High glucose-mediated oxidative stress impairs cell migration

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM Dglucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-AcetylCysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients

Topical Insulin Accelerates Wound Healing in Diabetes by Enhancing the AKT and ERK Pathways: A Double-Blind Placebo-Controlled Clinical Trial

Background: Wound healing is impaired in diabetes mellitus, but the mechanisms involved in this process are virtually unknown. Proteins belonging to the insulin signaling pathway respond to insulin in the skin of rats. Objective: The purpose of this study was to investigate the regulation of the insulin signaling pathway in wound healing and skin repair of normal and diabetic rats, and, in parallel, the effect of a topical insulin cream on wound healing and on the activation of this pathway. Research Design and Methods: We investigated insulin signaling by immunoblotting during wound healing of control and diabetic animals with or without topical insulin. Diabetic patients with ulcers were randomized to receive topical insulin or placebo in a prospective, double-blind and placebo-controlled, randomized clinical trial (NCT 01295177) of wound healing. Results and Conclusions: Expression of IR, IRS-1, IRS-2, SHC, ERK, and AKT are increased in the tissue of healing wounds compared to intact skin, suggesting that the insulin signaling pathway may have an important role in this process. These pathways were attenuated in the wounded skin of diabetic rats, in parallel with an increase in the time of complete wound healing. Upon topical application of insulin cream, the wound healing time of diabetic animals was normalized, followed by a reversal of defective insulin signal transduction. In addition, the treatment also increased expression of other proteins, such as eNOS (also in bone marrow), VEGF, and SDF-1 alpha in wounded skin. In diabetic patients, topical insulin cream markedly improved wound healing, representing an attractive and cost-free method for treating this devastating complication of diabetes.Sao Paulo Research Foundation (FAPESP)Sao Paulo Research Foundation (FAPESP)National Institute of Science and Technology (INCT)National Institute of Science and Technology (INCT)National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical-trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid-reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia. (C) 2009 ISDN. Published by Elsevier Ltd. All rights reserved.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq Brazil).Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia. (C) 2009 Elsevier Masson SAS. All rights reserved.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Brazil)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Oral administration of linoleic acid induces new vessel formation and improves skin wound healing in diabetic rats

Introduction Impaired wound healing has been widely reported in diabetes. Linoleic acid (LA) accelerates the skin wound healing process in non-diabetic rats. However, LA has not been tested in diabetic animals. Objectives We investigated whether oral administration of pure LA improves wound healing in streptozotocin- induced diabetic rats. Methods Dorsal wounds were induced in streptozotocin-induced type-1 diabetic rats treated or not with LA (0.22 g/kg b.w.) for 10 days. Wound closure was daily assessed for two weeks. Wound tissues were collected at specific time-points and used to measure fatty acid composition, and contents of cytokines, growth factors and eicosanoids. Histological and qPCR analyses were employed to examine the dynamics of cell migration during the healing process. Results LA reduced the wound area 14 days after wound induction. LA also increased the concentrations of cytokine-induced neutrophil chemotaxis (CINC-2 alpha beta), tumor necrosis factor-alpha (TNF-alpha) and leukotriene B-4 (LTB4), and reduced the expression of macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1). These results together with the histological analysis, which showed accumulation of leukocytes in the wound early in the healing process, indicate that LA brought forward the inflammatory phase and improved wound healing in diabetic rats. Angiogenesis was induced by LA through elevation in tissue content of key mediators of this process: vascular-endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT-2). Conclusions Oral administration of LA hastened wound closure in diabetic rats by improving the inflammatory phase and angiogenesis1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP446562/2014-9sem informação2012/10653-9; 2013/06810-

Oral Administration of Linoleic Acid Induces New Vessel Formation and Improves Skin Wound Healing in Diabetic Rats.

Impaired wound healing has been widely reported in diabetes. Linoleic acid (LA) accelerates the skin wound healing process in non-diabetic rats. However, LA has not been tested in diabetic animals.We investigated whether oral administration of pure LA improves wound healing in streptozotocin-induced diabetic rats.Dorsal wounds were induced in streptozotocin-induced type-1 diabetic rats treated or not with LA (0.22 g/kg b.w.) for 10 days. Wound closure was daily assessed for two weeks. Wound tissues were collected at specific time-points and used to measure fatty acid composition, and contents of cytokines, growth factors and eicosanoids. Histological and qPCR analyses were employed to examine the dynamics of cell migration during the healing process.LA reduced the wound area 14 days after wound induction. LA also increased the concentrations of cytokine-induced neutrophil chemotaxis (CINC-2αβ), tumor necrosis factor-α (TNF-α) and leukotriene B4 (LTB4), and reduced the expression of macrophage chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1). These results together with the histological analysis, which showed accumulation of leukocytes in the wound early in the healing process, indicate that LA brought forward the inflammatory phase and improved wound healing in diabetic rats. Angiogenesis was induced by LA through elevation in tissue content of key mediators of this process: vascular-endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT-2).Oral administration of LA hastened wound closure in diabetic rats by improving the inflammatory phase and angiogenesis

Effect of insulin on cellular and molecular mechanisms of wound healing in diabetes.

<p>Insulin induces activation of IR/SHC/ERK and IR/IRS/PI3K/AKT pathways in wound healing, which are canonical insulin signaling pathways. On the upper right-hand side, AKT is shown to increase VEGF (probably from macrophages, fibroblasts and epithelial cells) that will induce the phosphorylation and activation of eNOS in bone marrow, with consequent mobilization of EPCs to the circulation. SDF1α induces the homing of these EPC at the injury site, where they participate in neovasculogenesis. Insulin cream increased VEGF and SDF1α tissue expression in wound healing, and also increased eNOS phosphorylation in the bone marrow of an animal model of diabetes.</p

Time-course of IRS-1 and AKT expression following skin wounding in control (A, B) and diabetic animals (C, D).

<p>Tissue protein levels in the intact skin of control (CC) and intact skin of diabetic (DD) rats and in the wounded skin of control (WC) and wounded skin of diabetic animals (WD). Skin and wound extracts from control and diabetic rats were prepared, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036974#s2" target="_blank"><i>Materials and Methods</i></a>, four days after the wound incision. Tissue extracts were immunoblotted with (E) anti-IR antibody, (F) anti-IRS-1 antibody, (G) anti-IRS-2 antibody, (H) anti-SHC antibody, (I) anti-AKT antibody, and (J) anti-ERK1/2 antibody. Equal protein loading was confirmed by reblotting the membranes with anti-β-actin. Data were compared by ANOVA and Bonferroni post-test, and represented by the mean and standard deviation for each group of scanning densitometry of six different animals per group. *P<0.05 between groups.</p