Gender affects skin wound healing in plasminogen deficient mice.

The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin thickness and composition

The length of the hyperplastic epidermis and dermal wound gap is affected by gender.

<p>Histological analysis of H&E stained sections from fully reepithelialized wounds. (A–D) representative sections of healed wounds from Wt and Plg^−/− mice of both genders. The hyperplastic epidermis is underlined by green. (E & F) The length and average thickness of the hyperplastic epidermis. n = 9–11 for each group.</p

Decreased cell infiltration in fibrin rich areas of wound tissues.

<p>Fibrin(ogen) staining of skin wound sections. The epidermis is underlined by green, fibrin(ogen) is stained brown with NovaRED, and nuclei are stained blue with a hematoxylin counter stain. (A–D) representative stainings of wound tissues from Wt and Plg^−/− mice of both genders. (E & F) enlarged micrographs of areas boxed in B. (G & H) sections from 50% healed wounds in the back skin of male mice, which show the area between the advancing keratinocytes and the underlying muscle tissue Note the lack of cells in this region in the Plg^−/− mice. (I) quantifications of the total area of fibrin(ogen) positive lesions in the provisional matrix and quantifications of the number of cells within and outside this area. n = 9–11 for each group.</p

Ovariectomy does not induce a male like phenotype in plasminogen deficient female mice.

<p>A possible effect of ovarian derived hormones on skin wound healing in Plg^−/− mice was tested by excision of the ovaries before a 20 mm full thickness incisional skin wound was made. (A) Wound lengths were measured using digital calipers every other day until completely healed. No statistical significant difference was found between the different groups belonging to Wt mice. The difference between sham operated male and female Plg^−/− mice were statistically significant (p<0.05), while no significant difference was found between OVX operated Plg^−/− mice and the sham operated Plg^−/− mice. (B) An area under the curve analysis showed no significant effect of OVX in Plg^−/− mice, while the difference between genders was significant. (C) The percentage of mice with completely reepithelialized wounds presented as a function of time. The bar graph shows the average time to complete reepithelialization.</p

Gender does not affect the vessel concentration in fully reepithelialized wounds.

<p>CD34 stainings of fully reepithelialized wounds were performed to identify new vessels. (A–D) CD34 positive vessels were detected in the dermis proximal to the hyperplastic epidermis both in Wt and Plg^−/− mice of both genders. (E) Computerized staining analysis on whole wound sections reveal that Plg^−/− mice had a tendency to express less CD34 (area wise) than Wt mice while gender did not affect the level of CD34 staining.</p

Plasminogen deficiency does not affect the structure of naïve skin.

<p>The effect of Plg deficiency on the known differences in skin histology between genders was investigated using back skin from naïve Plg^−/− mice and Wt controls (8–12 weeks of age). n = 8–12 for each group. (A) The thickness of the dermis and hypodermis was determined by microscopy. (B) Representative skin sections from eight week old male and female mice.</p

Skin wound healing in plasminogen deficient mice is affected by gender.

<p>Skin wound healing was tested by measuring time to complete reepithelialization of a 20 mm long full skin thickness incisional wound on the back. (A) The wound length in FVB mice was determined every other day using digital calipers. A substantial difference was observed between male and female Plg^−/− mice (p<0.0001). (B) The area under the curve analysis revealed a significant difference between genders in the Plg^−/− mice. (C) The fraction of fully reepithelialized wounds is presented as a function of time. The bar graph shows the average time to complete reepithelialization. (D) A wound healing study identical to the one carried out in FVB mice were performed in C57Bl/6 mice. Similar results were obtained in C57Bl/6 mice with a significant difference between wound healing times in male and female Plg^−/− mice (p = 0.01). (E) Representative pictures of skin wounds in Wt and Plg^−/− male mice eight days after the incision.</p