Inhibition of Lipid Peroxidation Restores Impaired Vascular Endothelial Growth Factor Expression and Stimulates Wound Healing and Angiogenesis in the Genetically Diabetic Mouse

Abstract

Impaired wound healing is a well-documented phenomenon in experimental and clinical diabetes. Experimental evidence suggests that a defect in vascular endothelial growth factor (VEGF) regulation might be associated with wound-healing disorders. We studied the involvement of lipid peroxidation in the pathogenesis of altered VEGF expression in diabetes-related healing deficit by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db؉/ db؉ mice and their normal (db؉/؉m) littermates. Animals were then randomized to the following treatment: raxofelast (15 mg ⅐ kg -1 ⅐ day -1 i.p.), an inhibitor of lipid peroxidation, or its vehicle (DMSO/NaCl 0.9%, 1:1 vol: vol). The animals were killed on different days (3, 6, and 12 days after skin injury), and the wounded skin tissues were used for histological evaluation, for analysis of conjugated dienes (CDs), as an index of lipid peroxidation and wound breaking strength. Furthermore, we studied the time course of VEGF mRNA expression throughout the skin-repair process (3, 6, and 12 days after skin injury), by means of reverse transcriptasepolymerase chain reaction, as well as the mature protein in the wounds. Diabetic mice showed impaired wound healing with delayed angiogenesis, low breaking strength, and increased wound CD content when compared with their normal littermates. In healthy control mice, a strong induction of VEGF mRNA was found between day 3 and day 6 after injury, while no significant VEGF mRNA expression was observed at day 12 after injury. In contrast, VEGF mRNA levels, after an initial increase (day 3), were significantly lower in diabetic mice than in normal littermates, and light induction of VEGF mRNA expression was also present at day 12 after injury. Similarly, the wound content of the angiogenic factor was markedly changed in diabetic mice. Administration of raxofelast did not modify the process of wound repair in normal mice, but significantly improved the impaired wound healing in diabetic mice through the stimulation of angiogenesis, re-epithelization, and synthesis and maturation of extracellular matrix. Moreover, raxofelast treatment significantly reduced wound CD levels and increased the breaking strength of the wound. Lastly, the inhibition of lipid peroxidation restored the defect in VEGF expression during the process of skin repair in diabetic mice and normalized the VEGF wound content. The current study provides evidence that lipid peroxidation inhibition restores wound healing to nearly normal levels in experimental diabetes-impaired wounds and normalizes the defect in VEGF regulation associated with diabetes-induced skin-repair disorders. Diabetes 50:667-674, 2001

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse

“…The overproduction of free radicals in diabetes could also cause an impairment in the production and release of the powerful angiogenic factor VEGF. Experimental evidence from our laboratory have indicated that in genetically diabetic mice the mRNA levels of VEGF as well as the wound content of the mature angiogenic factor were severely reduced during the healing process [18]. Furthermore lipid peroxidation inhibition restored wound healing to nearly normal levels and normalized the defect in VEGF regulation associated with diabetes-induced skin-repair disorders [18].…”

“…To evaluate well-formed from poorlyformed capillary vessels the following parameters were considered: presence or absence of oedema, congestion, haemorrhage, thrombosis and intravascular or intervascular fibrin formation. The histological score adopted in this study was evaluated according to data regarding wound healing in experimental models [18,33].…”

“…Healing impairment is characterized by delayed cellular infiltration and granulation tissue formation, reduced angiogenesis, decreased collagen deposition and organization [14,15,16,17]. One of the mechanism underlying the healing impairment in diabetic mice is thought to result from a defect in VEGF regulation, during gene expression [18]. As a result, a reasonable approach to stimulate wound healing could be the topical application of growth factors.…”

Recombinant AAV vector encoding human VEGF165 enhances wound healing

“…15 Alternatively, growth factor production might be impaired at the gene expression level, as we have recently observed for VEGF in the genetically diabetic mouse. 16 Given these considerations, it stems evident that a reasonable approach to alter the microenvironment of the wounded tissue and to stimulate wound healing is the topical application of growth factors. Although the results in animal studies are encouraging in this respect, 17 topical treatment with growth factors in the clinical setting is limited by several factors, such as their short half-lives, their inactivation by wound proteases, their poor bioavailability from the utilized vehicles, and the consequent need for daily applications and high initial doses that might result in toxicity.…”

“…The histological score adopted in this study was evaluated according to literature data regarding wound healing in experimental models. 16 Immunohistochemistry Wounds collected at days 10 and 21 were fixed in 2% formaldehyde, embedded in paraffin, sectioned at 5 m thickness and mounted on to slides. Rehydrated serial paraffin sections were immersed in Tissue Unmasking Solution (Vector Laboratories, Burlingame, CA, USA) to reactivate hidden or masked epitopes and then placed in a microwave for 7 min at 350 W. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min.…”