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

The effects of recombinant human erythropoietin (rHuEPO) in diabetes-related healing defects were investigated by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJm ؉/؉ Lept db mice (db ϩ /db ϩ ) and their normoglycemic littermates (db ϩ/ϩ m). Animals were treated with rHuEPO (400 units/kg in 100 l s.c.) or its vehicle alone (100 l). Mice were killed on different days (3, 6, and 12 days after skin injury) for measurement of vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis, for monitoring angiogenesis by CD31 expression, and for evaluating histological changes. Furthermore, we evaluated wound-breaking strength at day 12. At day 6, rHuEPO injection in diabetic mice resulted in an increase in VEGF mRNA expression (vehicle ‫؍‬ 0.33 ؎ 0.1 relative amount of mRNA; rHuEPO ‫؍‬ 0.9 ؎ 0.09 relative amount of mRNA; P < 0.05) and protein wound content (vehicle ‫؍‬ 23 ؎ 5 pg/wound; rHuEPO ‫؍‬ 92 ؎ 12 pg/wound; P < 0.05) and caused a marked increase in CD31 gene expression (vehicle ‫؍‬ 0.18 ؎ 0.05 relative amount of mRNA; rHuEPO ‫؍‬ 0.98 ؎ 0.21 relative amount of mRNA; P < 0.05) and protein synthesis. Furthermore, rHuEPO injection improved the impaired wound healing and, at day 12, increased the wound-breaking strength in diabetic mice (vehicle ‫؍‬ 12 ؎ 2 g/mm; rHuEPO 21 ؎ 5 g/mm; P < 0.05). Erythropoietin may have a potential application in diabetes-related wound disorders.

Aims/hypothesis. We studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates (db+/+m). Methods. Animals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds. Results. We found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strenght of the wound and enhanced the wound content of VEGF. Conclusion/interpretation. Our study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes. [Diabetologia (2003) 46:546-555]

No abstract

In order to ascertain whether erythropoietin plays a role in early and late repair processes following ischaemic skin flap injury, a dorsal, caudally based skin flap was created in rats. The rats were successively divided into four groups. Group 1 was not treated. The other groups were treated with a subcutaneous administration of 0.9% NaCl saline solution (group 2), a subcutaneous administration of vehicle (group 3) or a subcutaneous administration of 300 IU/kg/day of recombinant human erythropoietin (group 4). We evaluated the possible relationships between neutrophil accumulation, myeloperoxidase activity and content in flap tissue, flap survival, flap temperature (using telethermography) and flap revascularization (using videocapillaroscopy). Necrosis in the flap was significantly less extensive in group 4 than in groups 1, 2 and 3. A significant increase in neutrophil infiltration occurred between the 1st and 24th hour in these groups, but this was not observed in group 4. These findings were confirmed by biochemical data of myeloperoxidase activity and malonyldialdehyde content. Between the 1st and 7th days, we recorded an increase of about 20% in flap temperature in groups 1, 2 and 3, whereas no significant variation was observed in group 4. On the 7th day, videocapillaroscopic findings showed an increase in the mean vascularization index in group 4. Our findings suggest that recombinant human erythropoietin administration can improve the wound healing process, in both early and late stages of injury, by reducing inflammatory response, increasing the density of capillaries in ischaemic flaps and allowing earlier repair of a damaged area.