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

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

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

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

Tumor-Preferential Induction of Immune Responses and Epidermal Cell Death in Actinic Keratoses by Ingenol Mebutate

<div><p>The rapid and strong clinical efficacy of the first-in-class, ingenol mebutate, against actinic keratosis (AK) has resulted in its recent approval. We conducted the first comprehensive analysis of the cellular and molecular mode of action of topical ingenol mebutate 0.05% gel in both AK and uninvolved skin of 26 patients in a phase I, single-center, open-label, within-patient comparison. As early as 1 day after application, ingenol mebutate induced profound epidermal cell death, along with a strong infiltrate of CD4⁺ and CD8⁺ T-cells, neutrophils, and macrophages. Endothelial ICAM-1 activation became evident after 2 days. The reaction pattern was significantly more pronounced in AK compared with uninvolved skin, suggesting a tumor-preferential mode of action. Extensive molecular analyses and transcriptomic profiling of mRNAs and microRNAs demonstrated alterations in gene clusters functionally associated with epidermal development, inflammation, innate immunity, and response to wounding. Ingenol mebutate reveals a unique mode of action linking directly to anti-tumoral effects.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01387711" target="_blank">NCT01387711</a></p></div

Ingenol mebutate 0.05% gel treatment causes rapid infiltration of T-cells, macrophages, and neutrophilic granulocytes.

<p>Biopsy specimens from all patients (<i>n</i> = 26) were subjected to histopathological evaluation. Skin tissues from the two treatment areas and all time points were assessed, and representative images are depicted. The panels depict hematoxylin & eosin staining as well as immunohistochemical staining for CD4⁺ T-lymphocytes, CD8⁺ T-lymphocytes, CD68⁺ macrophages/histiocytes, and myeloperoxidase (MPO⁺) neutrophils as indicated. The scale bar represents 100 μm.</p

Topical treatment with ingenol mebutate 0.05% gel affects expression of gene clusters relevant for inflammatory and wound healing responses, and induces a characteristic microRNA (miRNA) pattern.

<p>The profiles illustrated are heat-maps and 2-way hierarchical clusterings of deregulated genes of <b>(A)</b> the 95 most variable mRNAs, and <b>(B)</b> the 64 most variable miRNAs across samples (<i>n</i> = 24) for the first six patients in the study. Differentially expressed genes and miRNAs were identified by pair-wise comparison of the following groups: actinic keratosis day 0 (AK0) versus uninvolved skin day 0 (US0), AK2 versus AK0, US2 versus US0, and AK2 versus US2. The expression analysis included variance filtering (VAR >0.2), statistical significance test by analysis of variance (<i>P</i> < 0.01), expression cut-off (>2-fold), and the false-discovery-rate was controlled by the Benjamini-Hochberg procedure (q < 0.05). Microarray data can be found in the GEO repository (GSE63107). Arrows indicate mRNA genes and miRNAs that were selected for validation by quantitative polymerase chain reaction. The colors above the heat map indicate: US0 (dark blue, <i>n</i> = 6), US2 (light blue, <i>n</i> = 6), AK0 (green, <i>n</i> = 6), and AK2 (red, <i>n</i> = 6) samples, respectively. The red and green shades on the heatmap represent up- and down-regulated genes, respectively.</p

Ingenol mebutate 0.05% gel activates cutaneous blood vessels and induces epidermal cell death.

<p>Biopsy specimens from both treatment areas of all patients (<i>n</i> = 26) and all time points were assessed immunohistochemically for expression of CD20⁺ B-lymphocytes, ICAM-1 (CD54), and CD1a⁺ cells. Cleaved caspase 3 and TdT-mediated dUTP-biotin nick end labeling (TUNEL) reactivity indicating apoptotic responses were detected in five of these patients (arrows indicate examples of TUNEL positive epidermal cells). The figure depicts representative slides from all stainings as indicated. The scale bar represents 100 μm.</p