Figure 4

Severely delayed endothelial response during diabetes-impaired wound healing. A, Expression of the endothelial marker CD31 during wound healing is shown for Balb/c mice (wt) and diabetic mice (db/db) as indicated. Total cellular RNA (20 μg) from nonwounded and wounded back skin of Balb/c mice (wt) or diabetic mice (db/db) was analyzed by RNase protection assay at the indicated time points after injury for the presence of CD31 mRNA (left panels). Sixteen wounds (n = 16) from the backs of four animals were excised for each experimental time point and used for RNA isolation. ctrl skin refers to nonwounded skin of control and db/db mice. One representative experiment is shown; 1000 cpm of the hybridization probe were used as a size marker (probe). Expression of GAPDH mRNA is shown as a loading control. The degree of regulation as assessed by PhosphoImager analysis of the radiolabeled gels is shown schematically in B. For B, note that every experimental time point represents a total of 32 wounds (n = 32) from two independent animal experiments. C and D, Total protein (50 μg) from lysates of nonwounded and wounded back skin (Days 1, 3, 5, 7, and 13 after injury, as indicated) isolated from Balb/c mice (wt) and diabetic mice (db/db) as indicated were analyzed by immunoblotting for the presence of CD31 specific protein. Eight wounds (n = 8) from the backs of four animals were excised for each experimental time point and used for protein isolation. ctrl skin refers to nonwounded skin. CD31 protein is indicated by an arrow.