Low Baseline Expression of Fibrotic Genes in an Ex Vivo Human Skin Model is a Potential Indicator of Excessive Skin Scarring

Abstract

One of the challenges plastic surgeons face is the unpredictability of postoperative scarring. The variability of wound healing and subsequent scar formation across patients makes it virtually impossible to predict if a patient's surgery will result in excessive fibrosis and scarring, possibly amounting to keloids or hypertrophic scars. There is a need to find predictive molecular indicators of patients or skin location with high risk of excessive scarring. We hypothesized that baseline expression levels of fibrotic genes in the skin can serve as a potential indicator of excessive scarring.

Methods: An ex vivo model of skin fibrosis was used with abdominal and breast skin tissue from 45 patients undergoing breast reduction and/or abdominoplasty. Fibrosis was induced in skin explants in organ culture with transforming growth factor-β (TFGβ). Fibrotic gene response was assessed via quantitative real-time polymerase chain reaction and correlated with skin location, age, and baseline levels of fibrotic genes.

Results: The increase in TFGβ-induced fibronectin1 (FN1) gene expression in skin explants was significantly higher than for Collagen 1A1, alpha smooth muscle actin, and connective tissue growth factor. Also, FN1 expression positively correlated with donor age. Moreover, lower expression of the fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin correlated with a more pronounced fibrotic response, represented by higher induction levels of these genes.

Conclusions: Skin sites exhibit different baseline levels of profibrotic genes. Further, low baseline expression levels of fibrotic genes FN1, Collagen 1A1, and alpha smooth muscle actin, in donor skin may indicate a potential for excessive scarring of the skin.

Fig. 1.

Skin processing steps followed to set up organ culture. A, Skin was obtained from skin resection (abdominoplasty in image). B, Adipose tissue was removed to isolate the skin layer containing the epidermal and dermal layers. C, Equal-sized punches were taken from the skin layer using a 3-mm biopsy punch. D, Skin explants were placed in a 35-mm culture dish with cell culture media, forming an air–liquid interface, in which treatments with TGFβ or vehicle control were added.

Fig. 2.

TGFβ successfully established a fibrotic phenotype in ex vivo human skin in organ culture. RT-qPCR was used to measure TGFβ-induced FC in gene expression of COL1A1 (A), FN1 (B), CTGF (C), and ACTA2 (D) at 48 h, compared with vehicle control (normalized to 1), all normalized to the housekeeping gene GAPDH. Microscopic images of H&E-stained sections of skin explants treated with vehicle control (E) or TGFβ (F) for 120 h, with graphical presentation of their ECM bundle density (G). Data are shown as individual values along with the mean ± SEM. Scale bar = 100 µm. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

FN1 levels show a difference in baseline gene expression based on skin location. RT-qPCR was used to measure baseline gene expression of FN1 (A) and COL1A1 (B), both normalized to the housekeeping gene GAPDH, in breast skin vs abdominal skin. Data are shown as individual values along with the mean ± SEM. *P < 0.05.

Fig. 4.

FN1 and COL1A1 show differences in fibrotic responses based on skin location. RT-qPCR was used to measure TGFβ-induced FC in gene expression of FN1 (A) and COL1A1 (B), both normalized to the housekeeping gene GAPDH, in breast skin vs abdominal skin. Data are shown as individual values along with the mean ± SEM. *P < 0.05.

Fig. 5.

Collective data from breast and skin tissue show opposing trends in baseline expression versus fibrotic response of COL1A1, FN1, and ACTA2. Baseline expression (A) and fibrotic response (FC in expression) data (B) for COL1A1, FN1, ACTA2, and CTGF from both breast and abdominal tissue were pooled and compared. Data are shown as individual values along with the mean ± SEM. **P < 0.05, ****P < 0.0001.

Fig. 6.

Fibrotic responses of FN1, COL1A1, and ACTA2 are inversely correlated to their baseline expression. Correlation analysis was performed using Pearson correlation coefficient to investigate the relationship between baseline expression and fibrotic response (FC in expression) of FN1 (A), COL1A1 (B), and ACTA2 (C). P values in red show significance (P < 0.05).