGF+AF-MSC. MSC engraftment, histologic injury, and intestinal permeability were quantified. RESULTS There was increased MSC engraftment into injured compared to uninjured intestine for all experimental groups, with significantly increased engraftment for AF-MSC+HB-EGF compared to AF-MSC alone. Administration of HB-EGF and MSC improved intestinal histology and intestinal permeability after I/R injury. The greatest improvement was with combined administration of HB-EGF+AF-MSC. CONCLUSIONS Both HB-EGF alone and MSC alone can protect the intestines from I/R injury, with synergistic efficacy occurring when HB-EGF and AF-MSC are administered together.

Heparin-binding EGF-like growth factor (HB-EGF) therapy for intestinal injury: Application and future prospects.

Throughout the past 20 years, we have been investigating the potential therapeutic roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor family, in various models of intestinal injury including necrotizing enterocolitis (NEC), intestinal ischemia/reperfusion (I/R) injury, and hemorrhagic shock and resuscitation (HS/R). Our studies have demonstrated that HB-EGF acts as an effective mitogen, a restitution-inducing reagent, a cellular trophic factor, an anti-apoptotic protein and a vasodilator, via its effects on various cell types in the intestine. In the current paper, we have reviewed the application and therapeutic effects of HB-EGF in three classic animal models of intestinal injury, with particular emphasis on its protection of the intestines from NEC. Additionally, we have summarized the protective functions of HB-EGF on various target cells in the intestine. Lastly, we have provided a brief discussion focusing on the future development of HB-EGF clinical applications for the treatment of various forms of intestinal injury including NEC.

Heparin-binding epidermal growth factor-like growth factor improves intestinal barrier function and reduces mortality in a murine model of peritonitis

BACKGROUND The morbidity and mortality associated with bacterial peritonitis remain high. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent. The aim of this study was to evaluate the effect of HB-EGF in a model of murine peritonitis. METHODS HB-EGF(-/-) knockout (KO) mice and their HB-EGF(+/+) wild-type (WT) counterparts were subjected to sham operation, cecal ligation and puncture (CLP), or CLP with HB-EGF treatment (800 μg/kg IP daily). Villous length, intestinal permeability, intestinal epithelial cell (IEC) apoptosis, bacterial load in peritoneal fluid (PF) and mesenteric lymph nodes (MLN), inflammatory cytokine levels, and survival were determined. RESULTS After exposure to CLP, HB-EGF KO mice had significantly shorter villi (1.37 ± 0.13 vs 1.96 ± 0.4 relative units; P < .03), increased intestinal permeability (17.01 ± 5.18 vs 11.50 ± 4.67 nL/min/cm2; P < .03), increased IEC apoptotic indices (0.0093 ± 0.0033 vs 0.0016 ± 0.0014; P < .01), and increased bacterial counts in PF (25,313 ± 17,558 vs 11,955 ± 6,653 colony forming units [CFU]/mL; P < .05) and MLN (19,009 ± 11,200 vs 5,948 ± 2,988 CFU/mL/g; P < .01) compared with WT mice. Administration of HB-EGF to WT and HB-EGF KO mice exposed to CLP led to significantly increased villous length and decreased intestinal permeability, IEC apoptosis and bacterial counts in MLN (P < .05). Survival of HB-EGF KO mice subjected to CLP was significantly improved with administration of HB-EGF (P < .05). CONCLUSION HB-EGF gene KO increases susceptibility to peritonitis-induced intestinal injury, which can be reversed by administration of HB-EGF. These results support a protective role of HB-EGF in peritonitis-induced sepsis.

A technique for systemic mesenchymal stem cell transplantation in newborn rat pups

ABSTRACT Mesenchymal stem cells (MSC) have the potential to aid tissue regeneration. Intravenous (IV) MSC administration is currently being assessed following tissue injury. However, few studies have been performed to establish a safe and effective method of IV MSC infusion for newborns. We have established a safe, nontraumatic and effective technique for systemic MSC transplantation in newborn rats. Yellow-fluorescent-protein (YFP)-labeled MSC were characterized using MSC markers and their differentiation potential was confirmed. Rat pups were delivered by C-section on gestational day 21. The umbilical vein (UV) was cannulated and used for IV injection of MSC or saline control, which was performed under ultrasonographic imaging. An additional control group consisted of UV MSC injection in adult mice. Mean operating time, success rate of cannulation and death rate were recorded. YFP-MSC quantification in multiple organs was performed. Mean operating time was 3.9 ± 1.1 min. The success of UV MSC injection was 92.8%. The immediate and 24 hr delayed death rate for rat pups was significantly lower than that of adult mice (p < .05). No pups receiving saline injection died. After locating the patent foramen ovale (PFO) of newborn pups by ultrasonographic imaging, extra pulse-waves and wave-shape changes were detected when MSC were injected. The number of YFP-MSC was 15.8 ± 4.1 cells per visual field (CPVF) in the lungs, 2.9 ± 1.2 CPVF in the heart, and 19.8 ± 5.0 CPVF in the intestines. We conclude that IV MSC infusion through the UV is a convenient, safe, and effective method for systemic MSC transplantation in prematurely delivered newborn rats.

Heparin-binding EGF-like growth factor promotes neuronal nitric oxide synthase expression and protects the enteric nervous system after necrotizing enterocolitis

BackgroundNeonatal necrotizing enterocolitis (NEC) is associated with alterations of the enteric nervous system (ENS), with loss of neuronal nitric oxide synthase (nNOS)-expressing neurons in the intestine. The aim of this study was to investigate the roles of heparin-binding EGF-like growth factor (HB-EGF) in neural stem cell (NSC) differentiation, nNOS expression, and effects on ENS integrity during experimental NEC.MethodsThe effects of HB-EGF on NSC differentiation and nNOS production were determined using cultured enteric NSCs. Myenteric neuronal subpopulations were examined in HB-EGF knockout mice. Rat pups were exposed to experimental NEC, and the effects of HB-EGF treatment on nNOS production and intestinal neuronal apoptosis were determined.ResultsHB-EGF promotes NSC differentiation, with increased nNOS production in differentiated neurons and glial cells. Moreover, loss of nNOS-expressing neurons in the myenteric plexus and impaired neurite outgrowth were associated with absence of the HB-EGF gene. In addition, administration of HB-EGF preserves nNOS expression in the myenteric plexus and reduces enteric neuronal apoptosis during experimental NEC.ConclusionHB-EGF promotes the differentiation of enteric NSCs into neurons in a nitric oxide (NO)-dependent manner, and protects the ENS from NEC-induced injury, providing new insights into potential therapeutic strategies for the treatment of NEC in the future.

Heparin-binding epidermal growth factor-like growth factor restores Wnt/β-catenin signaling in intestinal stem cells exposed to ischemia/reperfusion injury.

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/β-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/β-catenin signaling was evaluated using immunofluorescent staining and Western blotting. RESULTS LGR5 expression and Wnt/β-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/β-catenin signaling. CONCLUSION These data show that HB-EGF preserves Wnt/β-catenin signaling in ISC after I/R injury.