Decorin Isoform Expression in HTS Fibroblasts: the effects of TGF-β1 and IFN-α2b

• Author / Creator

  Eremenko, Elizabeth

• Background: Hypertrophic scars (HTS) are a dermal fibroproliferative disorder that commonly occurs following burn injury. Due to the intricate nature of the wound healing process and an incomplete understanding of the molecular mechanisms involved in HTS formation, current treatments have significant limitations in their efficacy. HTS develop from the excessive synthesis and reduced degradation of structural proteins like collagen, and dysregulation of proteoglycans such as decorin in the extracellular matrix (ECM). Our group has demonstrated that decorin expression is significantly downregulated in HTS, deep dermal tissue, and thermally injured tissue, mitigating its ability to regulate pro-fibrotic transforming growth factor-beta 1 (TGF-β1) and normal fibrillogenesis. However, treatment of HTS fibroblasts with interferon-alpha 2b (IFN-α2b) has been shown to reduce excessive collagen synthesis and improve HTS by reducing serum TGF-β1 levels. The expression of decorin isoforms in HTS is currently unknown and the effects of TGF-β1 and IFN-α2b on decorin and decorin isoform expression are of great interest to our group. We hypothesize that there will be a differential expression of decorin and decorin isoforms in HTS fibroblasts relative to normal skin (NS) fibroblasts. By treating fibroblasts with TGF-β1 and/or IFN-α2b, TGF-β1 is expected to decrease the expression of decorin and decorin isoforms but increase type I collagen expression, whereas treatment with IFN-α2b will inhibit the pro-fibrotic effects of TGF-β1.
  Methods: The gene expression of decorin and decorin isoforms were quantified in HTS fibroblasts and site-matched NS fibroblasts and in superficial dermal fibroblasts (SF) and deep dermal fibroblasts (DF) using a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To determine the effects of TGF-β1 and IFN-α2b, fibroblasts were treated with either 10 ng/mL of TGF-β1 only, 2000 U/mL of IFN-α2b only, both TGF-β1 and IFN-α2b, or PBS as a control for 48 hours. The protein expression of decorin and type I collagen were quantified in fibroblasts with immunofluorescence (IF) staining. Decorin and procollagen type I protein secreted from the fibroblasts were measured in the cell culture media supernatant using an enzyme-linked immunosorbent assay (ELISA).
  Results: The mRNA expression of decorin and each decorin isoform (A1, A2, B, C, D, and E) were significantly reduced in HTS fibroblasts relative to NS. TGF-β1 significantly decreased the mRNA expression of decorin and specific decorin isoforms, whereas IFN-α2b had the opposite effect, in all fibroblast populations. IFN-α2b did not inhibit the effect of TGF-β1 decreasing decorin or decorin isoform mRNA expression in any of the fibroblast populations. TGF-β1 significantly increased the mRNA expression of COL1A1 in all fibroblast populations, the protein synthesis of type I collagen in NS fibroblasts, and the amount of procollagen type 1 present in the cell culture media in both HTS and site-matched NS fibroblasts. Although not significant, IFN-α2b increased the protein synthesis of decorin in HTS and site-matched NS fibroblasts. There were no statistically significant differences in decorin protein secreted into the cell culture media between the different treatment groups in HTS or site-matched NS fibroblasts. IFN-α2b did not significantly reduce the mRNA expression of COL1A1 in any fibroblast population, but IFN-α2b did significantly inhibit the effect of TGF-β1 on the mRNA expression of COL1A1 in SF. The protein expression of type I collagen and the procollagen type 1 secreted into the cell culture media were not significantly reduced after treatment with IFN-α2b and the effects of TGF-β1 were not significantly inhibited by IFN-α2b in HTS or site-matched NS fibroblasts.
  Conclusion: These data support that a further investigation into the structural and functional roles of decorin isoforms in HTS pathogenesis is warranted and that IFN-α2b is an important agent in reducing fibrotic outcomes.

• Subjects / Keywords

  • Hypertrophic Scarring
  • Decorin
  • Interferon-alpha 2b
  • Isoform
  • Transforming Growth Factor-Beta 1

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-ry2m-xj37

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.