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Analyzing the Wound Healing Deficit in Diabetic Mice
Xiaotian Wang, MD, Claire Mckeever, BA, Charles Patterson, BA, Paul Y. Liu, MD.
Dept. of Plastic Surgery, Rhode Island Hospital/Alpert Med School of Brown University, Providence, RI, USA.
PURPOSE: Healing of an open wound is delayed in diabetics. We examined re-epithelialization and contraction in diabetic (db/db) and non-diabetic (db/m) mouse wounds and compared the db/db and db/m dermal fibroblast contractility of collagen gels. We investigated gene expression of α-SMA, which functions in wound contraction; TGF-β transduction pathway, which regulates α-SMA expression, including TFG-β receptor type I, transcription factors SMAD 3 and 7; and analyzed gene expression of wound healing- related growth factors and receptors in dermal fibroblasts.
1. Wound healing: 57 db/db and 64 db/m mice were used. Dorsal paired 8 mm-diameter wounds were created on each mouse. From day 1 to 21 post-wounding, six wounds from each group were harvested daily, sectioned, and H&E stained. Wound histological diameters were measured between dermal edges and epithelial edges using ImageJ. A wound was healed when no histological epithelial gap was found.
2. Collagen gel contraction: Excised skin was cultured to obtain fibroblasts. The fibroblasts were seeded into three-dimension collagen gels, and size recorded for three weeks.
3. Gene expression: The gene expression levels of α-SMA, TGF-β, TGF-β type I receptor, SMAD-3 and SMAD-7 of the fibroblasts were analyzed using real-time RT-PCR relative quantitation assay. Levels of gene expression of bFGF, PDGF, VEGF-A and receptors bFGF-R, PDGF-RB, Flt-1 (VEGFR-1) were also analyzed. Differences in db/db and db/m fibroblast gene expression were analyzed by the Student’s t-test.
1. The contracted wound area in the db/m decreased constantly, while contraction of the wounds in the db/db was slower (p < 0.01). The db/m wounds healed by day 14; db/db by day 20. Comparing epithelialization of db/db and db/m, rates of epithelialization were similar in the first week after wounding, but more of the healing of the diabetic wound was via re-epithelialization.
2. Fibroblasts from the db/db mice failed to contract the collagen gels, while gels seeded with the non-diabetic fibroblasts shrank (p < 0.001) .
3. α-SMA gene expression was lower in the db/db fibroblasts (p < 0.05). No significant difference in expression of TGF-β was detected. A five-fold decrease in expression of TGF-beta type I receptor was noted in the diabetic animals (p<0.01. SMAD-3, a receptor-associated protein which serves as a substrate for TGF-Beta-R-I, shows a seven-fold decrease in expression in db/db (p<0.01). The down-regulation of SMAD-7 (p<0.01), a so-called inhibitory SMAD, could antagonize the signaling effects of other SMADs.
4. Gene expression of bFGF and bFGF-R decreased in db/db fibroblasts (p < 0.01). Similarly, PDGF-B and PDGF-RB gene expression was significantly lower in diabetic fibroblasts (p < 0.05 and p < 0.01). VEGF-A and VEGFR-1 gene expression was lower in db/db fibroblasts than in db/m fibroblasts (p < 0.001 and p < 0.05).
Impaired contraction is part of the reason diabetic wounds heal slowly. This may be due to decreased α−SMA and impaired SMAD signaling, but cytokines and sensitivity to them are also decreased.
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