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• W4247222014 abstract "Background & Aims: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. Methods: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. Results: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. Conclusions: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion. Background & Aims: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. Methods: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. Results: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. Conclusions: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion. Despite recent improvements in diagnostic and interventional health care, mortality rates in patients with intestinal ischemia/reperfusion (I/R) remain alarmingly high.1Brandt L.J. Boley S.J. AGA technical review on intestinal ischemia American Gastrointestinal Association.Gastroenterology. 2000; 118: 954-968Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar Loss of the barrier and immune functions of the intestine are major contributing factors leading to the deleterious complications of I/R such as multiple organ dysfunction syndrome.2El-Assal O.N. Besner G.E. Heparin-binding epidermal growth factor-like growth factor and intestinal ischemia-reperfusion injury.Semin Pediatr Surg. 2004; 13: 2-10Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Restitution is a critical healing process that restores intestinal barrier function early after injury. Three main mechanisms are involved in restitution, including cell migration,3Goke M. Podolsky D.K. Regulation of the mucosal epithelial barrier.Baillieres Clin Gastroenterol. 1996; 10: 393-405Abstract Full Text PDF PubMed Scopus (70) Google Scholar villous contraction,4Moore R. Carlson S. Madara J.L. Rapid barrier restitution in an in vitro model of intestinal epithelial injury.Lab Invest. 1989; 60: 237-244PubMed Google Scholar and the formation of tight intercellular junctions.5Podolsky D.K. Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair the best offense is a good defense.Am J Physiol. 1999; 277: G495-G499PubMed Google Scholar Restitution is temporally different from other forms of healing because it begins as early as 15 minutes after injury,6Takahashi M. Ota S. Shimada T. Hamada E. Kawabe T. Okudaira T. Matsumura M. Kaneko N. Terano A. Nakamura T. et al.Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.J Clin Invest. 1995; 95: 1994-2003Crossref PubMed Scopus (145) Google Scholar, 7Wilson A.J. Gibson P.R. Epithelial migration in the colon filling in the gaps.Clin Sci (Lond). 1997; 93: 97-108PubMed Google Scholar coinciding with a period of continuing tissue injury during reperfusion. Accordingly, for efficient restitution to occur, the remaining intestinal mucosal tissues must (1) survive the undergoing reperfusion injury and (2) execute the active repair processes of restitution. It is therefore not surprising that the intrinsic mechanism of healing by restitution is usually not enough to restore early functional recovery, as indicated by the high mortality rate after intestinal ischemia, often exceeding 90%.1Brandt L.J. Boley S.J. AGA technical review on intestinal ischemia American Gastrointestinal Association.Gastroenterology. 2000; 118: 954-968Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar Accordingly, therapeutic enhancement of restitution may rescue patients with intestinal ischemia from remote complications and may increase survival. Recently, it has become clear that the same factors that protect the mucosa from injury also play critical roles in facilitating repair of the epithelium after injury.5Podolsky D.K. Mucosal immunity and inflammation. V. Innate mechanisms of mucosal defense and repair the best offense is a good defense.Am J Physiol. 1999; 277: G495-G499PubMed Google Scholar Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family that was initially identified in the conditioned medium of cultured human macrophages.8Besner G. Higashiyama S. Klagsbrun M. Isolation and characterization of a macrophage-derived heparin-binding growth factor.Cell Regul. 1990; 1: 811-819Crossref PubMed Scopus (161) Google Scholar Expression of HB-EGF is significantly increased in response to tissue damage,9Cribbs R.K. Harding P.A. Luquette M.H. Besner G.E. Endogenous production of heparin-binding EGF-like growth factor during murine partial-thickness burn wound healing.J Burn Care Rehabil. 2002; 23: 116-125Crossref PubMed Scopus (62) Google Scholar hypoxia,10Jin K. Mao X.O. Sun Y. Xie L. Jin L. Nishi E. Klagsbrun M. Greenberg D.A. Heparin-binding epidermal growth factor-like growth factor hypoxia-inducible expression in vitro and stimulation of neurogenesis in vitro and in vivo.J Neurosci. 2002; 22: 5365-5373Crossref PubMed Google Scholar and oxidative stress11Frank G.D. Mifune M. Inagami T. Ohba M. Sasaki T. Higashiyama S. Dempsey P.J. Eguchi S. Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells role of metalloprotease and protein kinase C-delta.Mol Cell Biol. 2003; 23: 1581-1589Crossref PubMed Scopus (100) Google Scholar and after wounding of intestinal epithelial monolayers.12Ellis P.D. Hadfield K.M. Pascall J.C. Brown K.D. Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers involvement of mitogen-activated protein kinase cascades.Biochem J. 2001; 354: 99-106Crossref PubMed Scopus (49) Google Scholar This pattern of expression is consistent with a pivotal role for HB-EGF in I/R injury and repair processes. We have shown that HB-EGF protects intestinal epithelial cells from various lethal stimuli such as hypoxia and exposure to proinflammatory cytokines in vitro13Pillai S.B. Turman M.A. Besner G.E. Heparin-binding EGF-like growth factor is cytoprotective for intestinal epithelial cells exposed to hypoxia.J Pediatr Surg. 1998; 33 (discussion 978–979): 973-978Abstract Full Text PDF PubMed Scopus (47) Google Scholar, 14Michalsky M.P. Kuhn A. Mehta V. Besner G.E. Heparin-binding EGF-like growth factor decreases apoptosis in intestinal epithelial cells in vitro.J Pediatr Surg. 2001; 36: 1130-1135Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and increases survival in rats subjected to I/R injury in vivo.15Pillai S.B. Hinman C.E. Luquette M.H. Nowicki P.T. Besner G.E. Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury.J Surg Res. 1999; 87: 225-231Abstract Full Text PDF PubMed Scopus (58) Google Scholar The critical role of HB-EGF in postischemic regeneration has been shown in various organs, including the brain, kidney, and heart.10Jin K. Mao X.O. Sun Y. Xie L. Jin L. Nishi E. Klagsbrun M. Greenberg D.A. Heparin-binding epidermal growth factor-like growth factor hypoxia-inducible expression in vitro and stimulation of neurogenesis in vitro and in vivo.J Neurosci. 2002; 22: 5365-5373Crossref PubMed Google Scholar, 16Sakai M. Zhang M. Homma T. Garrick B. Abraham J.A. McKanna J.A. Harris R.C. Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury Isolation and localization of bioactive molecules.J Clin Invest. 1997; 99: 2128-2138Crossref PubMed Scopus (90) Google Scholar, 17Tanaka N. Masamura K. Yoshida M. Kato M. Kawai Y. Miyamori I. A role of heparin-binding epidermal growth factor-like growth factor in cardiac remodeling after myocardial infarction.Biochem Biophys Res Commun. 2002; 297: 375-381Crossref PubMed Scopus (24) Google Scholar HB-EGF is also a potent chemoattractant in various cell types.18Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing.J Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar, 19Faull R.J. Stanley J.M. Fraser S. Power D.A. Leavesley D.I. HB-EGF is produced in the peritoneal cavity and enhances mesothelial cell adhesion and migration.Kidney Int. 2001; 59: 614-624Crossref PubMed Scopus (43) Google Scholar Taken together, HB-EGF administration may represent a possible therapeutic strategy to promote restitution after intestinal I/R. Despite a recent increase in the number of studies of intestinal restitution, the intrinsic mechanism of restitution has remained poorly understood. Additionally, animal models have been largely replaced by in vitro intestinal wound healing models, which are invaluable tools for evaluation of possible mechanisms of restitution but do not show functional and overall structural intestinal recovery. Moreover, there is no current successful therapeutic method to enhance in vivo restitution. The current study was designed to evaluate the intrinsic mechanism of early intestinal healing after moderate ischemic injury in rats and the role of HB-EGF treatment in the structural and functional recovery of the intestinal barrier in vivo. The role of HB-EGF in intestinal healing was further characterized in scrape wounding of intestinal epithelial cell monolayers in vitro. Phospho—extracellular signal-regulated kinase (ERK) 1/2, total ERK1/2, phospho-Akt and Akt antibodies, LY294002, wortmannin, and U0126 were from Cell Signaling Technologies (Beverly, MA). Proliferating cell nuclear antibody (PCNA) was from Upstate Biotechnology (Lake Placid, NY), and β-actin antibody was from Sigma-Aldrich Corp (St Louis, MO). Alkaline phosphatase-linked anti-rabbit immunoglobulin G and anti-mouse immunoglobulin G were from Vector Laboratories (Burlingame, CA). Neutralizing anti-HB-EGF antibody was from R&D Systems Inc (Minneapolis, MN), and phospho-specific anti-EGF was from Biosource International, Inc (Camarillo, CA). AG1478, LY303511, and U0124 were from Calbiochem, EMD Biosciences Inc (La Jolla, CA). PD98059, CRM197, and protease inhibitor cocktail were from Sigma-Aldrich Corp. The Chemicon QCM 24-well cell migration assay was from Chemicon International (Temecula, CA). Rat intestinal epithelial (RIE-1) cells were kindly provided by Dr John Barnard (Columbus, OH). All experiments used human recombinant HB-EGF (rHB-EGF) corresponding to amino acids 74–148 of the mature HB-EGF protein that was produced in our laboratory in Escherichia coli by recombinant DNA technology and purified as previously described.20Davis K.M. Brigstock D.R. Johnson P.R. Crissman-Combs M.A. McCarthy D.W. Downing M.T. Besner G.E. Production of glycosylated heparin-binding EGF-like growth factor in HeLa cells using vaccinia virus.Protein Expr Purif. 1996; 8: 57-67Crossref PubMed Scopus (29) Google Scholar All procedures were approved by the Institutional Animal Care and Use Committee of the Children’s Research Institute. Male Sprague-Dawley rats weighing 250–300 g were fasted for 16–18 hours with access to water only. Under inhalation anesthesia using 2% isoflurane, a midline laparotomy was performed, followed by ligation of collaterals between the superior mesenteric, celiac, and inferior mesenteric arteries. The superior mesenteric artery was then occluded using 2 microvascular clamps for 45 minutes followed by reperfusion. Changes in arterial flow were assessed by morphologic intestinal changes and by intravital microscopy in randomly selected animals. Rats were divided into 3 groups: (1) the I/R plus HB-EGF treatment group, which received intraluminal injection at the jejunoileal junction of rHB-EGF (600 μg/kg) in 3 mL of 0.1% bovine serum albumin 25 minutes after the onset of ischemia; (2) the I/R group, which received intraluminal injection of vehicle only; and (3) the control group, which was subjected to identical procedures without vascular manipulation. Throughout the procedure, the abdominal cavity was kept temporarily closed with body temperature maintained at 37°C. Animals were killed by exsanguination under anesthesia. All animals recovered completely with no mortality. Three rats were used per group, with each experiment performed in duplicate. Serum lactate dehydrogenase (LDH) was evaluated using a kinetic assay as described previously.21Martinek R.G. A rapid ultraviolet spectrophotometric lactic dehydrogenase assay.Clin Chim Acta. 1972; 40: 91-99Crossref PubMed Scopus (101) Google Scholar Intestinal mucosal barrier function was assessed by mucosal-to-serosal clearance of fluorescein isothiocyanate-conjugated dextran (4 kilodaltons; FD-4) in everted intestinal ileal sacs incubated ex vivo as previously described with some modifications.22Liaudet L. Soriano F.G. Szabo E. Virag L. Mabley J.G. Salzman A.L. Szabo C. Protection against hemorrhagic shock in mice genetically deficient in poly(ADP-ribose)polymerase.Proc Natl Acad Sci U S A. 2000; 97: 10203-10208Crossref PubMed Scopus (180) Google Scholar In brief, after subjecting rats to 45 minutes of ischemia and 3 hours of reperfusion, the terminal 5 cm of ileum was harvested, lavaged using ice-cold saline, inverted inside out, and incubated in ice-cold Krebs’-Henseleit bicarbonate buffer at pH 7.4. The everted ileal sac was then suspended in a solution of FD-4/Krebs’-Henseleit bicarbonate buffer (40 μg/mL), representing the FD-4muc, and was continuously bubbled with 95% o2 and 5% co2 at 37°C for 30 minutes. The gut length (L) and diameter (D) were then measured, and the intraluminal Krebs’-Henseleit bicarbonate buffer (FD-4ser) was collected and measured (intraluminal volume). After dilution of the FD-4muc, both serosal and mucosal FD-4 were measured using a Bioassay Reader HTS 7000 (Perkin Elmer, Boston, MA) with excitation wave of 485 nm and emission measured at 520 nm. The mucosal-to-serosal FD-4 clearance was calculated using the following modified formula: Clearance (nL/min⁡/cm2)=FD-4ser×Intraluminal Volume (Original 0.6 mL+Absorbed Volume)Time in Minutes×FD-4muc×πLD Tissue sections were deparaffinized, rehydrated, and stained with H&E. Histologic scoring of the depth of tissue injury was performed according to Chiu et al.23Chiu C.J. McArdle A.H. Brown R. Scott H.J. Gurd F.N. Intestinal mucosal lesion in low-flow states I. A morphological, hemodynamic, and metabolic reappraisal.Arch Surg. 1970; 101: 478-483Crossref PubMed Scopus (1560) Google Scholar with modifications as follows: 0, no damage; 1, subepithelial space at villous tip; 2, loss of mucosal lining of the villous tip; 3, loss of less than half of the villous structure; 4, loss of more than half of the villous structure; and 5, transmural necrosis. Sections were evaluated blindly without prior knowledge of animal background. Tissue sections were deparaffinized, rehydrated, and placed in 0.5% periodic acid for 5 minutes. After washing, sections were incubated in Schiff reagent for 15 minutes at room temperature and counterstained using Harris hematoxylin. We propose simple morphologic criteria to identify restitution in vivo. The criteria are applied to well-aligned villi in periodic acid-Schiff—stained sections and include the following: (1) histologic features indicative of prior loss of mucosa resulting in subsequent villous contraction, with short, blunted, or concave villous tips compared with nondamaged or less-injured villi in the same histologic section; (2) restoration of the mucosal surface of injured villi with a single layer of flat, squamous enterocytes resulting from migration and flattening during restitution; and/or (3) restoration of mucosal continuity with a single cell layer containing 4 or more goblet cells in continuity, without intervening enterocytes. The last criterion is supported by evidence that goblet cells are more resistant to I/R injury compared with enterocytes.24Ikeda H. Yang C.L. Tong J. Nishimaki H. Masuda K. Takeo T. Kasai K. Itoh G. Rat small intestinal goblet cell kinetics in the process of restitution of surface epithelium subjected to ischemia-reperfusion injury.Dig Dis Sci. 2002; 47: 590-601Crossref PubMed Scopus (44) Google Scholar Accordingly, we observed that moderate I/R results in sparing of goblet cells that can then undergo restitution to form a single layer of multiple goblet cells without intervening enterocytes. RIE-1 and CaCo-2 cells were maintained in monolayer culture in Dulbecco’s modified Eagle medium supplemented with 5% or 20% fetal bovine serum, respectively, with 50 U/mL penicillin and 50 mg/mL streptomycin at 37°C in 5% co2. Cell wounding assays were performed as described with some modifications.25Ray R.M. McCormack S.A. Covington C. Viar M.J. Zheng Y. Johnson L.R. The requirement for polyamines for intestinal epithelial cell migration is mediated through Rac1.J Biol Chem. 2003; 278: 13039-13046Crossref PubMed Scopus (70) Google Scholar Briefly, cells were grown in 6-well plates until confluent and then incubated for 16 hours in serum-free medium. The bottom of the plates was marked by drawing 3 fine lines across the plate diameter, and then a wound was created perpendicular to the marked lines using a 100-μL sterile pipette tip. Photographs were recorded soon after wounding (0 hours) or at 4, 8, and 18 hours following wounding (post), and the width of the wound was measured across each of the 3 marked lines in each well (3 fields/well) using Axiovision 3.1 software (Carl Zeiss Inc, Thornwood, NY). Each experiment was performed 3 times in duplicate. The percent of wound healing was calculated as follows: Wound Healing % = (W0 h) − (Wpost), where W0 h and Wpost represent the average wound width per well (measured across the 3 marked lines) at time 0 and at either 4, 8, or 18 hours postwounding in 6 wells per group. A Chemicon QCM fluorometric cell migration assay, which uses the Boyden dual chamber principle to quantitatively assess cell migration through 8-μm pore size membranes, was performed according to the recommendations of the manufacturer to confirm the role of HB-EGF on cell migration. For experiments involving the use of specific inhibitors of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK1/2 activation, preliminary dose-response studies were performed to identify the lowest dose of each inhibitor that led to maximal inhibitory effects on HB-EGF-induced phosphorylation of the target molecule by Western blotting without detectable effects on cell viability. Those doses were subsequently used throughout subsequent experiments. RIE-1 cells were grown on a coverslip until confluent and maintained in serum-free media for 16 hours, and then a wound was created as described. After 3 hours, cells were fixed in 3.7% formaldehyde, treated in 0.1% Triton X-100 for 5 minutes, and stained with rhodamine phalloidin (1 μmol/L) in 1% bovine serum albumin for 30 minutes. Samples were imaged with a Zeiss LSM 510 META confocal imaging system (Carl Zeiss Inc.) across the preidentified marked lines as detailed previously. Total RNA was isolated from intestine using TRIzol reagent, Invitrogen Life Technologies (Carlsbad, CA), and 3 μg of total RNA was reverse transcribed by random priming in the presence of 200 U of Moloney murine leukemia virus reverse transcriptase at 42°C for 1 hour. The resulting single-stranded complementary DNA was used for subsequent polymerase chain reaction amplification using platinum Taq DNA polymerase. Each polymerase chain reaction mixture (50 μL) contained 1 μL of the resulting complementary DNA template, 1 μmol/L of primers, 200 μmol/L of deoxynucleoside triphosphates, and 1.5 mmol/L MgCl2 in 1× amplification buffer. After subjecting the reaction mixture to initial denaturation at 94°C for 2 minutes, samples were subjected to 31 cycles consisting of 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C, and a final extension at 72°C for 10 minutes. The sequences of the primers used in the study were as follows: HB-EGF (207 base pairs): forward 5′ GCC TCC TGT AAT TGC TCT GC 3′, reverse 5′ GCC AAA AAT CCT GGA GCA TA 3′; ErbB-1 (376 base pairs): forward 5′GAG CTC GTG GAA CCT CTC AC 3′, reverse 5′ GGG AGC CAA TGT TGT CCT TA 3′; ErbB-4 (331 base pairs): forward 5′ TGG AGG AAA GCC CTA TGA TG3′, reverse 5′ CTG GGG GAC CAA ATA TTC CT 3′; nardilysin C (307 base pairs): forward 5′ GAC TTG CTG GTG AAT GCT GA3′, reverse 5′ GGT CCC CGA CAT TAC TGA GA3′; and β-actin (349 base pairs): forward 5′ CTA AGG CCA ACC GTG AAA AG3′, reverse 5′ GCC ATC TCT TGC TCG AAG TC 3′. Protein lysates were prepared from cells or intestinal tissues using radioimmunoprecipitation assay lysis buffer containing 50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 1% sodium deoxycholate (Na DOC) and a complete protease inhibitor cocktail. Samples were centrifuged at 16,000 rpm for 10 minutes at 4°C, and protein concentration was assayed using BioRad protein assay reagent (Bio-Rad Laboratories Inc, Hercules, CA). Samples (50 μg protein/sample) were separated on 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis before transfer to nitrocellulose membranes. Membranes were blocked in 5% skim milk, incubated with primary antibody for 16 hours at 4°C, and then incubated with alkaline phosphatase-conjugated secondary antibody for 1 hour at room temperature. Membranes were washed with 0.1% Tween 20 in 20 mmol/L Tris buffer saline, pH 7.4. Protein bands were developed by enhanced chemifluorescence and then scanned with a Typhoon variable mood imager 9400 (Amersham Biosciences, Piscataway, NJ). All results are presented as mean ± SE. Statistical comparisons between all groups were performed using one-way analysis of variance. When we detected significant differences among the studied groups, further comparisons between each of 2 groups were performed using Student t test. P < .05 was considered statistically significant. Endogenous HB-EGF is induced in various organs in response to I/R injury.10Jin K. Mao X.O. Sun Y. Xie L. Jin L. Nishi E. Klagsbrun M. Greenberg D.A. Heparin-binding epidermal growth factor-like growth factor hypoxia-inducible expression in vitro and stimulation of neurogenesis in vitro and in vivo.J Neurosci. 2002; 22: 5365-5373Crossref PubMed Google Scholar, 26Homma T. Sakai M. Cheng H.F. Yasuda T. Coffey Jr, R.J. Harris R.C. Induction of heparin-binding epidermal growth factor-like growth factor mRNA in rat kidney after acute injury.J Clin Invest. 1995; 96: 1018-1025Crossref PubMed Scopus (130) Google Scholar We evaluated the effect of I/R on HB-EGF and its receptor expression in rat intestine. Intestinal I/R injury significantly enhanced the messenger RNA (mRNA) expression of endogenous HB-EGF (Figure 1A). Expression of ErbB-1 was increased 3 hours after reperfusion. HB-EGF also binds to the ErbB-4 receptor and to nardilysin C, which are both specifically involved in HB-EGF-induced cell migration.27Elenius K. Paul S. Allison G. Sun J. Klagsbrun M. Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation.EMBO J. 1997; 16: 1268-1278Crossref PubMed Scopus (321) Google Scholar, 28Nishi E. Prat A. Hospital V. Elenius K. Klagsbrun M. N-arginine dibasic convertase is a specific receptor for heparin-binding EGF-like growth factor that mediates cell migration.EMBO J. 2001; 20: 3342-3350Crossref PubMed Scopus (111) Google Scholar I/R increased the expression of both ErbB-4 and nardilysin C mRNA levels. These findings suggest that endogenous HB-EGF is involved in early postischemic regeneration after I/R. To elucidate the role of exogenous administration of HB-EGF during I/R injury, HB-EGF (600 μg/kg) was administered intraluminally 25 minutes after the induction of ischemia. Morphologic and histologic examination of the intestine revealed that I/R induced more damage in the ileum compared with the jejunum. Accordingly, subsequent examinations focused on changes in the ileum. Serum LDH was measured as a biochemical marker of tissue damage. LDH levels in HB-EGF-treated rats were not statistically different from untreated rats 3 hours after reperfusion. However, HB-EGF-treated rats had significantly lower LDH levels (181 ± 28 U/L) compared with untreated animals 6 hours after I/R (292 ± 30.9 U/L; P < .05) (Figure 1B). This suggests that HB-EGF treatment reduces the severity of reperfusion injury. We then assessed changes in histologic injury in the ileum within the first 3 hours after I/R. Immediately after ischemia, histologic injury scores in HB-EGF-treated rats (3.3 ± 0.1) were similar to those in untreated rats (3.5 ± 0.1) (Figure 1C). By 3 hours of reperfusion, both HB-EGF-treated (1.8 ± 0.15; P < .001) and untreated (2.6 ± 0.26; P < .05) animals had significantly lower injury scores compared with immediately after ischemia. Importantly, HB-EGF-treated rats showed significantly lower histologic injury scores compared with untreated animals at this point (P < .05). This suggests that early healing is involved in this rapid tissue recovery and that rHB-EGF enhances early recovery from I/R injury. To understand the mechanisms involved in rapid tissue recovery, we first examined the role of cell proliferation by evaluating the levels of PCNA in intestinal tissues. Importantly, PCNA expression was reduced during the first 2 hours of reperfusion in all animals compared with basal PCNA levels (Figure 2A), suggesting that increased cell proliferation was not responsible for the early healing seen with HB-EGF administration. To show the role of restitution in this process, we examined the intestine for morphologic evidence of restitution using the specific criteria outlined in Materials and Methods. At the end of ischemia, there were variable degrees of tissue damage. Frequently, goblet cells were found loosely attached to injured villi despite loss of all surrounding enterocytes (Figure 2B [b and c]), supporting their higher resistance to ischemic injury. One hour after reperfusion, restitution was evident in all animals. Villi were short and wide, with blunted or even concave tips due to villous contraction. The role of goblet cells in restitution was clear, with goblet cells frequently detected overlying previously injured villi in groups of 4 or more cells without intervening enterocytes (Figure 2B [d and e], arrowheads), a feature that was not detected in normal mucosa (Figure 2B [a]). Three hours after reperfusion, restitution continued, as evidenced by flat, short, contracted villi with increased epithelial lining. The number of goblet cells was reduced significantly at this point, suggesting the replacement" @default.
• W4247222014 created "2022-05-12" @default.
• W4247222014 creator A5033199846 @default.
• W4247222014 creator A5046256982 @default.
• W4247222014 date "2005-08-01" @default.
• W4247222014 modified "2023-10-09" @default.
• W4247222014 title "HB-EGF Enhances Restitution After Intestinal Ischemia/Reperfusion via PI3K/Akt and MEK/ERK1/2 Activation" @default.
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