epidermal-growth-factor and Ischemia

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

Polypeptide growth factors and gangliosides can both be considered as trophic agents involved in almost all stages of neural cell development, differentiation, survival, and pathology. In most cases their physiological roles are still not clear due to the considerable complexity in their regulation. Several growth factors [e.g., basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)] and one species of ganglioside (GM1) have been shown to exert interactions with each other and also to exhibit neuroprotective effects against retinal ischemia in vivo and cerebral excitotoxicity in vitro. Different experimental models are used to investigate their relevance to ischemic and excitotoxic conditions in the retina, and it is shown that (1) both bFGF and EGF show very effective neuroprotection for rat retinal neurones exposed to toxic levels of glutamate or its nonphysiological agonist kainate in vitro; (2) GM1 (10(-5M) used under the same conditions does not afford protection; (3) retinal glial cells also suffer morphological perturbations following glutamate or kainate treatment, but this effect is dependent on neuron-glial interactions, indicating the existence of intermediate neuron-derived messenger molecules; (4) these glial changes can be corrected by posttreatment with either bFGF or EGF in vitro; (5) using an in vivo animal model involving anterior chamber pressure-induced ischemia in adult rats, it is shown that either pretreatment by intraperitoneal injection of GM1, or posttreatment by intraocular injection of the same ganglioside, reduces significantly histological damage to inner nuclear regions; and (6) in cultured retinal Müller glial cells the existence of molecular and metabolic interactions between both types of trophic factors is demonstrated. Hence both these groups of trophic molecules show interesting features for retinal ischemic treatment.

Topics: Animals; Epidermal Growth Factor; Fibroblast Growth Factor 2; G(M1) Ganglioside; Gangliosides; Growth Substances; Ischemia; Neuroglia; Neurons; Neuroprotective Agents; Rats; Retina; Retinal Vessels

Polypeptide growth factors regulate kidney development, growth, and function and participate in processes of repair after renal injury. The use of one or more growth factors as therapeutic agents has been proposed in the settings of acute and chronic renal failure. In animal models of acute renal injury, the administration of epidermal growth factor, insulin-like growth factor I (IGF-I), or hepatocyte growth factor accelerates the restoration of kidney function and the normalization of histology post-acute renal injury and reduces mortality. The mechanisms by which the growth factors act in acute renal failure include the stimulation of anabolism, the maintenance of glomerular filtration, and the enhancement of tubular regeneration. IGF-I has been safely administered to humans with chronic renal failure. The growth factor enhances GFR and RPF in these individuals. Further studies will be required to establish a role for IGF-I or other growth factors as therapeutic agents for acute renal failure in humans and to define the utility of IGF-I as a medical therapy for chronic renal insufficiency.

Topics: Acute Kidney Injury; Animals; Epidermal Growth Factor; Glomerular Filtration Rate; Growth Substances; Hepatocyte Growth Factor; Insulin-Like Growth Factor I; Ischemia; Kidney; Kidney Failure, Chronic; Rats; Regeneration

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

Background/aim: This study aimed to investigate the effects of apoptosis-inducing Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP 3) and antiapoptotic epidermal growth factor (EGF) on the pathophysiology of experimental low-flow priapism. Materials and methods: Twenty-four adult Sprague-Dawley rats were divided into four equal groups. Group I was the control group. Ischemic priapism was induced for 4 h in Group II rats. In Group III, intraperitoneal EGF at 10 µg/kg was given for 7 days before induction of ischemic priapism for 4 h. In Group IV, intraperitoneal EGF at 20 µg/kg was given for 7 days before induction of ischemic priapism for 4 h. The western blot method was used to determine BNIP 3 expression levels and the TUNEL method was used to determine the apoptotic cells in the cavernosal tissue samples. Results: Although BNIP 3 expression levels were significantly higher in all three study groups compared to the controls, BNIP 3 was significantly higher in EGF-administered groups when compared to Group II (P < 0.05). The TUNEL score of group II was significantly higher than those of the other groups. Conclusion: Decreased apoptosis in cavernosal tissues obtained by antagonizing the apoptotic effect of BNIP 3 with EGF may facilitate the development of new conservative treatment methods via those pathways.

Topics: Animals; Apoptosis; Disease Models, Animal; Epidermal Growth Factor; Ischemia; Male; Membrane Proteins; Mitochondrial Proteins; Penile Erection; Penis; Priapism; Rats, Sprague-Dawley

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

Topics: Aldosterone; Animals; Apoptosis; Calcineurin; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Humans; Ischemia; Leukocytes, Mononuclear; Lymphokines; Male; Myocytes, Cardiac; Neovascularization, Physiologic; Nitric Oxide Synthase; Platelet Transfusion; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Proteins; Receptors, Angiotensin; Signal Transduction

Diabetes is increasing in the world and causes severe cardiovascular complications. Diabetes-induced limb ischemia leads to foot amputation and therapeutic remedies are urgently needed. Here we report that local injection of mesenchymal stem cells (MSCs) prestimulated with epidermal growth factor (EGF) restored blood flow and vasculogenesis in the ischemic hind-limb of type II diabetic (db(-)/db(-)) mice. Bone marrow cells from db(-)/db(-) mice are altered as evidenced by increased oxidative stress and reduced Akt and adhesion molecules when compared with control (db(-)/db(+)). Femoral artery ligation-induced ischemia was performed in the hind-limb of db(-)/db(-) and db(-)/db(+) mice for 28 days. Enhanced green fluorescent protein (EGFP)-MSCs stimulated+/-exogenous EGF for 24 h were injected locally into the ischemic muscle. Blood flow measured with MoorLDI-Laser and microangiography assessed with X-ray showed 100% recovery in db(-)/db(+) compared to 50% recovery in db(-)/db(-) mice. Interestingly, db(-)/db(-) mice had 60 and 96% blood flow recovery and 61 and 98% of vasculogenesis when treated with MSCs alone or MSCs modified with EGF, respectively. Western blot analysis of hind-limb muscles revealed an increase in Akt and vascular endothelial growth factor receptor phosphorylation and hypoxia-inducible factor) expression in db(-)/db(-) mice injected with MSCs or MSCs+EGF compared to db(-)/db(-) mice. Fluorescent microscopic images show that EGFP-MSCs differentiate into new microvessels. Adhesion and migration of MSCs on cultured endothelial cells were ICAM1-, VCAM1- and Akt-dependent mechanism and elevated when MSCs were prestimulated with EGF compared with nonstimulated MSCs. Our novel study data provide evidence that in type II diabetes, stimulated MSCs with EGF enhance the recovery of blood flow and angiogenesis.

Topics: Animals; Bone Marrow Cells; Cell Adhesion; Cell Movement; Diabetes Mellitus, Type 2; Endothelial Cells; Epidermal Growth Factor; Hindlimb; Hypoxia-Inducible Factor 1; Ischemia; Male; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred C57BL; Multipotent Stem Cells; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A

Bone marrow mononuclear cell (BM-MNC) implantation (BMI) for critical severe limb ischemia especially for Buerger's disease shows excellent clinical results but the mechanism of this treatment is still unknown. In this study, we investigated the changes in serum levels of angiogenesis-related factors after BMI treatment.. Twelve patients whose BMI treatments were clinically very effective was selected out of ninteen cases, nine patients had Buerger's disease, two patients had arteriosclerosis obliterans and one had systemic sclerosis. Venous bood from femoral vein or brachial vein of the recipient limbs of these patients.. Adrenomedulin (AM), soluble vascular cell adhesion molecule-1 (sVCAM-1), and C-reactive protein (CRP) serum levels 24 h after BMI treatment were significantly increased compared with those before BMI treatment (p < 0.05). Vascular endothelial growth factor (VEGF) serum levels after BMI treatment significantly increased between 1 week and 3 months after BMI treatment (p < 0.05). Nitric oxide (NO) serum levels after BMI treatment increased significantly 2 weeks after BMI treatment (p < 0.05). There was no correlation between the numbers of implanted cells and serum levels of measured angiogenesis-related factors that were significantly increased after BMI treatment.. It was concluded that the mechanism underlying BMI treatment consists of early and late phases. The early phase involves the direct action by implanted cells, and the late phase involves indirect paracrine action. In addition, it was considered that BMI treatment is effective when we implant a sufficient level of bone marrow (600 ml) to treat severe limb ischemia.

Topics: Adrenomedullin; Adult; Aged; Arterial Occlusive Diseases; C-Reactive Protein; Convalescence; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Follow-Up Studies; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Hepatocyte Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Ischemia; Leg; Male; Middle Aged; Nitric Oxide; Peripheral Vascular Diseases; Postoperative Period; Transplantation, Autologous; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

This study was designed to evaluate the effect of recombinant human erythropoietin (rhEPO), insulin-like growth factor-1 (rhIGF-1) and epidermal growth factor (rhEGF) on ischemia-induced hair cell loss in an organotypic cochlea culture. The apical, middle and basal parts of the organs of Corti (newborn rat, postnatal days 3-5) were exposed to ischemia (3.5 h) in glucose-free artificial perilymph (pO2 10-20 mmHg) with or without growth factors. Controls were exposed to normoxia. Twenty-four hours after the onset of ischemia, the cultures were stained using tetramethyl rhodamine isothiocyanate (TRITC) phalloidin (hair cells), propidium iodide (membrane integrity) and apoptosis detection kit (DNA-fragmentation). Ischemia (3.5 h) induced a hair cell loss of 20 and 40% in the middle and basal cochlear parts, respectively, and an increase of the numbers of PI-stained and DNA-fragmented nuclei (controls 0-1, ischemia 4-7 nuclei/100 microm). The basal part was more affected than the apical one. rhEPO and rhIGF-1 significantly attenuated the ischemia-induced hair cell loss by reducing processes involved in apoptosis and necrosis. rhEPO has been in clinical use for more than a decade and found to be well tolerated. Therefore, rhEPO could be an effective drug for the prevention of hearing loss via a hair cell protective mechanism.

Topics: Animals; Animals, Newborn; Apoptosis; Epidermal Growth Factor; Erythropoietin; Hair Cells, Auditory; Humans; In Vitro Techniques; Insulin-Like Growth Factor I; Ischemia; Necrosis; Organ of Corti; Rats; Recombinant Proteins

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent mitogen for smooth muscle cells and has been implicated in atherosclerosis, tissue regeneration after ischemia, vascular development, and tumor angiogenesis. We examined the hypothesis that HB-EGF participates in angiogenesis and collateral growth in ischemia.. During 3 weeks after femoral artery ligation, no attenuation occurred in recovery of hindlimb perfusion or distal saphenous artery flow in HB-EGF-null (HB-EGF(-/-)) versus wild-type mice. Lumen diameters of remodeled collaterals in gracilis muscle were similar by morphometry (87+/-8 versus 94+/-6 microm) and angiography, although medial thickening was reduced. Gastrocnemius muscle underwent comparable angiogenesis (41% and 33% increase in capillary-to-muscle fiber ratio). Renal renin mRNA, arterial pressure, and heart rate during anesthesia or conscious unrestrained conditions were similar between groups. These latter findings validate comparisons of perfusion data and also suggest that differences in arterial pressure and/or renin-angiotensin activity are not masking an otherwise inhibitory effect of HB-EGF absence. Four days after ligation, EGF receptor phosphorylation increased in muscle by 104% in wild-type but by only 30% in HB-EGF(-/-) mice. This argues against compensation by other EGF receptor ligands.. Our results suggest that HB-EGF is not required for arteriogenesis or angiogenesis in hindlimb ischemia.

Topics: Animals; Cardiomegaly; Epidermal Growth Factor; ErbB Receptors; Femoral Artery; Heparin-binding EGF-like Growth Factor; Hindlimb; Intercellular Signaling Peptides and Proteins; Ischemia; Ligation; Mice; Mice, Mutant Strains; Muscle, Skeletal; Neovascularization, Physiologic; Phenotype; Phosphorylation

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

Discovery of the common and ubiquitous molecular targets for the disruption of angiogenesis, that are independent of the characteristics of malignant tumors, is desired to develop the more effective antitumor drugs. In this study, we propose that the platelet-derived growth factor receptor-alpha (PDGFRalpha)-p70S6K signal transduction pathway in mesenchymal cells, which is required for functional angiogenesis induced by fibroblast growth factor-2, is the potent candidate. Using murine limb ischemia as a tumor-free assay system, we demonstrated that p70S6K inhibitor rapamycin (RAPA) targets mesenchymal cells to shut down the sustained expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), via silencing of the PDGFRalpha-p70S6K pathway. Irrespective of the varied expression profiles of angiogenic factors in each tumor tested, RAPA constantly led the tumors to dormancy and severe ischemia in the time course, even associated with upregulated expression of VEGF from tumors. Because RAPA showed only a minimal effect to hypoxia-related expression of VEGF in culture, these results suggest that RAPA targets the host-vasculature rather than tumor itself in vivo. Thus, our current study indicates that the PDGFRalpha-p70S6K pathway is an essential regulator for FGF-2-mediated therapeutic neovascularization, as well as for the host-derived vasculature but not tumors during tumor angiogenesis, via controlling continuity of expression of multiple angiogenic growth factors.

Topics: Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Hepatocyte Growth Factor; Hindlimb; Humans; Ischemia; Liver Neoplasms, Experimental; Male; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Nude; Mouth Neoplasms; Neoplasm Proteins; Neovascularization, Pathologic; Neovascularization, Physiologic; Platelet-Derived Growth Factor; Receptor, Platelet-Derived Growth Factor alpha; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stromal Cells

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

Topics: Animals; Blotting, Western; Epidermal Growth Factor; ErbB Receptors; Female; Ischemia; Kidney; Models, Animal; Pregnancy; Rats; Rats, Wistar; Uterus

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

The high-affinity 67-kd laminin receptor (67LR) is expressed by proliferating endothelial cells during retinal neovascularization. The role of 67LR has been further examined experimentally by administration of selective 67LR agonists and antagonists in a murine model of proliferative retinopathy. These synthetic 67LR ligands have been previously shown to stimulate or inhibit endothelial cell motility in vitro without any direct effect on proliferation. In the present study, a fluorescently labeled 67LR antagonist (EGF(33-42)) was injected intraperitoneally into mice and its distribution in the retina was assessed by confocal scanning laser microscopy. Within 2 hours this peptide was localized to the retinal vasculature, including preretinal neovascular complexes, and a significant amount had crossed the blood retinal barrier. For up to 24 hours postinjection, the peptide was still present in the retinal vascular walls and, to a lesser extent, in the neural retina. Non-labeled EGF(33-42) significantly inhibited pre-retinal neovascularization in comparison to controls treated with phosphate-buffered saline or scrambled peptide (P < 0.0001). The agonist peptide (Lam beta 1(925-933)) also significantly inhibited proliferative retinopathy; however, it caused a concomitant reduction in retinal ischemia in this model by promoting significant revascularization of the central retina (P < 0.001). Thus, 67LR appears to be an important target receptor for the modulation of retinal neovascularization. Agonism of this receptor may be valuable in reducing the hypoxia-stimulated release of angiogenic growth factors which drives retinal angiogenesis.

Topics: Animals; Epidermal Growth Factor; Hypoxia; Ischemia; Laminin; Mice; Mice, Inbred C57BL; Molecular Weight; Neovascularization, Pathologic; Peptide Fragments; Receptors, Laminin; Retinal Diseases; Retinal Vessels

We studied control of the epidermal growth factor (EGF) receptor and its ligands during kidney injury, since they may be importantly involved in repair.. The folic acid model of renal injury was used in these studies. Messenger RNA (mRNA) was evaluated by solution hybridization. Immunohistochemistry of transforming growth factor alpha (TGF-alpha) was also performed.. Twenty-four hours after folic acid induced acute renal injury, creatinine increased from 0.3 +/- 0.03 mg/dl in controls to 2.0 +/- 0.8 mg/dl in folic acid injured kidneys (n = 4, p < 0.03). mRNA for the EGF receptor was increased nearly sevenfold by 24 h, and mRNA for the receptor was increased as early as 1 h following folic acid treatment. EGF receptor ligand caused a profound downregulation of the receptors in proximal tubule basolateral membranes, but receptors returned rapidly to the membrane surface in injured kidneys. The mRNA levels for heparin-binding EGF and TGF-alpha, two EGF receptor ligands, increased within 1 h of injury. TGF-alpha mRNA increased from 1.0 +/- 0.09 (relative densitometry units) in control animals to 2.9 +/- 0.13 in folic acid treated rats at 24 h (n = 4, p < 0.01), and immunohistochemical staining for TGF-alpha increased in injured kidneys at distal nephron sites.. These studies indicate that upregulation of the EGF receptor is related to an increase in mRNA. The rapid return of receptors to the membrane surface following ligand stimulation may be useful in maintaining a mitogenic stimulus. Multiple EGF-like ligands may be important in activating the upregulated EGF receptor during repair from renal injury.

Topics: Acute Disease; Animals; Creatinine; Epidermal Growth Factor; ErbB Receptors; Folic Acid; Gene Expression; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Ischemia; Kidney Diseases; Ligands; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor alpha

Topics: Animals; Animals, Genetically Modified; Chronic Disease; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Ischemia; Kidney Diseases; Kidney Tubules; Nephrectomy

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

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

Genes differentially expressed after acute renal ischemic injury were identified using differential display-polymerase chain reaction (DD-PCR). Messenger RNA for calcyclin, a member of the S100 family of calcium-binding proteins, is increased in kidneys by 6 h following ischemic injury to rats compared with sham surgery. The level of calcyclin mRNA is increased 10-fold by 1 day postinjury and declines thereafter. In situ hybridization demonstrates little calcyclin mRNA in kidneys of sham-operated rats. However, calcyclin protein is present in glomeruli and distal tubules (DT). Compared with kidneys from sham-operated controls, both calcyclin mRNA and protein expression are increased at 1-3 days following ischemic injury in the thick ascending limb of Henle, the DT, and in damaged regenerating segments of proximal tubules. By 7 days postischemia there is a reduction in mRNA and protein expression. Calcyclin could play a role in the regulation of renal cell proliferation and regeneration in the recovery process after acute ischemic injury.

Topics: Animals; Calcium-Binding Proteins; Cell Cycle Proteins; Cloning, Molecular; Epidermal Growth Factor; In Situ Hybridization; Ischemia; Kidney; Male; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; S100 Calcium Binding Protein A6; S100 Proteins; Transcription, Genetic

Renal ischemic injury evokes an inflammatory response with increased cytokine and major histocompatibility complex (MHC) expression and a mild interstitial infiltrate. This "injury response" could contribute to the tendency of ischemically injured renal transplants to reject. The studies presented here evaluated the ability of recombinant human insulin-like growth factor-1 (rhlGF-1) given after renal injury to prevent renal inflammation. The left renal pedicle of CBA and BALB/c mice was clamped for 60 min, and rhlGF-1 (25, 50, 100 micrograms) was administered sc at 2, 24, 48, 72, and 96 h after reflow. Cytokine and MHC expression was monitored in the injured kidney, compared with the contralateral kidney. In untreated mice, a single episode of injury induced the expression of MHC mRNA and products and tumor necrosis factor-alpha (TNF-alpha) mRNA, and depressed preproepidermal growth factor (ppEGF) mRNA, for up to 5 wk. With immunohistology, epithelial Class I and II MHC expression was shown to be increased for 2 wk, and Class II positive interstitial cells were shown to be increased for up to 5 wk. The ischemically injured kidneys from mice treated with rhlGF-1 and examined at 5 days showed a dose-dependent normalization of all of the changes of the injury response. This included prevention of the increased expression of MHC and cytokines and the Class II positive interstitial cells, and restoration of ppEGF mRNA. Thus the complex and long-lasting increase in proinflammatory cytokines and MHC expression that follow renal ischemia can be interrupted by treatment with rhlGF-1 beginning 2 h after the injury. This therapy may have applications to the injury response in renal transplants.

Topics: Animals; Base Sequence; Blotting, Northern; Epidermal Growth Factor; Gene Expression Regulation; Graft Rejection; H-2 Antigens; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Ischemia; Kidney; Kidney Transplantation; Male; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Molecular Sequence Data; Nephritis; Polymerase Chain Reaction; Protein Precursors; Recombinant Proteins; Tumor Necrosis Factor-alpha

To define the relationship between renal epidermal growth factor (EGF) expression and thyroid hormones in acute renal failure, we performed an analysis of the renal thyroid hormone-EGF axis following acute ischemic renal injury in rats. Levels of mature EGF extractable from kidney were elevated 24 h postinjury, and levels of membrane-associated EGF precursor were reduced. Administration of triodothyronine (T3) to rats, either prior to or immediately following the induction of injury, did not further increase levels of extractable EGF. Levels of EGF mRNA in kidneys were reduced 24 h following acute ischemic damage and not affected by administration of T3. Enhanced production of mature EGF from EGF precursor occurred in membranes isolated from kidneys of rats 24 h postinjury compared with production in membranes from kidneys of normal rats. In addition, levels of thyroxine 5'-deiodinase activity in renal membranes were increased 24 h following injury. Levels of circulating total thyroxine (T4), free T4, and free T3 were reduced postischemic injury. Total T3 was unchanged. The administration of T3 to normal rats increased renal 5'-deiodinase activity and EGF precursor cleavage. Administration of propylthiouracil to rats inhibited renal 5'-deiodinase activity and prevented the increase in extractable EGF postischemic injury. We conclude that the increase in levels of mature EGF extractable from kidneys of rats postischemic injury results from enhanced activity of the serine protease that cleaves the EGF precursor. This activity may be stimulated by T3 produced in kidney. These alterations in renal T4 metabolism and EGF expression could serve to facilitate recovery of renal function following ischemia.

Topics: Acute Disease; Acute Kidney Injury; Animals; Epidermal Growth Factor; Hyperthyroidism; Iodide Peroxidase; Ischemia; Kidney; Male; Propylthiouracil; Rats; Rats, Sprague-Dawley; Renal Circulation; RNA, Messenger; Serine Endopeptidases; Thyroxine; Time Factors; Triiodothyronine

Growth factors such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and hepatocyte growth factor have been shown to accelerate the recovery from postischemic acute renal failure (ARF) with a concomitant increase in DNA synthesis. Interactions between growth factors have been demonstrated in a number of in vitro studies. This study examined the effect of exogenous IGF-1 on the DNA synthesis and EGF receptor (EGF-R) activation in postischemic rat kidneys. Thirty minutes after the relief of 30-minute total occlusion of the left renal artery in anesthetized 225 to 300 gm Sprague-Dawley rats, either IGF-1 (75 micrograms/kg) or normal saline solution (NS, 0.2 ml) was given by intravenous bolus, followed by twice daily subcutaneous injections of IGF-1 (50 micrograms/kg) or 0.2 ml NS for 4 days, respectively, in IGF-1-Tx) and NS treated (NS-Tx) groups (n = 8 each). On the day after the completion of treatment, inulin clearance (ml/kg/min) of the postischemic kidneys in the IGF-1-Tx group was significantly higher (p < 0.01) than inulin clearance of kidneys in the NS-Tx group. This was associated with improved kidney morphology. IGF-1 treatment also enhanced the labeling index of 5-bromo-2'-deoxyuridine (percent of stained tubule cells), a marker for active DNA synthesis, in the outer medulla of postischemic kidneys at 1 day and 2 days after the injury. EGF-R tyrosine phosphorylation (which reflects receptor activation) increased in postischemic kidneys in both NS-Tx (n = 5) and IGF-1-Tx (n = 3) groups 1 day after the injury as compared with nonischemic contralateral kidneys. In the IGF-1-Tx group there was also increased iodine 125-labeled EGF binding and EGF-R protein. Our results demonstrate a beneficial effect of IGF-1 on postischemic ARF. Furthermore, they suggest that EGF-R activation is involved in tubular regeneration and that IGF-1 may enhance EGF-R activation by increasing EGF-R expression.

Topics: Acute Kidney Injury; Analysis of Variance; Animals; Blood Pressure; Body Weight; DNA; Epidermal Growth Factor; ErbB Receptors; Insulin-Like Growth Factor I; Iodine Radioisotopes; Ischemia; Kidney Medulla; Kidney Tubules; Male; Phosphotyrosine; Rats; Rats, Sprague-Dawley; Regeneration; Renal Circulation; Tyrosine

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

Epidermal growth factor (EGF) is a small polypeptide belonging to a class of molecules that can mediate cell growth, differentiation, and acute phase responses. EGF mRNA is transcribed primarily in cells of the salivary gland and the kidney. We have found that the tissue and cellular specificities of EGF gene expression are controlled by a promoter region located upstream from the start of mRNA transcription. In a variety of experimentally induced forms of acute renal failure, the mRNA and protein levels for kidney EGF fall markedly and remain low for a prolonged period. This decrease was determined by nuclear runoff transcription to be a consequence of diminished transcription of the EGF gene rather than increased instability of the mRNA. Using transgenic mice, we found the effect of renal ischemia on EGF mRNA transcription to be a result of a disease-mediated interruption of the function of this upstream promoter region, presumably from alterations in the activity of one or more cellular trans-acting factors.

Topics: Acute Kidney Injury; Animals; Base Sequence; Epidermal Growth Factor; Ischemia; Kidney; Male; Mice; Mice, Transgenic; Molecular Probes; Molecular Sequence Data; Polymerase Chain Reaction; Promoter Regions, Genetic; Rats; Rats, Sprague-Dawley; Reference Values; Renal Circulation; Transcription, Genetic

We noted previously that ischemic acute tubular necrosis (ATN) induces local expression of MHC products in renal epithelium. The present investigations were conducted to establish the role of IFN-gamma in the regulation of MHC antigen expression in ATN and to explore the changes in cytokine and growth factor expression induced by ischemic renal injury. We produced unilateral ischemic ATN in mice by clamping the left renal pedicle. MHC class I and II steady state mRNA induction was assessed by northern blot analysis, and MHC product was quantified by the extent of binding of radiolabeled monoclonals to tissue homogenates. The steady state mRNA levels for IFN-gamma, IL-2, IL-10, and granulocyte-macrophage CSF were assessed by reverse transcriptase polymerase chain reaction and the levels for transforming growth factor-beta 1 and prepro-epidermal growth factor (ppEGF) were assessed by Northern blot analysis. In the injured kidneys, steady state mRNA levels for IFN-gamma, IL-2, IL-10, granulocyte-macrophage CSF, and transforming growth factor beta-1 were increased, whereas ppEGF mRNA was markedly decreased. The MHC expression was inhibited by treatment of mice with an anti-IFN-gamma mAb (R4-6A2). Murine EGF, administered in an attempt to accelerate recovery, did not reduce the cytokine and MHC changes. These data indicate that ischemic injury, and possibly other forms of injury, triggers a complex circuit of proinflammatory cytokines. This "injury response" could be relevant to clinical renal transplants, where ATN is associated with poor graft outcome.

Topics: Animals; Antibodies, Monoclonal; Base Sequence; Cytokines; Disease Models, Animal; DNA, Complementary; Epidermal Growth Factor; Histocompatibility Antigens; Interferon-gamma; Ischemia; Kidney; Kidney Tubular Necrosis, Acute; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Base Sequence; Cytokines; DNA Primers; Epidermal Growth Factor; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Inflammation; Interferon-gamma; Interleukin-10; Ischemia; Kidney; Major Histocompatibility Complex; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Molecular Sequence Data; Reference Values; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The reversibility of ischemic renal injury is dependent on epithelial cell regeneration and repopulation of the nephron. Renal cells produce and respond to many growth factors. In the rat, epidermal growth factor (EGF) is mitogenic for tubular cells and accelerates renal recovery after ischemia. We used a pig renal autotransplant model to evaluate the effect of exogenous EGF on renal recovery in a large animal more analogous to man. Group 1 animals underwent left autotransplant after 120 minutes of warm ischemia and received either a single intra-arterial dose of recombinant human EGF (EGF, 10(-7) M.) (N = 11) or vehicle alone (N = 6). Group 2 animals underwent left autotransplant after 72 hours of cold preservation with Collins' solution and received a similar intra-arterial dose plus a subcutaneous dose of EGF (0.5 ml. of 10(-3) M.) (N = 8) or vehicle alone (N = 6). Contralateral nephrectomy was performed in all animals. Daily creatinine measurements revealed no beneficial effect from EGF on recovery of renal function in Group 1 or 2 animals. Studies of EGF on pig proximal tubular cells demonstrated in vitro mitogenesis; autoradiography with 125I-EGF revealed binding of EGF throughout the kidney. Immunohistochemistry showed significant tubular cell proliferation in response to ischemic injury, without further enhancement from EGF. Thus, although exogenous EGF bound to pig kidney cells and stimulated cell proliferation, we were unable to demonstrate a clinically significant acceleration of recovery from ischemic injury.

Topics: Animals; Cell Division; Cold Temperature; Creatinine; Epidermal Growth Factor; Hot Temperature; Ischemia; Kidney; Kidney Transplantation; Male; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Swine

The time course for the increases in soluble renal epidermal growth factor (EGF) after ischemia has been established. These elevated levels of EGF have been compared with the degree of tissue injury as well as the extent of cell proliferation in the recovering tissue. Levels of soluble immunoreactive EGF (irEGF) in control animals were 9.74 +/- 1.1 ng/g wet wt (n = 4-8 for all values) and rose to 83.9 +/- 30 ng/g within 12 h after injury. Soluble irEGF content peaked at 88.8 +/- 15 ng/g at 24 h postinjury and returned to control values by 72 h. We previously reported that trypsin digestion of crude renal membranes (CRM) generates rat EGF that is indistinguishable from that isolated from the submandibular gland. Initial levels of trypsin-releasable membrane-associated irEGF were 439 +/- 26 ng/g. These levels fell to 46.6 +/- 9.6 ng/g at 48 h after injury. The total renal EGF demonstrated an 80% decline 48 h after injury but returned to 50% of the initial values after 72 h representing significant new synthesis of EGF-containing proteins between 48 and 72 h postinjury. Immunohistochemical staining of kidney paraffin sections for EGF immunoreactivity demonstrated staining intensities that paralleled the amount of irEGF in the trypsin-digested CRM fraction, suggesting that the membrane-associated irEGF is the predominant form detected by this technique. Regenerative hyperplasia subsequent to tubular insult was monitored by immunostaining nuclei of S phase cells after pulse labeling with the thymidine analogue 5-bromo-2'-deoxyuridine. Cell proliferation was particularly prominent in the outer stripe of outer medulla of kidneys exposed to ischemia and reached a maximum (19-fold higher than the baseline value) 48 h after reperfusion. Renal cell turnover returned to control values by day 7. The observation that the peak in soluble EGF levels (24 h) precedes the peak in tubular regeneration (48 h) by 24 h is consistent with the hypothesis that EGF is one of the mitogenic signals triggering regenerative hyperplasia after renal injury.

Topics: Acute Kidney Injury; Animals; Epidermal Growth Factor; Hyperplasia; Immunohistochemistry; Ischemia; Kidney; Kidney Tubules; Male; Necrosis; Rats; Rats, Sprague-Dawley; Regeneration; Renal Circulation

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

An initial event in cell response to growth factors is the change in the state of phosphorylation of a number of cellular proteins playing a role in cell proliferation. The effects of a single dose of exogenously administered epidermal growth factor (EGF) on renal serine/threonine protein kinases such as ribosomal protein S6 kinase(s) and protein kinase C (PKC) and on [3H]thymidine incorporation into tubule cell nuclei have been studied during the regenerative repair response after temporary renal ischemia in the rat, followed by reperfusion for up to 72 h. During the postischemic reflow, the PKC and S6 kinase activities increased at 24 and 72 h, respectively. EGF anticipated both increases: the PKC at 4 and the S6 kinase(s) at 24 h. Associated with this EGF-induced rise of S6 kinase activity, a significant increase in renal tubule cell proliferation was observed. These studies suggest the presence of a growth factor-activated serine/threonine phosphorylation cascade in the rat kidney participating in the regulation of cell growth during recovery from an ischemic insult.

Topics: Animals; Cell Division; Enzyme Activation; Epidermal Growth Factor; Ischemia; Kidney; Male; Protein Kinase C; Protein Serine-Threonine Kinases; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases; Subcellular Fractions; Thymidine

Kidneys of newborn (but not adult) mice are normally high permissive for polyomavirus (Py) infection and readily establish persistent infections. We have proposed that ongoing cellular differentiation, which occurs in newborn mice, may be necessary for a high level of in vivo Py replication (R. Rochford, J. P. Moreno, M. L. Peake, and L. P. Villarreal, J. Virol. 66:3287-3297, 1992). This cellular differentiation requirement may also be necessary for the reactivation of a persistent Py kidney infection and could provide an alternative to the accepted view that reactivation results from immunosuppression. To examine this proposal, the ability of adult BALB/c mouse kidneys to support primary acute Py infection or to reactivate previously established persistent Py infections after kidney-specific damage was investigated. Kidney damage was induced by both chemical (glycerol, cisplatin, or methotrexate) and mechanical (through renal artery clamping to produce unilateral renal ischemia) treatments. We also examined the effects of epidermal growth factor (EGF), which enhances the rate of kidney regeneration, on Py replication. Using histopathologic techniques, in situ hybridization for Py DNA, and immunofluorescence for Py VP1 production, we established that both chemical damage and damage through renal artery clamping of adult kidneys promoted high levels of primary Py replication in these normally nonpermissive cells. This damage also promoted the efficient reactivation of Py replication from persistently infected kidneys, in the absence of immunosuppression. EGF treatment significantly increased acute Py replication and also reactivation in damaged kidneys. These results support the view that ongoing cellular division and differentiation may be needed both for high levels of acute Py replication and for reactivation of persistent infections in vivo.

Topics: Acute Disease; Animals; Capsid; Capsid Proteins; Chronic Disease; Cisplatin; Epidermal Growth Factor; Epithelium; Fluorescent Antibody Technique; Glycerol; Ischemia; Kidney Tubules; Methotrexate; Mice; Mice, Inbred BALB C; Polyomavirus; Tumor Virus Infections; Virus Activation

We have established an in vitro model of ischemia incorporating the combination of anoxia with glucose deprivation, which is toxic to PC12 cells. In this model, nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) improve PC12 cell survival. K252a, a specific inhibitor of NGF-induced trk p140 autophosphorylation, did not alter the neuroprotection provided by EGF or bFGF, yet it completely abolished the protection provided by NGF. Activation of protein kinase A (PKA) with dibutyryl-cAMP also protected during ischemia, although it was not additive with the effect provided by growth factors. Furthermore, growth factors protected a PKA-deficient mutant as effectively as the parental cell line; thus, activation of PKA is protective against ischemia but is not necessary for the action of peptide growth factors. Neither the stimulation of protein kinase C (PKC) with acute phorbol ester treatment nor the downregulation of PKC with chronic high-dose phorbol ester treatment resulted in an altered response to growth factors in either the PC12 wild type or PKA-deficient mutant. Thus, protection by peptide growth factors depends on neither PKA nor PKC. Furthermore, downregulation of PKC alone was protective, indicating that PKC may contribute to toxicity. Interestingly, treatment with the kinase inhibitor H-7 was neuroprotective and may have enhanced the neuroprotective effect of NGF. In contrast, staurosporine, a broadly acting kinase inhibitor, inhibited the neuroprotective effect of NGF, but not of EGF or FGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Anisomycin; Cell Hypoxia; Cell Survival; Cyclic AMP-Dependent Protein Kinases; Epidermal Growth Factor; Fibroblast Growth Factor 2; Ischemia; Isoquinolines; Kinetics; Models, Biological; Neoplasm Proteins; Nerve Growth Factors; PC12 Cells; Phosphorylation; Piperazines; Protein Kinase C; Staurosporine; Time Factors

We have characterized the distribution of immunoreactive epidermal growth factor (irEGF) in control and ischemia-injured rat kidneys. Kidneys that had undergone ischemic injury contained levels of soluble irEGF that were six times those of uninjured kidneys. The predominant forms of soluble irEGF were native and des-Arg-epidermal growth factor (EGF), both of which are biologically active. Crude membrane fractions from whole kidneys were solubilized in Triton X-100 and tested for irEGF. Amounts of irEGF were slightly decreased in the ischemia-injured kidney membranes. However, when solubilized membrane fractions were digested with trypsin, which generates a single immunoreactive species which appears identical to native EGF, the amount of irEGF in control fractions increased 13-fold and the amount in injured fractions increased only 4-fold as measured by radioimmunoassay. To better characterize the membrane-associated irEGF, Triton X-100-solubilized membrane fractions from control animals were affinity purified and subjected to high-performance liquid molecular sieve chromatography. Three major peaks of material exhibited immunoreactivity to EGF antibodies, bound the EGF receptor, and stimulated [3H]thymidine incorporation in growth-arrested fibroblasts. Trypsin digestion of the two high-molecular-mass peaks enhanced these activities. The third peak eluted with native EGF and showed no change in activity with trypsin addition. We propose that EGF is released from membrane-associated EGF precursors and can then act in an autocrine or paracrine fashion to promote cell growth after ischemia-induced acute renal failure.

Topics: Acute Kidney Injury; Animals; Cell Membrane; Chromatography, High Pressure Liquid; Cytosol; DNA; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Ischemia; Kidney; Male; Octoxynol; Polyethylene Glycols; Protein Precursors; Radioimmunoassay; Radioligand Assay; Rats; Rats, Sprague-Dawley; Submandibular Gland; Trypsin

The 35-kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca(++)-dependent substrate for the EGF receptor tyrosine kinase, binds Ca++ and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole-kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays.

Topics: Animals; Annexin A1; Epidermal Growth Factor; Immunohistochemistry; Ischemia; Kidney; Kidney Tubular Necrosis, Acute; Mitosis; Rats; Reference Values; Reperfusion; Tissue Distribution

Epidermal growth factor (EGF) protects gastric mucosa against a variety of injurious agents, but the mechanism is unclear. Because the abnormal microvasculature of portal hypertensive (PHT) gastric mucosa is a major target of ischemia/reperfusion (I/R) injury, we used this model to assess EGF's protective role at the microvascular level.. Rats with PHT (staged portal vein ligation) received either EGF, 20 micrograms/kg, or saline solution intravenously, with or without indomethacin pretreatment (20 mg/kg subcutaneously). I/R was produced by withdrawing blood to systemic pressures of 30 mm Hg for 20 minutes and reinfusing it. Stomachs were excised 20 minutes later and evaluated for gross and histologic necrosis, microvascular permeability, mucosal ultrastructure and vimentin, and cyclooxygenase immunofluorescence.. In saline-treated rats, gross and histologic damage involved 46% +/- 3% of glandular mucosa and 23% +/- 3% of mucosal sections, respectively. Microvascular permeability was increased 43-fold over that of normal control rats. Vimentin immunofluorescence intensity was reduced to 36% +/- 4% that of normal control rats. EGF pretreatment reduced histologic necrosis to 2% +/- 1% (p less than 0.01). Microvascular permeability and vimentin intensity were almost normalized. Indomethacin partially reversed the mucosal protection induced by EGF.. EGF significantly reduces I/R injury to PHT gastric mucosa. Microvascular endothelia of PHT gastric mucosa are the major target of I/R injury and the site of EGF's protective action. Prostaglandins in part mediate EGF's protective action.

Topics: Animals; Capillaries; Endothelium, Vascular; Epidermal Growth Factor; Fluorescent Antibody Technique; Gastric Mucosa; Hypertension, Portal; Ischemia; Prostaglandins; Rats; Rats, Inbred Strains; Vimentin

Epidermal growth factor (EGF) receptors were detected in plasma membrane preparations of equine hoof wall laminar tissue at concentrations comparable to that of equine liver. Scatchard analysis of the equilibrium binding data suggested the presence of two classes of EGF binding sites in most of the controls (plasma membranes from clinically normal horses); a high-affinity class and a more numerous low-affinity class. The dissociation constant of the low-affinity class of EGF-specific receptors (KD = 1 x 10(-9)M) is in reasonable agreement with other values established for the EGF receptor. The variability between individual estimates for the KD of the high-affinity receptor class precluded an accurate estimate for those sites. A possible explanation is discussed. The high-affinity binding sites were uniformly absent in plasma membranes prepared from horses affected by chronic laminitis. Autoradiographic analysis localised the EGF receptors primarily to the secondary epidermal laminae, with an apparent greater density over the proliferative basal keratinocytes. Little label was associated with the dermal or the keratinised primary epidermal laminae. Tissue from horses with chronic laminitis had EGF receptors located uniformly over the hyperplastic epidermal keratinocytes. These data suggest that an EGF-mediated response may be involved in the hyperproliferative response that is characteristic of chronic laminitis.

Topics: Animals; Autoradiography; Binding Sites; Binding, Competitive; Cell Membrane; Chronic Disease; Epidermal Growth Factor; ErbB Receptors; Foot Diseases; Hoof and Claw; Horse Diseases; Horses; Ischemia; Liver; Spleen

Temporary renal ischemia is followed by increased DNA synthesis and cell division as the kidney restores the continuity of the renal epithelium. We sought to characterize some of the changes in proto-oncogene and growth factor expression during this proliferative response. Northern analysis of polyadenylated RNAs of kidney cortical and outer stripe of outer medullary tissue from male Sprague-Dawley rats was performed following release of renal hilar clamping of 50 minutes duration. Ischemia produced an increase in c-fos mRNA that reached a peak at one hour and declined rapidly to control levels by four hours after release of the clamp. A similar rapid increase and decrease in early growth response 1 (Egr 1) mRNA was noted. The response of these immediate early genes was typical of their response to mitogens, suggesting that they served a similar role in renal cell regeneration. Levels of c-Ki-ras and glyceraldehyde phosphate dehydrogenase mRNA were unchanged. Renal preproEGF mRNA decreased at two hours, was virtually absent by 24 hours and remained low for at least four days after ischemia. Urinary excretion of EGF fell immediately after release of ischemia and before the decline in preproEGF mRNA or SNGFR, suggesting post-transcriptional affects of ischemia on renal EGF production. EGF excretion returned to only 50% of control by day 21. Specific 125I-EGF binding increased in membrane fractions of cortex, outer medulla and inner medulla as early as 24 hours after release of the clamp. Cortical 125I-EGF binding increased in the proximal tubule but not in the glomerulus.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blotting, Northern; DNA; Epidermal Growth Factor; Gene Expression; Immunoblotting; Ischemia; Kidney; Male; Protein Precursors; Proto-Oncogenes; Rats; Rats, Inbred Strains; RNA, Messenger; Time Factors

To determine the timing and location of renal cell regeneration after ischemic injury to the kidney and to assess whether exogenous epidermal growth factor (EGF) enhances this regenerative repair process to accelerate recovery of renal function, experiments were undertaken in rats undergoing 30 min of bilateral renal artery clamp ischemia followed by reperfusion for varying time intervals. Renal cell regeneration, as reflected by incorporation of radiolabeled thymidine within the kidney, began between 24 to 48 h and reached a peak at 72 h after renal ischemia. As demonstrated by histoautoradiography, renal thymidine incorporation was essentially confined to tubule cells. Morphometric analysis of histoautoradiograph sections of renal tissue demonstrated that the majority of labeled cells were found in renal cortex, but some labeled cells were also located in the inner stripe of the outer medulla, suggesting that injury to medullary thick ascending limbs also occurs in this ischemic model. Exogenous EGF administration produced increases in renal thymidine incorporation compared with non-treated animals at 24, 48, and 72 h after ischemic injury. This accelerated DNA replicative process was associated with significantly lower peak blood urea nitrogen (BUN) and serum creatinine levels, averaging 63 +/- 20 and 3.1 +/- 0.4 mg/dl in EGF-treated ischemic rats compared with 149 +/- 20 and 5.1 +/- 0.1 mg/dl, respectively, in nontreated ischemic rats, and was also associated with a return to near normal BUN and serum creatinine levels in EGF-treated animals approximately 4 d earlier than that observed in nontreated animals. This report is the first demonstration that EGF accelerates the repair process of a visceral organ after an injurious insult.

Topics: Acute Kidney Injury; Animals; Autoradiography; Blood Urea Nitrogen; Constriction; Creatinine; DNA; Epidermal Growth Factor; Ischemia; Kidney; Kidney Tubules; Rats; Rats, Inbred Strains; Regeneration; Renal Artery

A great deal of knowledge has been gained concerning the activation of adenylate and guanylate cyclase in epidermal cells. Adenylate cyclase is activated by 4 different independent receptors-responding respectively to catecholamine (beta), to prostaglandins (E), to histamine (H2), and to adenosine and it phosphorylated derivatives. Upon activation, each of these receptors becomes unresponsive to further stimulation by its specific stimulator. Guanylate cyclase, on the other hand, is activated by histamine (H1) and epidermal growth factor (EGF). Unlike EGF, the histamine activation is extremely rapid (less than 5 minutes). Epidermal cells are permeable (leak) to cyclic GMP but not cyclic AMP. When the skin is traumatized or injured in any way (even by intradermal injection) there is a sudden catastrophic change in the intracellular levels of the cyclic nucleotides (and of ATP). Cyclic AMP rapidly rises to perhaps 5-10 times its normal resting level while cyclic GMP falls to 10-20% of its level in vivo. The rise in cyclic AMP is due to activation of adenylate cyclase while the fall in cyclic GMP is due in major part to activation of cyclic GMP phosphodiesterase (and perhaps the fall in ATP is due to activation of ATPase). The changes in ATP and cyclic AMP can be reversed by incubating the tissue in a buffered salt solution containing glucose, but this does not normalize the cyclic GMP content. The fall in cyclic GMP can be prevented by a phosphodiesterase inhibitor (IBMX ). This series of events has been called the "ischemia effect." However, it implies that a lack of oxygen is at fault, and that has not been shown to be the case. Its underlying cause and possible physiologic significance are not known. Do these changes in cyclic nucleotides have effects on epidermal proliferation? And does EGF? Agents which increase cyclic AMP do inhibit the epidermal outgrowth and mitotic activity of explant cultures of pig skin. Cyclic GMP does increase outgrowth at a particular concentration. Histamine, which elevates both cyclic nucleotides, has a biphasic action depending on its concentration. These findings imply that these nucleotides do act as one of the controls of epidermal proliferation. The action of cyclic GMP is not accompanied by detectably increased phosphorylation of epidermal proteins. On the other hand, EGF action which also enhances epidermal outgrowth is characterized by an increased protein phosphorylation that precedes any increase in cellular cyclic

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Animals; Cell Division; Cyclic AMP; Cyclic GMP; Enzyme Activation; Epidermal Growth Factor; Epidermis; Histamine; Humans; Ischemia; Phosphoproteins; Protein Kinases; Swine