Research ArticleWound Healing

Transcriptional signature primes human oral mucosa for rapid wound healing

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Science Translational Medicine  25 Jul 2018:
Vol. 10, Issue 451, eaap8798
DOI: 10.1126/scitranslmed.aap8798
  • Fig. 1 Comparison of paired oral and skin wounds in human subjects.

    (A) Table and schematic of spatiotemporal human biopsy sample collection (ClinicalTrials.gov #NCT01078467). Baseline biopsies were performed to create paired identically sized wounds in the oral mucosa (blue) and skin (orange) on day 1. Follow-up biopsies of the wound areas were collected on days 3 and 6 of healing in two different groups. (B) Representative hematoxylin and eosin (H&E) pictures of longitudinal sections of biopsies taken at day 1. Thirty healthy subjects were randomized in three groups (with 10 subjects on each group). SM, smooth muscle. (C) Representative pictures and quantitation of healing time course of oral wounds and skin wounds in group 1 after a 3-mm primary biopsy and of group 2 after a 5-mm secondary biopsy. Markings on the dental periodontal probe are in millimeters. Biopsy sites were demarcated using blue polypropylene sutures. Data are mean values, and error bars represent SDs. Number of samples for each group are as follows: 3-mm wounds, day 1, n = 29; day 3, n = 30; day 6, n = 20; day 9, n = 9; day 13, n = 9; day 15, n = 9; 5-mm wounds, day 3, n = 11; day 6, n = 20; day 9, n = 21; day 13, n = 21; day 15, n = 21. Nonparametric two-sided t test. (D) Graphs show the healing rate of 3-mm wounds in group 1 and of 5-mm wounds in group 2 (top) and percentage of contraction immediately after the 3-mm biopsy in group 1 and after the 5-mm biopsy in group 2 (bottom). n.s., not significant. *P < 0.05 by unpaired t test. Error bars represent SDs. (E) Representative H&E pictures of oral and skin wounds at days 3 and 6. Magnification of dotted boxes is shown on the right. In magnification, epithelium is marked with a dotted line.

  • Fig. 2 Wound-activated transcriptional networks are present in the unwounded oral mucosa.

    (A) Schematic representation of biopsy site in the mucosa of the cheek and posterior axillary region of the arm (left) and unsupervised clustering analysis of RNA-seq gene expression data of the 24 paired samples at days 1, 3, and 6. O, oral; S, skin. Numbers indicate matching subjects. Paired oral and skin samples were chosen randomly from four subjects for each day (24 total samples from 12 individual subjects) and were a mix of males and females. (B) Circular ideogram plot (CIRCOS) of the differential gene expression during wound healing (ANOVA). No significant differences were found in oral biopsies taken at day 6 versus day 1 (day 6/day 1). Ribbon connectors indicate the same genes present in different data sets. Number of genes with differential expression in each comparison: oral day 3 versus day 1 (day 3/day 1), 410 genes; skin day 3 versus day 1 (day 3/day 1), 1473 genes; skin day 6 versus day 1 (day 6/day 1), 1836 genes. See fig. S1A for explanation on CIRCOS plot. RPKM, reads per kilobase million; |FC|, absolute fold change. (C) Volcano plot indicating differential gene expression between unwounded (day 1) oral mucosa and skin. Dotted line region is magnified on the right panel, highlighting some of the most significantly up-regulated genes in the oral mucosa compared to the skin. P < 0.05 by paired t test. (D) Ingenuity Pathway Analysis (IPA) showing diseases and functions terms in up-regulated genes in the oral mucosa compared to the skin. (E) Unsupervised hierarchical clustering using a psoriasis gene signature with the gene expression of oral mucosa and skin data at baseline (day 1) and during wound healing (days 3 and 6).

  • Fig. 3 Oral keratinocytes show reduced differentiation during wound healing.

    (A) CIRCOS plot summarizing cross-reference of transcripts up-regulated during skin wound healing (skin day 3/day 1 and skin day 6/day 1) with those up-regulated in the oral mucosa with respect to the skin at basal conditions (oral day 1/skin day 1). The black line indicates genes up-regulated in the unwounded oral mucosa that are up-regulated during skin wound healing (250 unique genes; see table S2). Ribbon connectors indicate that the same genes are present in different data sets. Right: Graph indicates GO biological process terms enriched in the oral day 1/skin day 1 data set. Oral day 1/skin day 1, 760 genes; skin day 3/ day 1, 971 genes; skin day 6/day 1, 1089 genes. B&H, Benjamini and Hochberg’s; FDR, false discovery rate. (B) Relative mRNA expression of keratinization and epidermal cell differentiation markers throughout the wound healing process. (C) Representative pictures of unwounded (day 1) and wounded (days 3 and 6) oral mucosa and skin stained to show expression of the basal cell marker keratin 5 (K5, red) and differentiation markers keratin 4 (K4) for the oral mucosa and keratin 10 (K10) for the skin (green). Magnification of the dotted box is shown on the right of each picture. (D) Representative pictures of unwounded (day 1) and wounded (day 3) oral mucosa and skin stained to show expression of the basal marker cytokeratin 5 (K5, red) and activated epithelium keratin 6 (K6, green). (E) Representative pictures of unwounded (day 1) and wounded (days 3 and 6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, green) and proliferation marker proliferating cell nuclear antigen (PCNA; red). Quantification of number of cells positive for the proliferation marker PCNA per area (mm2). **P < 0.01 by unpaired t test, oral versus skin day 6. No asterisk means not statistically different between oral and skin. Data are mean values, and error bars represent SDs. n = 3 independent samples. For day 1, magnification shows the basal, unwounded expression of corresponding marker. For days 3 and 6, magnification shows the migratory tongue or wound area (I) or an adjacent epithelial area to the wound (II). D1, day 1; D3, day 3; D6, day 6.

  • Fig. 4 Inflammatory pathways are more active and sustained in skin wounds than in oral wounds.

    (A) IPA analysis showing diseases and functions terms found in differentially regulated genes during the wound healing process relative to day 1, including terms related to inflammatory processes. (B) CIRCOS plot showing the genes exclusively up-regulated during skin wound healing (black line) and GO biological process terms enriched in this data set. Ribbon connectors indicate that the same genes are present in different data sets. Oral day 3/day 1, 276 genes; skin day 3/day 1, 971 genes. (C) Relative mRNA expression of interleukins, chemokines, and other inflammatory regulators during wound healing. (D) Representative pictures of recruitment of immune cells during the wound healing process in the oral mucosa and the skin. Bottom: Quantification of recruitment of specific immune cell types during the wound healing process in the oral mucosa and the skin. *P < 0.05, **P < 0.01 by unpaired t test. No asterisk means not statistically different, comparisons between days 3 and 6 versus day 1 (D1) oral or skin, respectively. Data are mean values, and error bars represent SDs. n = 3. MPO, myeloperoxidase. (E) Representative images of unwounded (day 1) and wounded (day 3) oral mucosa and skin stained to detect expression of the basal marker keratin 5 (K5, red) and the immune modulators secretory leukocyte peptidase inhibitor (SLPI) (green) and ANXA1 (magenta). Magnification of dotted boxes is shown on the right of each picture.

  • Fig. 5 Transcriptional networks in oral keratinocytes contribute to rapid wound resolution.

    (A) Relative mRNA expression of genes consistently up-regulated in oral mucosa and oral keratinocytes, presented by fold change of the unwounded oral mucosa with respect to the unwounded skin (O1/S1) in the wound healing data set. (B) Western blot of PAX9, PITX2, PITX1, and SOX2 in primary cultures of human oral (NOK) and skin (NHEK) keratinocytes. n = 2. (C) Representative pictures of unwounded (day 1) and wounded (days 3 and 6) oral mucosa and skin stained to show expression of the basal marker keratin 5 (K5, red) and the indicated transcription factor (SOX2, PITX1, or PAX9; green). Magnification of the dotted boxes is shown on the right of each picture. For day 1, magnification shows the basal, unwounded expression of corresponding marker. For days 3 and 6, magnification shows the migratory tongue or wound area (I) or an adjacent epithelial area to the wound (II).

  • Fig. 6 Knockdown of oral signature genes reduces migratory capacity of primary oral keratinocytes.

    (A) Western blot of SOX2, PITX1, and total protein (RPS14) after transfection with respective siRNAs in NOK cells. (B) IPA analysis of RNA-seq data from NOK cells treated with siRNAs for SOX2 and PITX1: Diseases and functions terms related to migration and cell movement found in differentially regulated genes. P < 0.05 by IPA. (C) Relative cell migration distance of NOK cells transduced with siRNA for SOX2, PITX1, and siControl (siCON). Values were determined from three microscopic fields in n = 3 per group. **P < 0.01 by ANOVA, siSOX2 and siPITX1 versus control. Data are mean values, and error bars represent SDs. Scale bar, 100 μm.

  • Fig. 7 Overexpression of SOX2 and PITX1 increases migratory capacity of primary skin keratinocytes.

    (A) Representative pictures of NHEK cells transduced with indicated adenoviruses and stained to show overexpression of corresponding proteins. Scale bars, 20 μm. (B) GO biological process terms enriched in data sets of genes differentially regulated by PITX1 and SOX2 overexpression in NHEK cells. (C) Fold change of the expression of genes related to differentiation and response to biotic stimulus in NHEK cells transduced with PITX1 and SOX2, presented as log2 fold change (log FC) over green fluorescent protein (GFP) expression. (D) Migrating NHEK cells transduced with SOX2, PITX1, and GFP (control) by adenoviral delivery. Images were taken at 0 and 24 hours after removal of silicone insert. Values were determined by counting the number of migrating cells at 24 hours in six microscopic fields in n = 3 per group. **P < 0.01, *P < 0.05 by ANOVA, adSOX2 and adPITX1 versus control. Data are mean values, and error bars represent SDs.

  • Fig. 8 Conditional overexpression of SOX2 contributes to cutaneous wound healing.

    (A) Schematic representation of the experimental design used for evaluating cutaneous wound healing in K14CreERTM/LSL-SOX2 mice. Mice were treated with vehicle (ethanol) or tamoxifen for five consecutive days by topical application on dorsal skin (day −4 to day 0, red arrow). Wounds were created as a 6-mm full-thickness excisional dorsal skin wound by biopsy punch (day 1). (B) Representative images of unwounded skin stained to show expression of SOX2 (red) and the basal marker keratin 5 (K5, green) in K14CreERTM/LSL-SOX2 mice treated with vehicle or tamoxifen. DAPI (4′,6-diamidino-2-phenylindole) in blue. Epithelium is marked with dotted lines. (C) Photographs of the wound areas after topical treatment with vehicle or tamoxifen in K14CreERTM/SOX2 mice at 1, 3, 5, 7, and 9 days after wounding. Scale bar, 1 mm. (D) Percent wound area at each time point relative to the original wound area in K14CreERTM/LSL-SOX2 mice treated with vehicle or tamoxifen. Quantification of the wound areas in n = 7 wounds per group was performed using ImageJ software. *P < 0.05, tamoxifen versus vehicle at each day. Error bars represent SDs of seven wounds. (E) Representative H&E-stained sections of skin day 1 (unwounded) and day 5 (during wound healing, wound edges including epithelial tongue). Skin sections were from K14CreERTM/LSL-SOX2 mice after treatment with tamoxifen or vehicle. (F) Representative images of unwounded skin stained to show expression of PCNA+ proliferating epithelial cells (red). DAPI in blue. *P < 0.05 by unpaired t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/451/eaap8798/DC1

    Materials and Methods

    Fig. S1. Visualization of up-regulated genes in oral mucosa compared to the skin by CIRCOS plot.

    Fig. S2. Migration-related pathways in wound healing.

    Fig. S3. Control stainings and IVL expression and proliferation in oral and skin wounds.

    Fig. S4. Inflammatory pathways in wound healing.

    Fig. S5. Expression of oral signature genes in wound healing and in primary human oral and skin keratinocytes.

    Fig. S6. Knockdown of oral signature genes in primary oral keratinocytes.

    Fig. S7. Overexpression of SOX2 and PITX1 in primary skin keratinocytes.

    Fig. S8. Detection of GFP expression in K14CreER/LSL-SOX2 mice.

    Fig. S9. Proposed model indicating the main characteristics that improve wound healing in the oral mucosa.

    Table S1. Differentially regulated genes (oral versus skin day 1).

    Table S2. Genes expressed in the unwounded oral mucosa at steady state that are up-regulated in the skin during wound healing.

    Table S3. Significant GOs in wound-associated genes expressed in day 1 oral mucosa.

    Table S4. Individual subject-level data.

    References (4449)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Visualization of up-regulated genes in oral mucosa compared to the skin by CIRCOS plot.
    • Fig. S2. Migration-related pathways in wound healing.
    • Fig. S3. Control stainings and IVL expression and proliferation in oral and skin wounds.
    • Fig. S4. Inflammatory pathways in wound healing.
    • Fig. S5. Expression of oral signature genes in wound healing and in primary human oral and skin keratinocytes.
    • Fig. S6. Knockdown of oral signature genes in primary oral keratinocytes.
    • Fig. S7. Overexpression of SOX2 and PITX1 in primary skin keratinocytes.
    • Fig. S8. Detection of GFP expression in K14CreER/LSL-SOX2 mice.
    • Fig. S9. Proposed model indicating the main characteristics that improve wound healing in the oral mucosa.
    • Legends for tables S1 to S4
    • References (4449)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Differentially regulated genes (oral versus skin day 1).
    • Table S2 (Microsoft Excel format). Genes expressed in the unwounded oral mucosa at steady state that are up-regulated in the skin during wound healing.
    • Table S3 (Microsoft Excel format). Significant GOs in wound-associated genes expressed in day 1 oral mucosa.
    • Table S4 (Microsoft Excel format). Individual subject-level data.

    [Download Tables S1 to S4]

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