Positional differences in the wound transcriptome of skin and oral mucosa

Abstract

Background: When compared to skin, oral mucosal wounds heal rapidly and with reduced scar formation. Recent studies suggest that intrinsic differences in inflammation, growth factor production, levels of stem cells, and cellular proliferation capacity may underlie the exceptional healing that occurs in oral mucosa. The current study was designed to compare the transcriptomes of oral mucosal and skin wounds in order to identify critical differences in the healing response at these two sites using an unbiased approach.

Results: Using microarray analysis, we explored the differences in gene expression in skin and oral mucosal wound healing in a murine model of paired equivalent sized wounds. Samples were examined from days 0 to 10 and spanned all stages of the wound healing process. Using unwounded matched tissue as a control, filtering identified 1,479 probe sets in skin wounds yet only 502 probe sets in mucosal wounds that were significantly differentially expressed over time. Clusters of genes that showed similar patterns of expression were also identified in each wound type. Analysis of functionally related gene expression demonstrated dramatically different reactions to injury between skin and mucosal wounds. To explore whether site-specific differences might be derived from intrinsic differences in cellular responses at each site, we compared the response of isolated epithelial cells from skin and oral mucosa to a defined in vitro stimulus. When cytokine levels were measured, epithelial cells from skin produced significantly higher amounts of proinflammatory cytokines than cells from oral mucosa.

Conclusions: The results provide the first detailed molecular profile of the site-specific differences in the genetic response to injury in mucosa and skin, and suggest the divergent reactions to injury may derive from intrinsic differences in the cellular responses at each site.

Figure 1

More genes are significantly expressed in skin wounds than in the tongue wounds. One mm punch wounds were made on BALB/c mice skin and tongue. Wounds and surrounding tissues were harvested at 6 h, 12 h, and days 1, 3, 5, 7, and 10. Total RNA was extracted. Microarray was carried out using Affymetrix GeneChip arrays. A one-way ANOVA was used to identify significantly differentially expressed gene sets across all time points after wounding (FDR < 0.05). Figure shows the numbers of significantly expressed probes at each time point compared to normal tissues.

Figure 2

Numbers of tissue specific and commonly expressed probes in the skin and tongue wounds.

Figure 3

Clusters of differentially expressed probe sets in skin and tongue wounds. K-means clustering of filtered transcripts revealed 5 clusters of behaviors of differentially expressed probe sets in each type of wound that demonstrated similar expression patterns over the course of healing.

Figure 4

Results of k-means clustering projected into the plain of main principal component.

Figure 5

Differential response of skin and mucosal keratinocytes to IL-1β treatment. Primary skin and oral mucosal keratinocytes isolated from adult human were treated with recombinant human IL-1β for 24 hours. Relative mRNA expression (fold change) of IL-6 and TNF-α compared to medium treated cells was determined by real time PCR. * p < 0.01 between skin and mucosal keratinocytes for IL-6 and TNF-α (n = 3).

Figure 6

Wound closure is more rapid in oral wounds. Pictures shown are representatives of H&E stained 1 mm skin and tongue wounds 24 hours after wounding. Wounded areas are circled.

Figure 7

Time line of specific aspects of wound healing in mucosa and skin. The relative timing and abundance of inflammatory cytokines, neutrophils, macrophages, and angiogenesis is shown for mucosal and skin wounds, as well as the time of wound closure (arrows). Mucosal wounds close much more rapidly than skin; all other aspects of repair are temporally similar at the two sites. Overall, the magnitude of the response is much less in mucosa, suggesting that mucosa heals by a simpler restoration process than the inflammatory repair process observed in adult skin. Adapted from references [1,2].