epidermal-growth-factor and Reperfusion-Injury

Intestinal ischemia/reperfusion (I/R) injury affects patients of different ages, especially premature babies and the elderly. The outcome after intestinal I/R is often dismal, which may be attributed to loss of the barrier and immune functions of the intestines, as well as development of secondary injury in remote organs. The available treatment for advanced gut ischemia mandates extensive resection, which may cause growth retardation in infants and nutritional problems in the elderly. Throughout the past decade we have been investigating the potential therapeutic role of heparin-binding epidermal growth factor-like factor (HB-EGF) in intestinal I/R. The mitogenic and chemoattractant functions of HB-EGF formed the initial rationale for our investigations. In addition, HB-EGF is a potent antiapoptotic protein that enables cells and tissues exposed to different apoptotic stimuli to survive hypoxic, oxidative, and nutritional stresses. HB-EGF is known to have a vital role in wound healing and postischemic regeneration in different organs. In the current review, we summarize the results of our findings of the beneficial effects of HB-EGF in intestinal I/R, supported by additional evidence from the literature and an explanation of different possible mechanisms of its actions. Collectively, the data strongly suggest a potential therapeutic role for the use of HB-EGF to treat intestinal ischemic diseases such as I/R and necrotizing enterocolitis.

Topics: Animals; Apoptosis; Cytoprotection; Endothelium, Vascular; Epidermal Growth Factor; ErbB Receptors; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Intestines; Oxidative Stress; Regeneration; Reperfusion Injury; RNA, Messenger

For the past decade, an attempt has been made by many research groups to define the roles of the growing number of Bcl-2 gene family proteins in the apoptotic process. The Bcl-2 family consists of pro-apoptotic (or cell death) and anti-apoptotic (or cell survival) genes and it is the balance in expression between these gene lineages that may determine the death or survival of a cell. The majority of studies have analysed the role/s of the Bcl-2 genes in cancer development. Equally important is their role in normal tissue development, homeostasis and non-cancer disease states. Bcl-2 is crucial for normal development in the kidney, with a deficiency in Bcl-2 producing such malformation that renal failure and death result. As a corollary, its role in renal disease states in the adult has been sought. Ischaemia is one of the most common causes of both acute and chronic renal failure. The section of the kidney that is most susceptible to ischaemic damage is the outer zone of the outer medulla. Within this zone the proximal tubules are most sensitive and often die by necrosis or desquamate. In the distal nephron, apoptosis is the more common form of cell death. Recent results from our laboratory have indicated that ischaemia-induced acute renal failure is associated with up-regulation of two anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-XL) in the damaged distal tubule and occasional up-regulation of Bax in the proximal tubule. The distal tubule is a known reservoir for several growth factors important to renal growth and repair, such as insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF). One of the likely possibilities for the anti-cell death action of the Bcl-2 genes is that the protected distal cells may be able to produce growth factors that have a further reparative or protective role via an autocrine mechanism in the distal segment and a paracrine mechanism in the proximal cells. Both EGF and IGF-1 are also up-regulated in the surviving distal tubules and are detected in the surviving proximal tubules, where these growth factors are not usually synthesized. As a result, we have been using in vitro methods to test: (i) the relative sensitivities of renal distal and proximal epithelial cell populations to injury caused by mechanisms known to act in ischaemia-reperfusion; (ii) whether a Bcl-2 anti-apoptotic mechanism acts in these cells; and (iii) whether an autocrine and/or paracrine growth factor mechanism is initiated. The following rev

Topics: Acute Kidney Injury; Animals; Apoptosis; Epidermal Growth Factor; Genes, bcl-2; Growth Substances; Humans; Insulin-Like Growth Factor I; Ischemia; Kidney; Necrosis; Nephrons; Reperfusion Injury

Free jejunal flap reconstruction is the main treatment for patients after pharyngo-oesophagectomy. Flaps are unavoidably subjected to ischaemia and reperfusion (I/R) during preparation. Enteral nutrition has been shown to improve the recovery of injured intestine, although the precise underlying mechanism remains unclear. This study was aimed to determine whether early enteral 5% glucose infusion is beneficial for the recovery of flap. Further, the possibility that enteral glucose infusion induces altered mucosal responses was evaluated.. Patients, who underwent free jejunal flap reconstructions after pharyngo-oesophagectomy, were enrolled. An externalised monitor loop was made to observe the viability of flap and to collect intestinal fluid. Control patients (n = 11) received peripheral parenteral nutrition for seven post-operation days. For early enteral-fed patients (n = 12), in addition to fluid infusion, administration of 5% glucose (25 ml h(-1)) via a jejunostomy tube was initiated 6h after surgery. Blood, flap fluid and mucosal specimens were harvested. Plasma and flap luminal levels of interleukin (IL)-6, IL-10, epidermal growth factor (EGF) and secretory immunoglobulin A (sIgA) were measured. Further, mucosal morphology was examined.. There were no significant differences in either plasma or luminal concentrations of IL-6, IL-10 and sIgA at different time points between groups. The luminal EGF level in the control group reduced markedly from the 3rd postoperative day, contrasting with a well-maintained level in the early enteral-fed group. No significant difference in mucosal histology between groups was observed.. Early enteral glucose infusion does not significantly benefit the ischaemia-reperfusion-injured flap; however, it does preserve EGF levels in the flap lumen.

Topics: Adult; Enteral Nutrition; Epidermal Growth Factor; Esophagectomy; Female; Follow-Up Studies; Glucose; Humans; Jejunostomy; Jejunum; Male; Pharyngectomy; Plastic Surgery Procedures; Reperfusion Injury; Treatment Outcome

The aim of the study was to observe the neuroreparative effect of electroacupuncture in rats with cerebral ischemia-reperfusion injury, and to explore the difference in the therapeutic effect of acupuncture on different acupoint groups after cerebral ischemia-reperfusion.. Experimental rats were randomly divided into: sham operation group, model group, electroacupuncture group, rehabilitation group, and Diankang group (electroacupuncture + rehabilitation training). There were 24 rats in each group, and the focal cerebral ischemia-reperfusion model was established by Zea-Longa suture method. After modeling, it took 4 hours to electroacupuncture at Baihui and Dazhui points, which was used to observe the changes of nerve function in rats with signs of keel nerve function defect. Protein expression was detected by immunohistochemistry.. Compared with the model group, the EA 3d, 7d, 10d groups and the rehabilitation group had no significant difference in promoting the expression of Nestin (p>0.05). There was a significant difference (p<0.01). After cerebral ischemia-reperfusion injury, the expression of bFGF and EGF on the ischemic side was stronger. The peak of bFGF expression appeared earlier, and the peak of EGF expression appeared later. The expression of bFGF and EGF in cerebral ischemic cortex at different time points of ischemia in electroacupuncture group, rehabilitation group and Diankang group was increased, and the response was enhanced. The effect of Diankang group on the upregulation of bFGF and EGF was more significant (p<0.01, p<0.05).. Under the influence of different effects, Diankang is superior to simple treatment in improving ischemic neurological dysfunction. This may be related to the fact that Diankang can promote the proliferation of neural stem cells and the expression of neurotrophic factors on the ischemic side of the rat brain.

Topics: Animals; Brain Ischemia; Cerebral Infarction; Electroacupuncture; Epidermal Growth Factor; Ischemia; Nestin; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The purpose of this study was to determine whether the epidermal growth factor receptor (EGFR), which is a classical receptor tyrosine kinase, is involved in the protective effect of morphine against ischemia/reperfusion (I/R)-induced myocardial mitochondrial damage.. Isolated rats hearts were subjected to global ischemia followed by reperfusion. Cardiac H9c2 cells were exposed to a simulated ischemia solution followed by Tyrode's solution to induce hypoxia/reoxygenation (H/R) injury. Triphenyltetrazolium chloride (TTC) was used to measure infarct size. The mitochondrial morphological and functional changes were determined using transmission election microscopy (TEM), mitochondrial stress assay, and mitochondrial swelling, respectively. Mitochondrial fluorescence indicator JC-1, DCFH-DA, and Mitosox Red were used to determine mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS) and mitochondrial superoxide. A TUNUL assay kit was used to detect the level of apoptosis. Western blotting analysis was used to measure the expression of proteins.. Treatment of isolated rat hearts with morphine prevented I/R-induced myocardial mitochondrial injury, which was inhibited by the selective EGFR inhibitor AG1478, suggesting that EGFR is involved in the mitochondrial protective effect of morphine under I/R conditions. In support of this hypothesis, the selective EGFR agonist epidermal growth factor (EGF) reduced mitochondrial morphological and functional damage similarly to morphine. Further study demonstrated that morphine may alleviate I/R-induced cardiac damage by inhibiting autophagy but not apoptosis. Morphine increased protein kinase B (Akt), extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation, which was inhibited by AG1478, and EGF had similar effects, indicating that morphine may activate Akt, ERK, and STAT-3 via EGFR. Morphine and EGF increased intracellular reactive oxygen species (ROS) generation. This effect of morphine was inhibited by AG1478, indicating that morphine promotes intracellular ROS generation by activating EGFR. However, morphine did not increase ROS generation when cells were transfected with siRNA against EGFR. In addition, EGFR activity was markedly increased by morphine, but the effect of morphine was reversed by naltrindole. These results suggest that morphine may activate EGFR via δ-opioid receptor activation.. Morphine may prevent I/R-induced myocardial mitochondrial damage by activating EGFR through δ-opioid receptors, in turn increasing RISK and SAFE pathway activity via intracellular ROS. Moreover, morphine may reduce myocardial injury by regulating autophagy but not apoptosis.

Topics: Animals; Epidermal Growth Factor; ErbB Receptors; Mitochondria, Heart; Morphine; Myocytes, Cardiac; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; Quinazolines; Rats; Reactive Oxygen Species; Receptors, Opioid; Reperfusion; Reperfusion Injury; RNA, Small Interfering; Superoxides; Tyrphostins

Peptic ulcer episodes cause damage to the stomach and intestine, with inflammatory cell infiltration and oxidative stress as the main players. In this study, we investigated the potential of anthocyanidin malvidin for preventive and curative peptic ulcer treatment. The anthocyanidin effects were examined in gastric ulcer mouse models induced by ethanol, non-steroidal anti-inflammatory drugs (NSAIDs), ischemia-reperfusion (IR), acetic acid and duodenal ulcer induced by polypharmacy. Expression levels of oxidative and inflammatory genes were measured to investigate the mechanism of anthocyanin activity. At a dose of 5 mg·kg

Topics: Acetic Acid; Animals; Anthocyanins; Antioxidants; Biomarkers; Cyclooxygenase 1; Disease Models, Animal; Duodenum; Epidermal Growth Factor; Ethanol; Gastric Mucosa; Gene Expression Regulation; Indomethacin; Inflammation; Male; Matrix Metalloproteinase 9; Mice; Oxidative Stress; Peptic Ulcer; Polypharmacy; Protective Agents; Reperfusion Injury; Stomach Ulcer; Tight Junctions; Wound Healing

To investigate the function and expression of the PGE2 receptors EP1-4 in rat retinal ischemia-reperfusion (I/R) injury and to determine the regulatory role of resveratrol (RES) in this process.. In vitro, we stimulated primary astrocytes extracted from the optic disc of rats with epidermal growth factor (EGF) and RES, and detected the location of EP1-4 expression with immunofluorescence. The expression of antiglial fibrillary acidic protein (GFAP), EGF receptor (EGFR), inducible NOS (iNOS), and EP1-4 in astrocytes was detected with western blotting. In vivo, we established an I/R injury model and RES treatment model with Sprague-Dawley rats. Changes in the thickness of the inner retina were observed with hematoxylin and eosin (H&E) staining. EP1-4 localization in the retina was observed with immunohistochemistry. The expression of COX-2, iNOS, and EP1-4 in the control and model groups was detected with western blotting.. In this study, immunofluorescence and immunohistochemistry showed that EP1-4 are expressed in astrocytes and the rat retina. EGF stimulation increased the expression of EGFR, iNOS, EP1, EP2, and EP4 in astrocytes. The expression of EP1-4 was statistically significantly increased on the third day after model induction, and EP1-4 expression decreased to normal levels on day 7. EGF and RES mediated the decrease in the expression of EP2. RES treatment significantly reduced retinal damage and RGC loss, as demonstrated by the relatively intact tissue structure on day 7 observed with H&E staining. Moreover, inflammation was associated with this I/R injury model, as demonstrated by the early induction of proinflammatory mediators, and this inflammation was significantly attenuated after RES treatment.. These results indicate that the COX-2/PGE2/EPs pathway is involved in retinal damage and astrocyte inflammation. In addition, the results suggest that the neuroprotective effects of RES may be associated with decreased production of inflammatory mediators. These results suggest that the PGE2 receptor may be a key factor in the treatment of neurodegenerative diseases, and that RES may be used as a possible therapeutic strategy for glaucoma.

Topics: Animals; Astrocytes; Cyclooxygenase 2; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Inflammation; Male; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Optic Disk; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin E; Reperfusion Injury; Resveratrol; Retina; Signal Transduction

Topics: Apelin; Epidermal Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Reperfusion Injury; Signal Transduction

Damage and loss of retinal ganglion cells (RGCs) can cause visual impairment. The underlying molecular mechanisms that mediate RGC death in ischemic retinal diseases are still unclear. In this study, we sought to understand the neuroprotective effect of rapamycin, the selective inhibitor of mTORC1, on RGC survival and the cellular mechanics that mediate this effect. Recent studies have reported that the epidermal growth factor (EGF) receptor shows an increase in expression in astrocytes after injury, and this receptor can promote their transformation into reactive astrocytes. Our results, along with previous works from others, show the colocalization of phosphor-EGF receptors with the astrocyte marker glial fibrillary acidic proteins in reactive astrocytes in the injured retina. In our in vitro studies, using primary astrocyte cultures of the optic nerve head of rats, showed that rapamycin significantly blocked EGF-induced mTOR signaling mainly through the PI3K/Akt pathway in primary astrocytes, but not through the MAPK/Erk pathway. Additionally, rapamycin dramatically inhibited the activation of mTOR signaling in our ratinal ischemia-reperfusion (I/R) injury model in vivo. Astrocyte activation was assessed by immunostaining retinal flat mounts or cross sections with antibody against GFAP, and we also used western blots to detect the expression of GFAP. Taken together, these results revealed that rapamycin decreases the activation of astrocytes after retinal ischemia-reperfusion injury. Furthermore, rapamycin can improve retinal RGC survival in rats during I/R, as detected by FluoroGold labeling. Our data reveals the neuroprotective effects of rapamycin in an experimental retina injury model, possibly through decreasing glial-dependent intracellular signaling mechanisms for suppressing apoptosis of RGCs. Our study also presents an approach to targeting reactive astrocytes for the treatment of optic neurodegenerations.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Astrocytes; Blotting, Western; Cell Survival; Cells, Cultured; Epidermal Growth Factor; Glial Fibrillary Acidic Protein; Male; Neuroprotective Agents; Optic Disk; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Intestinal ischemia/reperfusion is a major problem which may lead to multiorgan failure and death. The aim of the study was to evaluate the effects of epidermal growth factor (EGF) on apoptosis, cell proliferation, oxidative stress and the antioxidant system in intestinal injury induced by ischemia/reperfusion in rats and to determine if EGF can ameliorate these toxic effects. Intestinal ischemia/reperfusion injury was produced by causing complete occlusion of the superior mesenteric artery for 60 min followed by a 60-min reperfusion period. Animals received intraperitoneal injections of 150 μg/kg human recombinant EGF 30 min prior to the mesenteric ischemia/reperfusion. Mesenteric ischemia/reperfusion caused degeneration of the intestinal mucosa, inhibition of cell proliferation, stimulation of apoptosis and oxidative stress in the small intestine of rats. In the ischemia/reperfusion group, lipid peroxidation was stimulated accompanied by increased intestinal catalase and glutathione peroxidase activities, however, glutathione levels and superoxide dismutase activities were markedly decreased. EGF treatment to rats with ischemia/reperfusion prevented the ischemia/reperfusion-induced oxidative injury by reducing apoptosis and lipid peroxidation, and by increasing antioxidant enzyme activities. These results demonstrate that EGF has beneficial antiapoptotic and antioxidant effects on intestinal injury induced by ischemia/reperfusion in rats.

Topics: Animals; Apoptosis; Caspase 3; Epidermal Growth Factor; Glutathione; Ischemia; Jejunum; Male; Malondialdehyde; Oxidative Stress; Rats, Sprague-Dawley; Reperfusion Injury

Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.

Topics: Animals; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Gene Expression Regulation; Hepatocytes; Liver; Liver Regeneration; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Physiologic; Reperfusion Injury; Signal Transduction; Vascular Endothelial Growth Factor Receptor-1

Recruited macrophages play a critical role in liver repair after acute liver injury. Leukotriene B4 (LTB4) is a potent chemoattractant for macrophages. In this study, we investigated the role of LTB4 receptor type 1 (BLT1) in liver repair during hepatic ischemia/reperfusion (I/R) injury. BLT1-knockout mice (BLT1(-/-)) or their wild-type counterparts (WT) were subjected to partial hepatic I/R. Compared with WT, BLT1(-/-) exhibited delayed liver repair and hepatocyte proliferation accompanied by a 70% reduction in the recruitment of macrophages and a 70-80% attenuation in hepatic expression of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1). Disruption of BLT1 signaling also reduced the expression of EGF by 67% on recruited macrophages expressing VEGFR1 in the injured liver. Treatment of WT mice with an EGF-neutralizing antibody delayed liver repair and reduced macrophage recruitment, compared with control immunoglobulin G (IgG). BLT1 signaling enhanced the expression of VEGF, VEGFR1, and EGF in isolated peritoneal macrophages in vitro. These results indicate that BLT1 signaling plays a role in liver repair after hepatic I/R through enhanced expression of EGF in recruited macrophages and that the development of a specific agonist for BLT1 could be useful for liver recovery from acute liver injury.

Topics: Animals; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Fluorescent Antibody Technique; Gene Expression; Hepatocytes; Liver; Macrophages; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, Leukotriene B4; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Administration of exogenous epidermal growth factor (EGF) improves testicular injury after acute ischemia-reperfusion (IR) stress, but the molecular basis is poorly understood. The role of endogenous EGF in testicular recovery and the underlying intracellular signaling pathways involved were herein investigated. In mice, testicular IR injury significantly enhanced the expression level of endogenous Egf at the very beginning of reperfusion. Expression of EGF receptor (Egfr (ErbB1)) was accordingly upregulated 3  h after reperfusion. Deprivation of majority of circulated EGF by sialoadenectomy aggravated testicular detriment (especially in pachytene spermatocytes), enhanced germ cell apoptosis, and thereafter resulted in impaired meiotic differentiation after IR insult. Mechanistically, endogenous EGF signaling appeared to be indispensable for the proper maintenance of Sertoli germ cells anchoring junction dynamics during the early testicular recovery. We also provided the in vitro evidences in a well-established rat Sertoli germ cell co-cultures model that the pro-survival effect of endogenous EGF on germ cells in response to testicular IR insult is mediated, at least in part, via the phosphatidylinositol 3-kinase/pAkt pathway. Collectively, our results suggest that the augment of endogenous EGF during the early testicular recovery may act on top of an endocrinous cascade orchestrating the intimate interactions between Sertoli cells and germ cells and may operate as indispensable defensive mechanism in response to testicular IR stress. Future studies in this field would shed light on this complicated pathogenesis.

Topics: Adherens Junctions; Animals; Cell Adhesion; Cell Membrane Permeability; Cells, Cultured; Epidermal Growth Factor; Female; Germ Cells; Male; Mice; Mice, Inbred C57BL; Models, Biological; Rats; Recovery of Function; Reperfusion Injury; Sertoli Cells; Testis; Time Factors

The 'new penumbra' concept imbues the transition between injury and repair at the neurovascular unit with profound implications for selecting the appropriate type and timing of neuroprotective interventions. In this conceptual study, we investigated the protective effects of pigment epithelium-derived factor (PEDF) and compared them with the properties of epidermal growth factor (EGF) in a rat model of ischemia-reperfusion injury. We initiated a delayed intervention 3 hours after reperfusion using equimolar amounts of PEDF and EGF. These agents were then administered intravenously for 4 hours following reperfusion after 1 hour of focal ischemia. Magnetic resonance imaging indices were characterized, and imaging was performed at multiple time points post reperfusion. PEDF and EGF reduced lesion volumes at all time points as observed on T2-weighted images (T2-LVs). In addition PEDF selectively attenuated lesion volume expansion at 48 hours after reperfusion and persistently modulated blood-brain barrier (BBB) permeability at all time points. Intervention with peptides is suspected to cause edema formation at distant regions. The observed T2-LV reduction and BBB modulation by these trophic factors is probably mediated through a number of diverse mechanisms. A thorough evaluation of neurotrophins is still necessary to determine their time-dependent contributions against injury and their modulatory effects on repair after stroke.

Topics: Animals; Blood-Brain Barrier; Brain Edema; Disease Models, Animal; Epidermal Growth Factor; Eye Proteins; Male; Nerve Growth Factors; Rats; Reperfusion Injury; Serpins; Stroke; Time Factors

Epidermal growth factor (EGF) is an attractive and promising therapeutic application for intestinal disorders. The current study examined its influence on proliferation and restoration after ischemia-reperfusion (I/R) injury in rat small intestine. Six groups were performed: sham operation (Con); ischemia for 30 min with subsequent reperfusion for 30 min (I/R); I/R injured with 500 μg/kg EGF injected 5 min before ischemia (Pre-l); I/R injured with 50 μg/kg EGF injected 5 min before ischemia (Pre-s); I/R injured with 500 μg/kg EGF injected 5 min after reperfusion (Post-l); and I/R injured with 50 μg/kg EGF injected 5 min after reperfusion (Post-s). Intestinal histological damage, crypt cell proliferation degree, mucosal permeability, tight junction proteins expression, and levels of inflammation factors were studied for each group. Compared with the I/R group, administration of EGF in the Pre-l, Pre-s, and Post-l groups all presented a significant proliferation effect. The levels of FD4, IL-6, and TNF-α were dramatically decreased in all EGF-treated groups. Histological destruction was improved and TJs recovery was notably accelerated in all EGF-treated groups except the Post-s group. D-lactate concentration was only diminished in the Pre-l group. These results suggest that mucosally applied EGF can promote intestinal proliferation and improve restoration after I/R injury. EGF intraluminal administration is an effective treatment against intestinal I/R injury.

Topics: Animals; Cell Proliferation; Epidermal Growth Factor; Inflammation; Interleukin-6; Intestinal Mucosa; Intestines; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tight Junction Proteins; Tight Junctions; Tumor Necrosis Factor-alpha

Ischemia-reperfusion (IR) injury is a common cause of acute renal failure. Bone marrow (BM)-derived mesenchymal stromal cells (MSC) delivered after renal IR are renoprotective, but knowledge of the protective mechanism is still in development. This investigation analyzed the protective molecular mechanisms of MSC, in particular relating to modulated oxidative stress.. In vivo and in vitro models of renal IR were analyzed with and without MSC. In vivo, adult male Sprague-Dawley rats were subjected to 40-min unilateral renal IR. Rat BM-derived MSC were administered at 24 h post-IR (IR + MSC). Other groups had IR but no MSC, or MSC but no ischemia (all groups n = 4). Apoptosis, inflammation, oxidative stress and reparative signal transduction molecules or growth factors were studied 4 days post-IR. In vitro, protection by MSC against oxidative stress (0.4 mm hydrogen peroxide) was investigated using rat renal tubular epithelial cells (NRK52E) with or without MSC in co-culture (tissue culture trans-well inserts), followed by similar analyses to the in vivo investigation.. In vivo, kidneys of IR + MSC animals had significantly increased cell proliferation/regeneration (cells positive for proliferating cell nuclear antigen, expression of epidermal growth factor), increased heme-oxygenase-1 (improved cell survival, anti-oxidant) and decreased 8-OHdG (decreased oxidative stress). In vitro, MSC delivered with oxidative stress significantly decreased apoptosis and Bax (pro-apoptotic protein), and increased mitosis and phospho-ERK1/2, thereby minimizing the damaging outcome and maximizing the regenerative effect after oxidative stress.. The benefits of MSC, in IR, were primarily pro-regenerative, sometimes anti-apoptotic, and novel anti-oxidant mechanisms were identified.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Bone Marrow Cells; Cell Line; Cell Proliferation; Epidermal Growth Factor; Gene Expression; Heme Oxygenase-1; Inflammation; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Oxidative Stress; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; Signal Transduction

To investigate effects of combination of erythropoietin (EPO) and epidermal growth factor (EGF) on renal ischaemia and on reactive oxygen species in a rat model.. In all, 90 male Sprague-Dawley rats were allocated into five groups of 18, designated: Sham; treated with right nephrectomy only; Control, subjected to left renal ischaemia for 45 min with no treatment; EPO-treated, as the control but with EPO pretreatment; EGF-treated, as the control but with EGF pretreatment; EPO + EGF-treated, as the control but with EPO and EGF pretreatment. Renal function, histopathology and malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) levels in kidneys were assessed at 1, 2 and 7 days after ischaemia.. All rats except the controls had a significant improvement in serum creatinine, creatinine clearance and fractional excretion of Na(+) ; all three were significantly better in EPO + EGF group than in all other groups Histopathological examination showed marked structural damage in control rats. The tubular damage was least in the EPO + EGF group. The control group had a significant increase in MDA level and a significant decrease in SOD and GSH, while the EPO + EGF group had a marked significant reduction in MDA and increase in GSH and SOD.. The protection against ischaemia/reperfusion injury might be maximal when EPO and EGF are administered concomitantly, and their protective effect might be partly due to their antioxidant effects.

Topics: Analysis of Variance; Animals; Antioxidants; Drug Therapy, Combination; Epidermal Growth Factor; Erythropoietin; Kidney; Male; Random Allocation; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.

Topics: Animals; Benzofurans; Blotting, Western; Cell Line; Computational Biology; Epidermal Growth Factor; ErbB Receptors; HSP27 Heat-Shock Proteins; Mitochondrial Proteins; Muscle Proteins; Protein Binding; Proteomics; Rats; Reperfusion Injury; Signal Transduction

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hypoxia-inducible neuroprotective protein that also stimulates proliferation of neuronal precursor cells. In this study, we investigated the possible role of HB-EGF in ischemia and reperfusion injury by measuring the changes in its mRNA expression following focal cerebral ischemia. We also examined neural damage after a middle cerebral artery occlusion (MCAO) and reperfusion in ventral forebrain specific HB-EGF knockout (KO) mice. The levels of HB-EGF mRNA in the cerebral cortex of wild-type (WT) mice were significantly increased 3-24 h after MCAO and reperfusion. Cerebral infraction in HB-EGF KO mice was aggravated at 1 day and 6 days after MCAO and reperfusion compared with WT mice. The number of terminal deoxynucleotidyl transferase (TdT)-mediated dNTP nick end labeling (TUNEL) and an oxidative stress marker, 8-hydroxy-2'-deoxyguanosine (8-OHdG) positive cells, were higher in HB-EGF KO mice than in WT mice. On the other hand, fewer bromodeoxyuridine (BrdU) positive cells were found in the subventricular zone in HB-EGF KO mice compared with WT mice. These results indicate that HB-EGF may play a pivotal role in ischemia and reperfusion injury and that endogenously synthesized HB-EGF is necessary for both the neuroprotective effect and for regulation of cell proliferation in the subventricular zone.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult Stem Cells; Analysis of Variance; Animals; Brain Infarction; Bromodeoxyuridine; Cerebral Ventricles; Deoxyguanosine; Disease Models, Animal; Epidermal Growth Factor; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Intercellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Prosencephalon; Reperfusion Injury; RNA, Messenger; Transforming Growth Factor alpha

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF.

Topics: Adult; Aged; Animals; Cells, Cultured; Disease Models, Animal; Epidermal Growth Factor; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Kidney; Kidney Transplantation; Male; Mice; Middle Aged; Pyrimidines; Pyrroles; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Up-Regulation; Young Adult

Recovery from ischemic acute kidney injury requires the replacement of damaged tubular cells. This repair process involves epidermal growth factor (EGF) synthesized in medullary the thick ascending limbs (mTAL) of Henle. Atrial natriuretic peptide (ANP), a hormone synthesized by the cardiac atria, increases glomerular filtration rate and renal medullary blood flow. However, the effects of ANP on renal recovery after I/R-induced renal injury remain unclear. We therefore examined whether human ANP enhances recovery from I/R-induced renal injury by reducing damage to EGF-producing kidney cells in a rat model. Male Wistar rats weighing 200-240 g were observed for 48 h after reperfusion following 45-min renal ischemia. Rats were intravenously administered alpha-human ANP (alpha-hANP) at 0.2 microg/kg/min beginning immediately after ischemia and continuing for 2 h after reperfusion. Outer medullary blood flow (OMBF), EGF mRNA, serum blood urea nitrogen (BUN) and creatinine levels as indicators of glomerular function were measured, while urinary N-acetyl beta-D-glucosaminidase (NAG) was used as a specific indicator of proximal tubular function. OMBF was increased by alpha-hANP after reperfusion and maintained significantly higher mRNA level of EGF in the kidney 24 h after reperfusion. I/R-induced increases in serum concentrations of BUN and creatinine and urinary concentrations of NAG were also reduced by alpha-hANP, with improved histopathological changes, including acute tubular necrosis at 24-48 h after reperfusion. This report is the first to demonstrate that alpha-hANP accelerates recovery following renal ischemic insult by reducing the damage to EGF-producing kidney cells.

Topics: Acute Kidney Injury; Animals; Atrial Natriuretic Factor; Blood Urea Nitrogen; Creatinine; Epidermal Growth Factor; Humans; Kidney; Kidney Diseases; Male; Rats; Rats, Wistar; Renal Circulation; Reperfusion Injury; Urea

This study examined the effects of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) supplementation to University of Wisconsin solution (UW) in steatotic and nonsteatotic livers during cold storage. Hepatic injury and function were evaluated in livers preserved for 24 hours at 4 degrees C in UW and in UW with EGF and IGF-I (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. AKT was inhibited pharmacologically. In addition, hepatic injury and survival were evaluated in recipients who underwent transplantation with steatotic and nonsteatotic livers preserved for 6 hours in UW and UW with EGF and IGF-I (separately or in combination). The results, based on isolated perfused liver, indicated that the addition of EGF and IGF-I (separately or in combination) to UW reduced hepatic injury and improved function in both liver types. A combination of EGF and IGF-I resulted in hepatic injury and function parameters in both liver types similar to those obtained by EGF and IGF-I separately. EGF increased IGF-I, and both additives up-regulated AKT in both liver types. This was associated with glycogen synthase kinase-3beta (GSK3(beta)) inhibition in nonsteatotic livers and PPAR gamma overexpression in steatotic livers. When AKT was inhibited, the effects of EGF and IGF-I on GSK3(beta), PPAR gamma, hepatic injury and function disappeared. The benefits of EGF and IGF-I as additives in UW solution were also clearly seen in the liver transplantation model, because the presence of EGF and IGF-I (separately or in combination) in UW solution reduced hepatic injury and improved survival in recipients who underwent transplantation with steatotic and nonsteatotic liver grafts. In conclusion, EGF and IGF-I may constitute new additives to UW solution in steatotic and nonsteatotic liver preservation, whereas a combination of both seems unnecessary.

Topics: Adenosine; Allopurinol; Animals; Cell Survival; Cold Ischemia; Disease Models, Animal; Epidermal Growth Factor; Fatty Liver; Glutathione; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Insulin; Insulin-Like Growth Factor I; Liver; Liver Transplantation; Organ Preservation; Organ Preservation Solutions; Perfusion; PPAR gamma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Raffinose; Rats; Rats, Zucker; Recombinant Proteins; Reperfusion Injury; Time Factors

Hepatic steatosis is a major risk factor in ischemia-reperfusion (I/R). IGF-binding proteins (IGFBPs) modulate IGF-I action by transporting circulating IGF-I to its sites of action. Epidermal growth factor (EGF) stimulates IGF-I synthesis in vitro. We examined the effect of IGF-I and EGF treatment, separately or in combination, on the vulnerability of steatotic livers to I/R. Our results indicated that I/R impaired IGF-I synthesis only in steatotic livers. Only when a high dose of IGF-I (400 microg/kg) was given to obese animals did they show high circulating IGF-I:IGFBP levels, increased hepatic IGF-I levels, and protection against damage. In lean animals, a dose of 100 microg/kg IGF-I protected nonsteatotic livers. Our results indicated that the combined administration of IGF-I and EGF resulted in hepatic injury parameters in both liver types similar to that obtained by IGF-I and EGF separately. IGF-I increased egf expression in both liver types. The beneficial role of EGF on hepatic I/R injury may be attributable to p38 inhibition in nonsteatotic livers and to PPAR gamma overexpression in steatotic livers. In conclusion, IGF-I and EGF may constitute new pharmacological strategies to reduce the inherent susceptibility of steatotic livers to I/R injury.

Topics: Animals; Epidermal Growth Factor; Fatty Liver; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Liver; p38 Mitogen-Activated Protein Kinases; PPAR gamma; Rats; Rats, Zucker; Reperfusion Injury

Intestinal ischemia/reperfusion (I/R) injury is believed to be the major initiator of the systemic inflammatory response syndrome. As a result of intestinal I/R, the gut becomes a major source of inflammatory cytokine production. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is cytoprotective after intestinal I/R and down-regulates pro-inflammatory cytokine production in vitro. We now examine the effects of HB-EGF on pro-inflammatory cytokine expression in vivo.. Rats were randomized into three groups: sham-operated, superior mesenteric artery occlusion (SMAO) for 90 min followed by 8 h of reperfusion (I/R), and I/R with intraluminal administration of HB-EGF 25 min after the initiation of ischemia (I/R + HB-EGF). Serum was drawn at 2, 4, 6, and 8 h post reperfusion for determination of cytokine protein levels using a bioplex suspension array system. Additional animals underwent the same ischemic protocol followed by 30 and 60 min of reperfusion with harvesting of ileal mucosa. Ileal pro-inflammatory cytokine gene expression was determined using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for TNF-alpha, IL-6, and IL-1beta.. HB-EGF decreased TNF-alpha, IL-6, and IL-1beta serum protein levels at 4, 6, and 8 h after intestinal I/R injury. In addition, HB-EGF decreased local intestinal mucosal mRNA expression of TNF-alpha, IL-6, and IL-1beta 30 and 60 min after intestinal injury.. We conclude that pro-inflammatory cytokine expression is increased both locally and in the systemic circulation after intestinal I/R and that the administration of HB-EGF significantly reduces intestinal I/R-induced pro-inflammatory cytokine expression in vivo.

Topics: Animals; Cytokines; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Inflammation Mediators; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Interleukin-6; Intestinal Mucosa; Intestines; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tumor Necrosis Factor-alpha

The gut is highly susceptible to injury after hemorrhagic shock and resuscitation (HS/R) because of progressive mesenteric hypoperfusion. The aim of the current study was to evaluate the effect of heparin-binding EGF-like growth factor (HB-EGF) on mesenteric microcirculatory blood flow and intestinal injury in rats subjected to HS/R.. HS/R was induced in adult rats, with some rats receiving HB-EGF (600 mug/kg) IV at the onset of resuscitation (HS/R+HB-EGF) and others receiving vehicle only (HS/R). FITC-dextran was administered intra-arterially to evaluate mesenteric microcirculation, and intestinal damage and restitution were evaluated histologically. Data were expressed as mean +/- SE, with P < .05 considered statistically significant.. Microcirculatory blood flow was significantly reduced 1 hour after HS/R. HS/R+HB-EGF rats had significantly increased microcirculatory flow compared with HS/R rats at 1 hour (4.5 +/- 0.43 vs 2.64 +/- 0.46, P < .05) and 3 hours (8.04 +/- 1.58 vs 2.89 +/- 0.63, P < .05) after HS/R. HS/R+HB-EGF rats had significantly less intestinal damage compared with HS/R rats 3 hours after resuscitation (2.04 +/- 0.5 vs 3.08 +/- 0.5, P < .05), along with significantly fewer incompetent (nonresurfaced, nonhealed) villi, which is indicative of improved restitution.. HB-EGF significantly improved postresuscitation microcirculatory blood flow in rats subjected to HS/R, associated with significantly decreased intestinal damage and increased restitution. These results suggest that HB-EGF may be a useful therapeutic agent that improves intestinal blood flow in patients with intestinal injury secondary to hemorrhagic shock.

Topics: Animals; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Intestines; Male; Microcirculation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Resuscitation; Shock, Hemorrhagic; Specific Pathogen-Free Organisms; Splanchnic Circulation

Novel therapies for the treatment of MOF (multiple organ failure) are required. In the present study, we examined the effect of synthetic GHRP-6 (growth hormone-releasing peptide-6) on cell migration and proliferation using rat intestinal epithelial (IEC-6) and human colonic cancer (HT29) cells as in vitro models of injury. In addition, we examined its efficacy when given alone and in combination with the potent protective factor EGF (epidermal growth factor) in an in vivo model of MOF (using two hepatic vessel ischaemia/reperfusion protocols; 45 min of ischaemia and 45 min of reperfusion or 90 min of ischaemia and 120 min of reperfusion). In vitro studies showed that GHRP-6 directly influenced gut epithelial function as its addition caused a 3-fold increase in the rate of cell migration of IEC-6 and HT29 cells (P<0.01), but did not increase proliferation ([3H]thymidine incorporation). In vivo studies showed that, compared with baseline values, ischaemia/reperfusion caused marked hepatic and intestinal damage (histological scoring), neutrophilic infiltration (myeloperoxidase assay; 5-fold increase) and lipid peroxidation (malondialdehyde assay; 4-fold increase). Pre-treatment with GHRP-6 (120 microg/kg of body weight, intraperitoneally) alone truncated these effects by 50-85% (all P<0.05) and an additional benefit was seen when GHRP-6 was used in combination with EGF (1 mg/kg of body weight, intraperitoneally). Lung and renal injuries were also reduced by these pre-treatments. In conclusion, administration of GHRP-6, given alone or in combination with EGF to enhance its effects, may provide a novel simple approach for the prevention and treatment of MOF and other injuries of the gastrointestinal tract. In view of these findings, further studies appear justified.

Topics: Animals; Cell Movement; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; Growth Hormone-Releasing Hormone; Humans; Intestinal Mucosa; Lipid Peroxidation; Male; Multiple Organ Failure; Neutrophil Infiltration; Oligopeptides; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Tumor Cells, Cultured

Hepatic ischemia-reperfusion injury is an inevitable consequence during liver surgery. The outcome is particularly poor in cirrhotic livers, which are more prone to hepatic ischemia-reperfusion injury. We aim to study whether FTY720 could attenuate hepatic ischemia-reperfusion injury both in normal and in cirrhotic livers. We applied a 70% liver-ischemia (60 min) model in rats with normal or cirrhotic livers. FTY720 was given 20 min before ischemia and 10 min before reperfusion (1 mg/kg, i.v.). Liver tissues and blood were sampled at 20 min, 60 min, 90 min, 6 h and 24 h after reperfusion for detection of MAPK-Egr-1, Akt pathways and caspase cascade. Hepatic ultrastructure and apoptosis were also compared. FTY720 significantly improved liver function in the rats with normal and cirrhotic livers. Akt pathway was activated at 6 and 24 h after reperfusion. FTY720 significantly down-regulated Egr-1, ET-1, iNOS and MIP-2 accompanied with up-regulation of A20, IL-10, HO-1 and Hsp70. MAPK (Raf-MEK-Erk) pathway was down-regulated. Hepatic ultrastructure was well maintained and fewer apoptotic liver cells were found in the FTY720 groups. In conclusion, FTY720 attenuates ischemia-reperfusion injury in both normal and cirrhotic livers by activation of cell survival Akt signaling and down-regulation of Egr-1 via Raf-MEK-Erk pathway.

Topics: Animals; Apoptosis; Blotting, Western; Chemokine CXCL2; Chemokines, CXC; DNA Primers; Down-Regulation; Endothelin-1; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Fingolimod Hydrochloride; Gene Expression Regulation; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hepatocytes; HSP70 Heat-Shock Proteins; Immunosuppressive Agents; In Situ Nick-End Labeling; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-10; Liver; Male; MAP Kinase Signaling System; Microscopy, Electron; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; p38 Mitogen-Activated Protein Kinases; Propylene Glycols; Proteins; raf Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sphingosine; Time Factors; Up-Regulation

To investigate the role of P-selectin, intercellular adhesion molecule-1 (ICAM-1) and dendritic cells (DCs) in liver/kidney of rats with hepatic/renal ischemia-reperfusion injury and the preventive effect of anti-P-selectin lectin-EGF domain monoclonal antibody (anti-PsL-EGFmAb) on the injury.. Rat models of hepatic and renal ischemia-reperfusion were established. The rats were then divided into two groups, one group treated with anti-PsL-EGFmAb (n = 20) and control treated with saline (n = 20). Both groups were subdivided into four groups according to reperfusion time (1, 3, 6 and 24 h). The sham-operated group (n = 5) served as a control group. DCs were observed by the microscopic image method, while P-selectin and ICAM-1 were analyzed by immunohistochemistry.. P-selectin increased significantly in hepatic sinusoidal endothelial cells and renal tubular epithelial cells 1 h after ischemia-reperfusion, and the expression of ICAM-1 was up-regulated in hepatic sinusoid and renal vessels after 6 h. CD1a(+)CD80(+)DCs gradually increased in hepatic sinusoidal endothelium and renal tubules and interstitium 1 h after ischemia-reperfusion, and there was the most number of DCs in 24-h group. The localization of DCs was associated with rat hepatic/renal function. These changes became less significant in rats treated with anti-PsL-EGFmAb.. DCs play an important role in immune pathogenesis of hepatic/renal ischemia-reperfusion injury. Anti-PsL-EGFmAb may regulate and inhibit local DC immigration and accumulation in liver/kidney.

Topics: Animals; Antibodies, Monoclonal; Dendritic Cells; Epidermal Growth Factor; Immunohistochemistry; Intercellular Adhesion Molecule-1; Kidney; Lectins; Liver; Male; P-Selectin; Protein Structure, Tertiary; Rats; Rats, Wistar; Reperfusion Injury

We have previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an intestinal cytoprotective agent. The current study examined whether HB-EGF is effective as salvage therapy as well as prophylactic therapy for intestinal ischemia-reperfusion (I/R) injury, whether intravenous administration is as effective as intraluminal administration, and whether increased benefits are seen with increasing dose.. Total midgut I/R injury in rats was achieved by occlusion of a first-order branch of the superior mesenteric artery for 60 minutes, followed by reperfusion for 6 hours. Rats were treated with HB-EGF 5 minutes before ischemia, halfway through the ischemic event, or 5 minutes after ischemia. Route of administration was tested by administering HB-EGF either intraluminally or intravenously. Seven different doses of HB-EGF were tested.. Heparin-binding, EGF-like growth factor protected the intestine from injury when administered before injury and was also effective when administered during ischemia or even after injury. Intraluminal administration of HB-EGF was superior to intravenous administration. Increasing doses of HB-EGF resulted in a greater cytoprotective effect.. These data demonstrate that HB-EGF acts as an effective intestinal cytoprotective agent when administered intraluminally not only before injury, but also during injury and, most importantly, even after intestinal injury has already occurred. These findings support a basis for the prophylactic use of intraluminal HB-EGF in high-risk patients, as well as for the administration of HB-EGF to salvage patients in whom an intestinal insult has already occurred.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Infusions, Intravenous; Intercellular Signaling Peptides and Proteins; Intestines; Ischemia; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Risk Factors; Salvage Therapy

Epidermal growth factor (EGF) modulates Leydig cell proliferation, steroidogenesis, spermiogenesis, and Sertoli cell activity. It plays an important role in repairing ischemia-reperfusion injury in different tissues. The aim of this study was to evaluate the effects of sustained and local administration of EGF on improving bilateral testicular tissue after torsion. A total of 57 Wistar albino rats were used. For the EGF transport system, 1x2 cm gelatin films containing 2 microg EGF were used. Torsion was created by rotating the right testis 720 degrees in a clockwise direction for 4 h in all groups except the control group. Then, in the torsion group, bilateral orchiectomy was performed. After returning the torsioned ipsilateral testes to their normal state, the bilateral testes were wrapped by 1x2 cm unloaded gelatin films in the gelatin (G7 and G21) groups and, by 2 microg EGF loaded gelatin films in the EGF 7 and EGF 21 groups. The testes were removed on the seventh and 21st days, respectively, for biochemical and histological examination. Histologically, Johnsen's spermatogenesis criteria and mean seminiferous tubule diameter (MSTD) measurements were used. The EGF7 group did not show significant loss of Sertoli cells, while in the G7 group the number of these cells decreased. The ipsilateral ischemic testis of the EGF21 group showed Leydig cell hyperplasia, and the contralateral non-ischemic testes in this group were similar to the control group. In the G21 group, the bilateral testes showed Sertoli cell only syndrome in some sections, and most of the cells were undergoing apoptosis. The mean spermatogenesis scores and MSTD in the EGF7 and EGF21 groups were higher than in the G7 and G21 groups ( P<0.05). Malondialdehyde levels were significantly lower in the EGF groups than in the G groups ( P<0.05). Glutathione peroxidase (GSH-Px) levels in the G21 group were significantly higher than in the EGF21 group. Our study shows that local and sustained EGF release after testicular torsion improves bilateral testicular injury. EGF administration may be a new treatment choice for bilaterally injured testis after detorsion without removing the twisted testis.

Topics: Animals; Apoptosis; Epidermal Growth Factor; Glutathione Peroxidase; Hyperplasia; Leydig Cells; Male; Malondialdehyde; Orchiectomy; Rats; Rats, Wistar; Reperfusion Injury; Seminiferous Tubules; Sertoli Cells; Spermatic Cord Torsion; Spermatogenesis; Testis; Time Factors

This study examined whether heparin-binding epidermal growth factor (EGF) like growth factor (HB-EGF), a proven intestinal cytoprotective molecule, exerts its protective effects through modulation of adhesion molecule expression and inflammatory cell infiltration, important pathogenic mediators of ischemia/reperfusion (I/R) injury.. Total midgut I/R injury in rats was achieved by occlusion of the superior mesenteric artery for 90 minutes followed by reperfusion. Rats were treated intraluminally with 600 microg/kg HB-EGF or with PBS 45 minutes after the onset of ischemia. Four- or 24-hours post-I/R, ileum was harvested and processed for immunhistochemical detection of P-/E-selectins, intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1), and polymorphonuclear cells (PMN)/macrophages (MPhi).. P-/E-selectins were significantly induced in vascular endothelia 4 hours after I/R injury compared with normal intestine. HB-EGF treatment significantly down-regulated the expression of P-/E-selectins. I/R-injured intestine displayed overexpression of ICAM-1 and VCAM-1, which were significantly down-regulated by HB-EGF treatment. Lastly, I/R injury caused significant infiltration of PMN and MPhi into wounded tissue 24 hours after I/R compared with normal intestine. HB-EGF treatment significantly decreased PMN and MPhi infiltration into the injured tissue.. HB-EGF intestinal cytoprotection is mediated, in part, by down-regulation of expression of adhesion molecules and infiltration of PMN and MPhi after intestinal I/R injury.

Topics: Animals; Cell Adhesion Molecules; Chemotaxis, Leukocyte; Constriction; Depression, Chemical; E-Selectin; Endothelium, Vascular; Epidermal Growth Factor; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; Intercellular Adhesion Molecule-1; Intercellular Signaling Peptides and Proteins; Intestines; Ischemia; Macrophages; Male; Mesenteric Artery, Superior; Neutrophils; P-Selectin; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Vascular Cell Adhesion Molecule-1

Lipoxins are lipoxygenase-derived eicosanoids with anti-inflammatory and proresolution bioactivities in vitro and in vivo. We have previously demonstrated that the stable synthetic LXA4 analog 15-epi-16-(FPhO)-LXA4-Me is renoprotective in murine renal ischemia/reperfusion injury, as gauged by lower serum creatinine, attenuated leukocyte infiltration, and reduced morphologic tubule injury.. We employed complementary oligonucleotide microarray and bioinformatic analyses to probe the transcriptomic events that underpin lipoxin renoprotection in this setting.. Microarray-based analysis identified three broad categories of genes whose mRNA levels are altered in response to ischemia/reperfusion injury, including known genes previously implicated in the pathogenesis of ischemia/reperfusion injury [e.g., intercellular adhesion molecule-1 (ICAM-1), p21, KIM-1], known genes not previously associated with ischemia/reperfusion injury, and cDNAs representing yet uncharacterized genes. Characterization of expressed sequence tags (ESTs) displayed on microarrays represents a major challenge in studies of global gene expression. A bioinformatic annotation pipeline successfully annotated a large proportion of ESTs modulated during ischemia/reperfusion injury. The differential expression of a representative group of these ischemia/reperfusion injury-modulated genes was confirmed by real-time polymerase chain reaction. Prominent among the up-regulated genes were claudin-1, -3, and -7, and ADAM8. Interestingly, the former response was claudin-specific and was not observed with other claudins expressed by the kidney (e.g., claudin-8 and -6) or indeed with other components of the renal tight junctions (e.g., occludin and junctional adhesion molecule). Noteworthy among the down-regulated genes was a cluster of transport proteins (e.g., aquaporin-1) and the zinc metalloendopeptidase meprin-1 beta implicated in renal remodeling.. Treatment with the lipoxin analog 15-epi-16-(FPhO)-LXA4-Me prior to injury modified the expression of many differentially expressed pathogenic mediators, including cytokines, growth factors, adhesion molecules, and proteases, suggesting a renoprotective action at the core of the pathophysiology of acute renal failure (ARF). Importantly, this lipoxin-modulated transcriptomic response included many genes expressed by renal parenchymal cells and was not merely a reflection of a reduced renal mRNA load resulting from attenuated leukocyte recruitment. The data presented herein suggest a framework for understanding drivers of kidney injury in ischemia/reperfusion and the molecular basis for renoprotection by lipoxins in this setting.

Topics: Acute Kidney Injury; ADAM Proteins; Animals; Antigens, CD; Claudin-1; Claudin-3; Claudins; DNA, Complementary; Epidermal Growth Factor; Kidney; Lipoxins; Membrane Proteins; Metalloendopeptidases; Mice; Oligonucleotide Array Sequence Analysis; Reperfusion Injury; RNA, Messenger; Transcription, Genetic

Acute pancreatitis is accompanied by the enhanced expression of EGF in the pancreas and the administration of EGF was found to exhibit the beneficial effect on edematous cerulein-induced pancreatitis. Therefore, we decided to determine the influence of EGF on necro-hemorrhagic pancreatitis induced by ischemia and reperfusion (I/R). Acute pancreatitis was induced in rats by restricting the pancreatic blood flow (PBF) in the inferior splenic artery for 30 min using microvascular clips. EGF was administered three times daily (10 microg/kg per dose s.c.) starting immediately after the clips removal. Rats were sacrificed on day 1, 3, 5, 10 and 21 following ischemia. PBF was measured using a laser Doppler flowmeter. Morphological signs of pancreatitis, as well as the levels of plasma amylase, lipase, interleukin-1beta and interleukin-10 concentration and pancreatic cell proliferation were examined.. Ischemia with reperfusion caused acute necro-hemorrhagic pancreatitis with a histological and biochemical manifestation of pancreatic damage, followed by a spontaneous regeneration. The administration of EGF caused the reduction in the histological signs of pancreatic damage, such as necrosis, edema and leukocyte infiltration, and accelerated the pancreatic repair. Also, EGF treatment significantly attenuated the reduction in pancreatic blood flow and DNA synthesis. The activity of plasma amylase and lipase, as well as plasma interleukin-1beta and interleukin-10 concentrations were decreased in EGF treated animals.. EGF exerts beneficial influence on the course of I/R induced pancreatitis and this effect seems to be related to the reduction in the activation of pro-inflammatory interleukin cascade, the improvement of PBF, and the increase in pancreatic cell growth.

Topics: Amylases; Animals; DNA; Epidermal Growth Factor; Interleukin-1; Interleukin-10; Ischemia; Lipase; Male; Pancreas; Pancreatitis; Rats; Rats, Wistar; Regional Blood Flow; Reperfusion Injury

To measure the effect of pre-ischemic administration of intraluminal epidermal growth factor on the changes in intestinal permeability induced by 30 mins of superior mesenteric artery occlusion, followed by 2 hrs of reperfusion.. Prospective, randomized, placebo-controlled experimental study.. University basic science research laboratory.. Healthy, young, adult, male Sprague-Dawley rats.. A 10-cm segment of small intestine was isolated and studied in situ in rats that were anesthetized with fentanyl and mechanically ventilated. Intestinal ischemia-reperfusion injury was induced by temporary occlusion of the superior mesenteric artery for 30 mins, followed by 2 hrs of reperfusion. Three groups were studied: time controls with a sham operation, saline-treated ischemia-reperfusion, and epidermal growth factor-treated ischemia-reperfusion. Epidermal growth factor, 100 ng/min, was infused intraluminally, beginning 30 mins before and continued until 40 mins after ischemia.. Intestinal permeability was measured for each 10-min time period by using chromium-labeled EDTA. Histopathologic injury was assessed by light microscopy. After superior mesenteric artery occlusion, intestinal permeability increased approximately ten-fold and was sustained for 2 hrs of reperfusion in saline-treated rats. Pretreatment with epidermal growth factor significantly reduced the permeability changes during reperfusion by >60% compared with saline-treated animals (p <.05). Histopathologic sections revealed apparently more extensive loss of epithelial cells and mucosal disruption in saline-treated intestine compared with epidermal growth factor-treated intestine.. Pre-ischemic administration of intraluminal epidermal growth factor significantly protects against intestinal ischemia-reperfusion injury.

Topics: Animals; Epidermal Growth Factor; Intestinal Mucosa; Intestine, Small; Male; Permeability; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Ischemia/reperfusion of mesenteric vessels is a useful model for acute vascular insufficiency and the early stages of multiorgan failure, conditions associated with high morbidity and mortality. Epidermal growth factor (EGF) is a potent mitogen that shows potential for use in intestinal injury. We therefore examined its influence on this model. Male Sprague-Dawley rats received human recombinant EGF (2 mg/kg i.p., n = 14) or saline (n = 16); 25 minutes before arterial clamping of the superior mesenteric artery (ischemic period) for 60 minutes followed by a final 60-minute reperfusion period. Additional rats were not operated on (controls, n = 7) or had sham operation (laparotomy only, n = 10). Ischemia/reperfusion caused macroscopic damage affecting 56%, 51 to 67% (median, interquartile range), of small intestinal length and intraluminal bleeding. Malondialdehyde levels (free radical marker) increased eightfold compared to nonoperated animals (2400, 2200 to 2700 micro mol/mg protein versus 290, 250 to 350 micro mol/mg protein, P < 0.01) and myeloperoxidase levels (marker for inflammatory infiltrate) increased 15-fold (3150, 2670 to 4180 U/g tissue versus 240, 190 to 250 U/g tissue, P < 0.01). Pretreatment with EGF reduced macroscopic injury to 11%, 0 to 15%; prevented intraluminal bleeding; and reduced malondialdehyde and myeloperoxidase levels by approximately 60% and 90% (all P < 0.01 versus non-EGF-treated). Mesenteric ischemia/reperfusion also damaged the lungs and kidneys and increased serum tumor necrosis factor-alpha levels (circulating cytokine activity marker). EGF pretreatment also reduced these changes. These studies provide preliminary evidence that EGF is a novel therapy for the early treatment or prevention of intestinal damage and multiorgan failure resulting from mesenteric hypoperfusion.

Topics: Animals; Epidermal Growth Factor; Humans; Injections, Intraperitoneal; Intestines; Male; Mesenteric Arteries; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestinal epithelial cells from anoxia/reoxygenation in vitro, and to protect the intestines from ischemia/reperfusion (I/R) injury in vivo. The goal of the present study was to determine whether the cytoprotective effects of HB-EGF were due, in part, to its ability to decrease reactive oxygen species (ROS) production. Human whole blood, polymorphonuclear leukocytes, and monocytes, as well as rat intestinal epithelial cells, were exposed to stimuli designed to produce an oxidative burst in these cells. Treatment of the cells with HB-EGF led to a significant decrease in oxidative burst production. In vivo, total midgut I/R injury in rats led to increased ROS production, which was markedly decreased by HB-EGF treatment. Histochemically, I/R injury led to increased ROS production, which was significantly decreased with HB-EGF treatment. HB-EGF cytoprotection is due, in part, to its ability to decrease ROS production. Future studies will determine the mechanisms by which HB-EGF exerts these effects.

Topics: Animals; Epidermal Growth Factor; Free Radicals; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Luminol; Monocytes; Neutrophils; Rats; Reactive Oxygen Species; Reperfusion Injury; Respiratory Burst; Tetradecanoylphorbol Acetate

Epidermal growth factor (EGF) plays an important role in tubular regeneration in kidneys with ischemia/reperfusion (I/R) injury. This study was undertaken to evaluate the influence of cyclosporine A (CsA) or FK506 on mature EGF expression and tubular regeneration in rat kidneys with I/R injury.. Two separate studies were performed. First, the expression of EGF and tubular regeneration was observed in rat kidneys with I/R injury on days 1, 2, 3, 5, and 7. Second, the dose-dependent response of EGF expression and tubular regeneration to CsA (5, 10, and 20 mg/kg) or FK506 (0.25, 0.5, and 1.0 mg/kg) was observed in rat kidneys with I/R injury. I/R injury was induced by clamping both renal arteries for 45 min, and CsA or FK506 was injected just after release of vascular clamps. Rats were sacrificed on day 1 for evaluation of EGF expression, and on day 2 for evaluation of BudU-positive cells. Renal function, tubular injury score, EGF expression assessed by immunoblotting, levels of CsA and FK506 in whole blood, and immunostaining for BrdU was studied.. EGF expression was maximal on day 1 (cortex, 29-fold; medulla, 31-fold compared with sham-operated controls), and renal tubular regeneration measured with the number of BrdU-positive cells was maximal on days 2 and 3 in kidney with I/R injury, and thereafter the level of EGF and the number of BrdU-positive cells decreased progressively. CsA or FK506 treatment to ischemic rat kidneys reduced the expression of EGF and the number of BrdU-positive cells in a dose-dependent manner.. CsA or FK506 treatment delays recovery from acute tubular necrosis, and this may be associated with decreased EGF expression by CsA or FK506.

Topics: Animals; Antimetabolites; Bromodeoxyuridine; Cyclosporine; Epidermal Growth Factor; Immunosuppressive Agents; Kidney Tubules; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tacrolimus

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a known mitogenic, chemotactic, and cytoprotective growth factor for epithelial cells, was examined to see whether it could protect intestinal barrier function and decrease bacterial translocation (BT) after ischemia/reperfusion (I/R) injury.. In vitro, tight junctional integrity of intestinal epithelial cells (IEC-6) cells was evaluated by measuring transepithelial electric resistance (TEER), and monolayer permeability was evaluated by translocation of Escherichia coli C25. In vivo, crypt cell proliferation was assessed by 5-bromodeoxyuridine incorporation with calculation of a proliferative index (PI), and BT was evaluated by culture of mesenteric lymph nodes.. In vitro, anoxia damaged tight junctional integrity and increased permeability of IEC-6 cell monolayers, events that were reversed completely by treatment of the cells with HB-EGF. Twenty-four hours after I/R injury in vivo, crypt cell proliferation index (PI) decreased significantly from 35.6 +/- 4.5 to 17.8 +/- 3.4. Administration of HB-EGF preserved crypt cell activity with PI of 34.9 +/- 4.1, similar to that of normal ileum. None of the normal or sham-operated animals showed BT, whereas BT occurred in 87.5% of I/R-injured rats. In animals exposed to I/R but treated with HB-EGF, BT was decreased significantly to 12.5%.. HB-EGF preserves proliferation of crypt cells, maintains integrity of epithelial cells, and subsequently decreases enteric BT after I/R injury.

Topics: Animals; Bacterial Translocation; Epidermal Growth Factor; Gram-Negative Bacteria; Heparin-binding EGF-like Growth Factor; Ileum; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Male; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Tight Junctions

The immunosuppressive effect of rapamycin is mediated by inhibition of interleukin-2-stimulated T cell proliferation. We report for the first time that rapamycin also inhibits growth factor-induced proliferation of cultured mouse proximal tubular (MPT; IC(50) ~1 ng/ml) cells and promotes apoptosis of these cells by impairing the survival effects of the same growth factors. On the basis of these in vitro data, we tested the hypothesis that rapamycin would impair recovery of renal function after ischemic acute renal failure induced in vivo by renal artery occlusion (RAO). Rats given daily injections of rapamycin or vehicle were subjected to RAO or sham surgery. Rapamycin had no effect on the glomerular filtration rate (GFR) of sham-operated animals. In rats subjected to RAO, GFR fell to comparable levels 1 day later in vehicle- and rapamycin-treated rats (0.25 +/- 0.08 and 0.12 +/- 0.05 ml. min(-1). 300 g(-1), respectively) (P = not significant). In vehicle-treated rats subjected to RAO, GFR increased to 0.61 +/- 0.08 ml. min(-1). 300 g(-1) on day 3 (P < 0.02 vs. day 1) and then rose further to 0.99 +/- 0.09 ml. min(-1). 300 g(-1) on day 4 (P < 0.02 vs. day 3). By contrast, GFR did not improve in rapamycin-treated rats subjected to RAO over the same time period. Rapamycin also increased apoptosis of tubular cells while markedly reducing their proliferative response after RAO. Furthermore, rapamycin inhibited activation of 70-kDa S6 protein kinase (p70(S6k)) in cultured MPT cells as well as in the renal tissue of rats subjected to RAO. We conclude that rapamycin severely impairs the recovery of renal function after ischemia-reperfusion injury. This effect appears to be due to the combined effects of increased tubular cell loss (via apoptosis) and profound inhibition of the regenerative response of tubular cells. These effects are likely mediated by inhibition of p70(S6k).

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Survival; Cells, Cultured; Cisplatin; Enzyme Activation; Epidermal Growth Factor; Glomerular Filtration Rate; Immunosuppressive Agents; Kidney Transplantation; Kidney Tubules, Proximal; Lysophospholipids; Mice; Mice, Inbred C57BL; Nephrectomy; Recovery of Function; Reperfusion Injury; Ribosomal Protein S6 Kinases; Sirolimus

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestine from ischemia/reperfusion (I/R) injury in vivo and to down-regulate inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in intestinal epithelial cells in vitro. The present study was undertaken to investigate whether HB-EGF could modulate the iNOS/NO axis after total midgut I/R injury in rats. I/R injury induced a significant increase in iNOS gene expression (quantified by real-time RT-PCR) and protein production (detected by western blots), as well as elevation of serum NO levels (measured by chemiluminescence assay). Nitrotyrosine (NT) and iNOS production colocalized immunohistochemically, with positive staining found mainly in villous and crypt epithelial cells, as well as ganglion cells. Intraluminal administration of HB-EGF 45 min after the start of a 90-min ischemic interval significantly decreased I/R-induced iNOS gene expression and protein production, as well as serum NO levels. Immunohistochemically, HB-EGF administration led to elimination of iNOS and NT staining in crypt epithelial cells and ganglion cells, with only weak staining that remained in villous epithelial cells. Thus, HB-EGF protects the intestine from I/R injury, at least partially, through down-regulation of the iNOS/NO/NT pathway, a mechanism that is central to I/R injury in multiple organ systems.

Topics: Animals; Blotting, Western; Down-Regulation; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Intestines; Luminescent Measurements; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Ribosomal; Transcription, Genetic

In order to estimate a regenerative response in the early phase after renal ischemia-reperfusion in rat, we examined the time course of the activation of epidermal growth factor receptor (EGFR) as a response of signal transduction pathway after 45 min ischemia in kidney. The activation of EGFR was observed 5-30 min after the start of reperfusion. Simultaneously, superoxide anion/hydrogen peroxide generated in the mitochondrial fraction was elevated during the same period. On the other hand, the level of EGF decreased in a time-dependent manner. These results suggested that superoxide anion/hydrogen peroxide generated during the ischemia-reperfusion other than EGF could act as an activator for the EGFR. In summary, the activation of EGFR is important as a regenerative response at an early stage after the start of reperfusion in ischemic kidney.

Topics: Acute Kidney Injury; Animals; Blotting, Western; Epidermal Growth Factor; ErbB Receptors; Kidney; Male; Mitochondria; Oxygen; Phosphorylation; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Ischemia followed by reperfusion leads to severe organ injury and dysfunction. Inflammation is considered to be the most important cause of tissue injury in organs subjected to ischemia. The mechanism that triggers inflammation and organ injury after ischemia remains to be elucidated, although different causes have been postulated. We investigated the role of apoptosis in the induction of inflammation and organ damage after renal ischemia. Using a murine model, we demonstrate a relationship between apoptosis and subsequent inflammation. At the time of reperfusion, administration of the antiapoptotic agents IGF-1 and ZVAD-fmk (a caspase inactivator) prevented the early onset of not only renal apoptosis, but also inflammation and tissue injury. Conversely, when the antiapoptotic agents were administered after onset of apoptosis, these protective effects were completely abrogated. The presence of apoptosis was directly correlated with posttranslational processing of the endothelial monocyte-activating polypeptide II (EMAP-II), which may explain apoptosis-induced influx and sequestration of leukocytes in the reperfused kidney. These results strongly suggest that apoptosis is a crucial event that can initiate reperfusion-induced inflammation and subsequent tissue injury. The newly described pathophysiological insights provide important opportunities to effectively prevent clinical manifestations of reperfusion injury in the kidney, and potentially in other organs.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blood Urea Nitrogen; Caspases; Chemotaxis, Leukocyte; Cysteine Proteinase Inhibitors; Cytokines; Depression, Chemical; Drug Administration Schedule; Epidermal Growth Factor; Humans; In Situ Nick-End Labeling; Insulin-Like Growth Factor I; Ischemia; Kidney; Male; Mice; Neoplasm Proteins; Nephritis; Peroxidase; Protein Processing, Post-Translational; Recombinant Proteins; Reperfusion Injury; RNA-Binding Proteins

A major contributor to the development and progression of ischemia-reperfusion (IR)-induced acute renal failure (ARF) is the loss of functioning tubular epithelial cells by means of various cell deletion or death processes. Although the term "acute tubular necrosis" is still used to describe the pathology of ARF, this is a misnomer because apoptotic cell death, as well as necrosis, occurs [1, 2] along with desquamation and loss of viable epithelial cells [3]. Apoptosis was first described in renal disease in 1987 in an animal model of hydronephrosis [4]. In ARF, with reference to only the death processes, the relative contribution of necrosis or apoptosis possibly depends on the extent of the initiating events. For example, after prolonged total renal ischemia, necrosis or "accidental cell death" occurs from the resultant negation of the cell's energy and protein levels. In apoptosis, the cells use their own energy processes and proteins to die, and often the initiating ischemia is more mild [5]. Finally, despite prolonged ischemia, within the heterogeneous renal cell populations there are those that are more sensitive to ischemia, such as the proximal straight tubule and to some extent the thick ascending limb (TAL) of the loop of Henle. It may be hypothesized that these cells tend to undergo necrosis in comparison with the less sensitive segments that undergo apoptosis. Because apoptosis is gene driven, its identification is important because of the possibility of its modulation via molecular controls. However, despite these new concepts of ARF, patient death remains high, at approximately 30 to 50% of ARF cases. Recovery from ARF depends not only on the replacement or regeneration of cells deleted by death, the theme of many recent studies, but also on protection of cells from death. Both processes are dependent on many of the cellular and molecular controls that have evolved in multicellular organisms to manage normal development, differentiation and growth processes, but that then become involved in the pathogenesis and progression of many renal diseases, including ARF.

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-X Protein; Body Weight; Cell Division; Cell Survival; Epidermal Growth Factor; Epithelial Cells; Insulin-Like Growth Factor I; Loop of Henle; Male; Necrosis; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; Transforming Growth Factor beta

We have shown previously that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is cytoprotective for intestinal epithelial cells exposed to hypoxia in vitro. We now examine the effects of HB-EGF on the recovery of small intestine from ischemic injury in vivo.. Segmental intestinal ischemia of 60-min duration was produced in adult rats by occlusion of a first-order branch of the superior mesenteric artery. Recombinant HB-EGF (100 microg) was injected intraluminally into the proximal small bowel after 45 min of ischemia in experimental animals, and buffered saline was injected in control animals. Animals were sacrificed after 48 h, and the affected bowel was resected, processed, and examined microscopically, with histologic grading of the ischemic injury. Additional animals were allowed to recover for up to 1 month to evaluate mortality differences.. Intraluminal administration of HB-EGF resulted in significantly decreased extent and severity of ischemia/reperfusion injury, with significantly decreased grade of injury in the HB-EGF-treated compared with nontreated animals (average injury grade 0.66 compared with 2.44, respectively). Moreover, the mortality rate was significantly lower in the HB-EGF-treated animals compared with nontreated animals (0% vs 25%, respectively). HB-EGF-treated animals had increased weight gain in the postischemia recovery period.. We conclude that HB-EGF, given intraluminally, reduces both the amount and the severity of ischemia/reperfusion injury in the small bowel, reduces the mortality associated with intestinal ischemia, and may enhance intestinal recovery. The in vitro and in vivo cytoprotective effects of this growth factor suggest that it may, in the future, be clinically useful in treating patients with intestinal ischemia.

Topics: Acute Disease; Animals; Cytoprotection; Epidermal Growth Factor; ErbB Receptors; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Intestines; Ischemia; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Several peptide growth factors, including EGF, are known to protect endothelium from oxygen-related damage or ischemia-reperfusion, in vitro experiments show that such protective effect involves endogenous endothelium-related factors like nitric oxide and prostanoids. However, in vivo demonstrations of a possible role in related vascular diseases are lacking. In our experiments, human EGF and fraction C, a 3-10 kDa oligosaccharidic fraction from an aqueous extract of Triticum vulgare, known as growth promoters for several cell types including endothelial cells, were found protective against ischemic necrosis of the mouse tail induced by i.v. k-carrageenin plus endothelin-1. After i.p. injection, peak activities were observed at 10 micrograms/kg EGF and 2 mg/kg fraction C. Pretreatment with L-NAME reduced protection in a dose-dependent manner. Addition of indomethacin increased the effect of L-NAME, suggesting that both nitric oxide and eicosanoids are involved in the protective effect of EGF and fraction C.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; Ischemia; Male; Mice; Necrosis; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Plant Extracts; Reperfusion Injury; Tail; Tritium

We have recently reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA is induced in the rat kidney after acute ischemic injury. The present studies were designed to investigate whether bioactive HB-EGF protein is also produced in response to renal injury induced by either ischemia/reperfusion or aminoglycosides. Heparin-binding proteins were purified from kidney homogenates by heparin affinity column chromatography using elution with a 0.2-2.0 M gradient of NaCl. A single peak of proteins that eluted at 1.0-1.2 M NaCl was detected in the postischemic kidney within 6 h of injury. This eluate fraction stimulated DNA synthesis in quiescent Balb/c3T3, RIE, and NRK-52E cell lines, all of which are responsive to the epidermal growth factor family of mitogenic proteins. The EGF receptor of A431 cells was also tyrosine phosphorylated by this eluate peak. Furthermore, immunoblotting with a polyclonal antibody against rat HB-EGF indicated that the eluate peak contained immunoreactive proteins of 22 and 29 kD mol wt, consistent with the reported sizes of the secreted form and membrane anchored form of HB-EGF, respectively. Immunohistochemical studies revealed that HB-EGF was produced predominantly in distal tubules in kidneys injured either by ischemia/reperfusion or aminoglycoside administration. We also found that during metanephric development immunoreactive HB-EGF was detected in the ureteric bud as early as E14.5 and persisted in structures arising from the ureteric bud throughout embryogenesis. These results suggest that in response to acute injury, HB-EGF is produced predominantly in distal tubules and that endogenous HB-EGF may be an important growth factor involved in renal epithelial cell repair, proliferation, and regeneration in the early stages of recovery after acute renal injury, as well as in nephrogenesis.

Topics: Aminoglycosides; Animals; Cells, Cultured; Chromatography, Affinity; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Immunoblotting; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Kidney; Male; Phosphorylation; Precipitin Tests; Rats; Rats, Sprague-Dawley; Regeneration; Reperfusion Injury; RNA, Messenger

Urinary immunoreactive epidermal growth factor (EGF) levels decrease, and renal immunoreactive EGF levels increase in rats with ischaemic acute renal failure (ARF). We investigated the immunohistochemical localization of EGF and EGF receptor in rabbits with ischaemic ARF to clarify the significance of renal EGF. Male New Zealand White rabbits underwent right nephrectomy prior to a 60 min renal artery clamp. At 3, 6, 24, 48, 72 and 96 h after ischaemia, serum urea nitrogen and serum creatinine were determined. Guinea pig anti-rabbit EGF antibody and monoclonal anti-EGF receptor antibody were used for the primary incubation. EGF was immunolocalized to the ascending limb of Henle and the distal convoluted tubule in the normal right kidneys. However, in the post ischaemic left kidneys at 6, 24, 48 and 72 h, immunoreactivity of EGF was associated with proximal tubules. In the normal kidneys, antibody to EGF receptor reacted with distal tubules and collecting ducts. In the ischaemic kidneys, EGF receptor was localized in the basolateral membrane in the proximal tubules. The expression of EGF and EGF receptor in renal tubules may play an important role in repair following ischaemic renal damage.

Topics: Acute Kidney Injury; Animals; Biomarkers; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Kidney; Kidney Tubules, Proximal; Male; Necrosis; Rabbits; Reperfusion Injury; Time Factors

We previously reported that following bilateral acute tubular necrosis (ATN) profound changes in the intrarenal insulin-like growth factor-I axis occurs which are unrelated to altered nutritional intake. In this current report we studied rats with unilateral ATN to assess whether these changes reflect a response to acute injury or the accompanying uremia. Compared to the contralateral kidney, the injured kidney showed an increase in IGF-I receptor number without a change in IGF-I receptor mRNA levels, a decrease in IGF-I mRNA and IGF-I protein levels, a decrease in growth hormone (GH) receptor mRNA abundance and receptor binding. There was also a decrease in IGF binding protein-2, -3 and -5 mRNA levels together with a fall in protein products. Since this unilateral ATN model excludes the influence of uremia and reduced nutritional intake, we surmised that these changes reflect a direct response to injury. Next, because of the reduced GH receptor binding noted above and the reported decrease in epidermal growth factor (EGF) expression in ATN, we tested the thesis that the low kidney IGF-I mRNA levels in ATN are partly due to a relative or absolute deficiency of these hormones. Administration of EGF or GH promptly increased ATN kidney IGF-I mRNA levels to control kidney values, lending support to the thesis. The response to EGF also suggests that the salutary effect of EGF treatment in ATN may partly be mediated by stimulating IGF-I production.

Topics: Animals; Antibody Specificity; Blotting, Western; Cell Membrane; Endopeptidases; Epidermal Growth Factor; Gene Expression; Growth Hormone; Insulin-Like Growth Factor I; Kidney; Male; Rats; Rats, Sprague-Dawley; Receptor, IGF Type 1; Receptors, Somatotropin; Reperfusion Injury; RNA, Messenger

Previous studies have suggested that EGF or other members of the EGF family of mitogenic proteins are involved in proliferation of renal tubular epithelial cells occurring during recovery from injury to the kidney. The present studies examined whether expression of mRNA for the recently identified heparin-binding EGF-like growth factor (HB-EGF) is regulated in response to renal injury induced by either ischemia/reperfusion or mercuric chloride. Increased expression of HB-EGF mRNA was demonstrated in the post-ischemic kidney within 45 min of unilateral ischemia/reperfusion in the rat. Induction of HB-EGF mRNA occurred only when ischemia was followed by reperfusion, and was not eliminated by removal of blood cells from the post-ischemic kidney by saline perfusion. In situ hybridization with 35S-labeled antisense riboprobes of HB-EGF indicated that compared with control, there was increased HB-EGF mRNA expression in the 6 h post-ischemic kidney in the inner cortex and outer medulla in a patchy distribution, with the greatest expression in the inner stripe of the outer medulla. Expression occurred primarily in tubular epithelial cells. Recombinant human HB-EGF stimulated [3H]-thymidine incorporation in both primary cultures of rabbit proximal tubule cells and NRK 52E normal rat kidney epithelial cells, with potency similar to that of EGF. Induction of HB-EGF mRNA was observed in tubules freshly isolated from rat renal cortex or outer medulla when the tubules were subjected to reoxygenation after incubation in anoxic conditions. The nephrotoxin, mercuric chloride, also caused induction of HB-EGF mRNA both in vivo and in isolated rat cortical tubules. The anoxia/reoxygenation-induced expression of HB-EGF mRNA in isolated tubules was inhibited by the free radical scavengers, di- and tetra-methylthiourea, indicating involvement of reactive oxygen species. These findings indicate that HB-EGF mRNA is inducible in the kidney in vivo by acute tubular injury and suggest that HB-EGF may act as an autocrine/paracrine growth factor involved in proliferation of tubular epithelial cells and repair of the kidney.

Topics: Animals; Cell Hypoxia; Cells, Cultured; Epidermal Growth Factor; Free Radical Scavengers; Gene Expression Regulation; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Ischemia; Kidney; Kidney Cortex; Kidney Tubules, Proximal; Male; Mercuric Chloride; Rabbits; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; RNA, Messenger

We examined the thrombolytic properties of a novel modified human tissue plasminogen activator (PA) (E6010), in which cysteine 84 is replaced by serine, and which has a prolonged biologic half-life (t1/2). We compared the thrombolytic efficacy of continuous intracoronary (i.c.) infusion of E6010 with that of recombinant human tissue PA (rt-PA) in a canine model with copper coil-induced 1-h-old coronary artery thrombi and also compared the relation between thrombolytic efficacy and plasma clearance represented by pharmacokinetic parameters of i.c.-infused E6010 and rt-PA. Sixty-minute E6010 and rt-PA i.c. infusions were compared. The thrombolytic effects of i.c.-infused E6010 and rt-PA, represented by time to reperfusion (TR), reperfusion rate at 60 min (RR), and reocclusion rates at 60 min after reperfusion (OR) were as follows. E6010: Dose 0.06, 0.15, 0.3 (mg/kg/h); TR 25 +/- 10, 15 +/- 10, 13 +/- 5 (min); RR 100, 100, 100 (%); and OR 0, 0, 17 (%), respectively. Recombinant t-PA: Dose 0.06, 0.15, 0.3 (mg/kg/h); TR 47 +/- 12, 18 +/- 17, 14 +/- 4 (min); RR 50, 75, 100 (%); and OR 100, 33, 33 (%), respectively. These findings indicate that E6010 has more potent thrombolytic activity than rt-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha-2-Antiplasmin; Animals; Antibodies, Monoclonal; Coronary Thrombosis; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibrinogen; Fibrinolytic Agents; Half-Life; Humans; Infusions, Intra-Arterial; Plasminogen; Recombinant Proteins; Reperfusion Injury; Tissue Plasminogen Activator

Epidermal growth factor (EGF) protects gastric mucosa against many injurious factors, but no study has examined whether EGF may protect against ischemia-reperfusion (I-R)-induced gastric mucosal injury. We assessed the effect of EGF pretreatment on the extent of ischemia-reperfusion-induced gastric mucosal damage in rats. Assessment of injury and protection included: the extent of macroscopic necrosis; qualitative and quantitative histology with measurement of deep mucosal necrosis; microvascular permeability after injection of fluorescein-conjugated albumin; and transmission electron microscopy. After I-R, saline-pretreated rats (placebo group) had macroscopic necrosis involving 40 +/- 6% of total gastric mucosal area. Histology revealed exfoliation of the surface epithelial cells, mucosal hemorrhages, microvascular injury, and extensive deep mucosal necrosis involving 7 +/- 5% of mucosal strips. Microvascular permeability assessed by fluorescein-conjugated albumin was significantly increased to 327 +/- 29% of that in normal rats (without ischemia-reperfusion). Transmission electron microscopy showed severe microvascular injury. EGF pretreatment significantly reduced gross mucosal necrosis to 17 +/- 6% and deep histologic mucosal necrosis to 2 +/- 1% (both p < 0.01 versus saline pretreated). Integrity of the mucosal microvessels was preserved and microvascular permeability was close to normal. This study demonstrates that EGF significantly reduces ischemia-reperfusion injury to the rat gastric mucosa and that this effect of EGF may be mediated by its protection of the mucosal microvessels.

Topics: Animals; Capillaries; Epidermal Growth Factor; Gastric Mucosa; Ischemia; Male; Microscopy, Electron; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury