Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity

In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed

PECAM1(+)/Sca1(+)/CD38(+) vascular cells transform into myofibroblast-like cells in skin wound repair.

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1(+)/Sca1(+) vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1(+)/Sca1(+)/CD38(+) cells proliferate upon wounding and could give rise to α-SMA(+) myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1(+)/Sca1(+)/CD38(+) vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1(+)/Sca1(+)/CD38(+) cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma

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