Expression of perivascular and endothelial cell-specific markers in wound repair.

<p>Analysis of PECAM1, desmin, α-SMA and Sca1 expression in the wounded skin. (A) Dorsal view of full thickness wounds on the back skin of mice one (D1), seven (D7) and 14 (D14) days post injury. Representative H&E-stained cryosections of selected wounds (arrowhead) during inflammation (D1), granulation (D7) and remodeling (D14) are shown. (B-D) Immunostaining of (B) PECAM1/desmin, (C) PECAM1/α-SMA or (D) PECAM1/Sca1 expression at the different stages of wound healing. The individual monochrome signals for PECAM1, desmin, α-SMA and Sca1 are shown in overviews. Squares within the images represent closeups of overlays for the PECAM1/desmin, PECAM1/α-SMA PECAM1/Sca1 stainings (B-D). Bars 1 cm (A, top), 1 mm (A, lower panel), 100 µm (B).</p

<i>In situ</i> detection of the CD38 receptor in the maturing skin and wound.

<p>(A) Expression of PECAM1 and CD38 in cryosections of newborn (nb), three weeks (3 wk) and three months (3 mo) old skin was detected by immunofluorescence microscopy. The fluorescence signal for CD38 (top row) and the overlay with PECAM1 is shown. (B) Confocal microscopy analysis of PECAM1/CD38 expression in three weeks old skin. PECAM1⁺ vessels lacking CD38 expression are indicated (arrowheads). (C) Localization of PECAM1 and CD38 expression in cryosections of wounds one (D1), seven (D7) and 14 days (D14) post injury. The fluorescence signal for CD38 (top row), the overlay with PECAM1 and higher magnifications of the wounded area are shown (A, C, squares). Bars 100 µm (A, C), 50 µm (B).</p

Distribution of PECAM1 and Sca1 protein on skin- and wound-derived cells.

<p>(A) Flow cytometry analysis of Sca1 and PECAM1 expression in CD45^- non-hematopoietic cells isolated from newborn (nb), three weeks (3 wk) and three months (3 mo) old dermis. Sca1⁺ (1, red box), PECAM1⁺ (2, green ellipse) and PECAM1⁺/Sca1⁺ (3, black ellipse) cell populations are highlighted. (B) Dot plots of Sca1 and PECAM1 expression in cell suspensions isolated from full thickness wounds one (D1), seven (D7) and 14 days (D14) post injury of eight weeks old mice. Percentage of positive cell populations at the different time points of flow cytometry analysis (lower panel) is given with standard deviation and significant changes were determined using the unpaired two-tailed student’s T-test (n≥3, **p≤0.01, n.s = not significant).</p

Identification of PECAM1⁺/CD38⁺ cells in human basal cell carcinomas (BCC).

<p>(A) H&E-stained cryosection of a BCC. (B–D) Confocal microscopy analysis of PECAM1, CD38 and α-SMA expression in two BCC biopsies (B–C, D). (B) Overview (top row) of the highly vascularized PECAM1⁺ stroma. Squares within the merged image indicate the magnified region (lower row). PECAM1^low/CD38⁺ cells are marked (arrowheads). Bars 100 µm (A), 50 µm (B, C), 10 µm (D).</p

Myofibroblast-like cell formation and modulation of CD38 receptor activity. (A)

<p>Representative cell cycle analysis of skin- and wound-derived Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺ cells seven days post injury using propidium iodide (PI) stain in flow cytometry analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053262#pone.0053262.s001" target="_blank">figure S1</a>). The relative percentage of cells in G1 (green), S (ochre) and G2 (blue) are highlighted. (B) Flow cytometric detection of α-SMA in wound-derived Sca1⁺, PECAM1⁺/Sca1⁺ and PECAM1⁺ cells seven days post injury (n = 7 mice). (C) Immunofluorescence analysis of α-SMA expression in cultured wound-derived Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺ cells. Nucleoli were detected using DAPI stain. (D) Morphometric analysis of wounds in immunodeficient mice stimulated with rat anti-CD38 or isotype matched antibodies (n = 4). Distances between edges of the panniculus carnosus (δ pc), hair follicles (δ A) and the area of the granulation tissue (g) were determined. Statistics: unpaired two-tailed student’s T-test (*p≤0.05, **p≤0.01). Bars 100 µm (C), 500 µm (D).</p

Expression profile of isolated Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺

<p><b>cells.</b> Marker expression in the sorted cell populations isolated from dermis or wounds seven days post injury. (A) Relative mRNA expression levels of perivascular (<i>Desmin</i>, <i>Pdgfrb</i>, <i>Angpt1</i>), endothelial (<i>Angpt2</i>, <i>Tie2</i>, <i>Pecam1</i>), progenitor cell-specific markers (<i>Sca1</i>, <i>Cd34</i>) and of the CD38 receptor (<i>Cd38</i>) were determined by semiquantitative RT-PCR in non-hematopoietic Sca1⁺, PECAM1⁺ and PECAM1⁺/Sca1⁺ cell populations. The band intensities of the electrophoretic gels were processed and quantified using Image J software. The relative expression intensity of the individual genes compared to <i>Gapdh</i> is given. The lack of Sca1 or Pecam1 expression in sorted PECAM1⁺ or Sca1⁺ cells demonstrated the purity of the cell fractions. (B) Flow cytometric detection of TIE2, CD34, CD38 expression at the cell surface of Sca1⁺ (red), PECAM1⁺ (green) and PECAM1⁺/Sca1⁺ (black) cells isolated from dermis or full thickness wounds seven days post injury.</p