Comparative analysis of wounds from oral mucosa versus skin reveals novel therapeutic targets for rapid wound healing [PreClinical]

1. Comparative profiling of wounds induced in the oral mucosa versus skin revealed expression of genes that prime the oral mucosa for wound healing.

2. Overexpression of one of these genes , SOX2, resulted in accelerated cutaneous wound healing in both cultured skin keratinocytes and in mice.

Evidence Rating Level: 2 (Good)

Study Rundown: The overall process of wound resolution is well known to follow a stereotyped process consisting of four major phases: hemostasis, inflammation, proliferation, and remodeling.  It has long been observed that oral wound heal at a faster rate than skin wounds. This study aimed to determine the differences that exist between oral wound healing versus skin wound healing with the goal of identifying novel therapeutic targets to improve and accelerate wound resolution.

The authors recruited thirty healthy nonsmokers as study subjects. Each subject received a 3-mm punch biopsy both in the mucosa of the cheek and in the posterior axillary region of the arm, and was followed for 15 days. Several subjects received follow-up biopsies at later time points (either at day 3 or day 6). Tracking of wound diameter over time indicated that wounds in the oral mucosa healed significantly faster than skin wounds.

Paired oral and skin samples were then used for gene expression (RNA-seq) analysis. These analyses revealed an overall up-regulation of gene activity in the skin wounds between day 1 and day 6, indicating that the skin wounds had not completely healed by this time point. In contrast to the skin wounds, the transcriptional profiles of the oral biopsies revealed signatures of wound healing at day 3 that had resolved by day 6. Furthermore, there were relatively few changes in gene expression during oral healing, whereas a large number of genes were differentially expressed during skin wound healing. From these results, the authors hypothesized that transcriptional regulatory networks responsible for accelerated healing in the oral mucosa are already present in the unwounded state in this tissue. Supporting this hypothesis, 250 genes were identified that were highly expressed in unwounded oral mucosa, but only upregulated in the skin during wound healing.

Finally, the authors identified transcripts that were overexpressed in human oral mucosa as well as in mouse oral mucosa in vivo, and in human and mouse oral keratinocytes. Eight genes were identified four of which encode transcriptional regulators. Knockdown of these oral signature genes with siRNA resulted in a reduction of the migratory capacity of primary oral keratinocytes, whereas overexpression of SOX2 and PITX1 increased the migratory capacity of primary skin keratinocytes. Consistent with these results, conditional overexpression of SOX2 accelerated cutaneous wound healing in mice. These results suggest that genetic or pharmacologic approaches to increase the expression or activity of transcription factors such as SOX2 may be a viable strategy to accelerate wound healing.

Click here to read about wound healing in the oral mucosa versus skin

Relevant Reading: Wound repair and regeneration

In-Depth [clinical and animal trial]: Thirty participants were recruited for enrollment in this clinical study. Inclusion criteria for enrollment included (1) nonsmoking status (2) only occasional (social) alcohol consumption, and pregnancy was an exclusion criterion. A 3-mm punch biopsy was used to create a wound in the mucosa of the check and posterior axillary region of the arm for each subject. All subjects received an oral and skin biopsy at the day 1 time point. The thirty subjects were then randomized into three equally sized groups. Group 1 subjects received one set of biopsies at their day 1 time point only, group 2 subjects received a second set of biopsies at their day 3 time point, and group 3 subjects received a second set of biopsies at their day 6 time point. For RNA-seq analyses paired oral and skin samples were chosen randomly from four subjects for each day.

The number of differentially expressed genes between conditions was examined. Differentially expressed genes were genes with an RPKM > 1, q < 0.05 and |fold-change| > 2. By these criteria, between skin day 3 and skin day 1 samples, 1473 genes were differentially expressed. Between skin day 6 and skin day 1 samples, 1836 genes were differentially expressed. In contrast, significantly fewer genes were differentially expressed between oral day 3 and oral day 1 samples (410 genes). No significant differences were found in oral biopsies taken at day 6 versus day 1. RNAseq data and staining for inflammatory markers also indicated that inflammatory pathways are more active and sustained in skin wounds than in oral wounds.

Messenger RNAs consistently upregulated in oral mucosa and oral keratinocytes were identified. Western blot of proteins corresponding to these mRNAs was performed in human oral (NOK) and skin (NHEK) keratinocytes. siRNA knockdown of two oral signature genes, SOX2 or PITX1 was performed in NOK cells and confirmed with Western blot and was shown to significantly reduce migratory capacity of primary oral keratinocytes by >20% in each case (P < 0.01). Overexpression of either SOX2 or PITX1 in NHEK cells increased the migratory capacity of these cells by ~200% (P < 0.01).

K14CreERTM/LSL-SOX2 mice were generated for conditional overexpression of SOX2. Mice were treated with vehicle (ethanol) (n=7) or tamoxifen (n=7) for five consecutive days. Cutaneous wounds were created as 6-mm dorsal skin wounds by punch biopsy and tracked over the course of 11 days. Tamoxifen treated mice overexpressing SOX2 had improved wound healing as quantified by wound area (%) over time (P < 0.05).

Image: PD

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