Figure 5.

a: Factors capable of fibronectin breakdown in acute wounds are reduced with estrogen treatment. Fibronectin degradation is decreased in acute wounds of aged humans treated with estrogen compared to placebo. Degradation bands appear as pale regions in the gel. Lane 1, female-estrogen; 2, male-estrogen; 3, female-placebo; 4, male-placebo; 5, female skin; 6, 10 ng human neutrophil elastase. NS, normal skin. Band intensities were quantified using image analysis. Results are depicted as means ± SD. Each bar represents the average of band intensities from n = 5 subjects and thus the gel is a general representation. b: Fibronectin immunostaining is markedly increased at day 7 in estrogen-treated wounds. A marked increase in immunostaining for fibronectin was observed in the wounds of the estrogen-treated subjects for both males and females, compared to placebo. E, epidermis. In b no epithelium was present over the wound but photograph of section was taken below clot. Original magnification, ×25. c: Protease responsible for fibronectin degradation confirmed as elastase in acute wound tissue. Immunoblotting with an anti-human neutrophil elastase antibody confirmed that day 7 healing wounds in placebo-treated groups contained elastase, which localized to the degradation band seen in a parallel zymogram. Experiment shown is representative of n = 8 males, n = 8 females. Tissue from placebo groups contained >50 ng elastase/20 μg dry weight, whereas tissue from estrogen-treated groups contained <50 ng/20 μg dry weight. The sensitivity of this assay was >80 ng elastase, whereas the zymography is known to be a more sensitive assay for protease detection (in Figure 4A ▶ 10 ng elastase can be detected). M, males; F, females; +, estrogen; −, placebo. d: Elastase activity in acute wound tissue is reduced with estrogen treatment. Elastase activity was quantified using a synthetic elastase substrate degradation assay. *P < 0.05; n = 5 for each group. Elastase activity is expressed as ng/ml activity per 20 μg dry weight.