Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Leask, Andrew

Affiliation

University of Saskatchewan

Abstract

Fibrotic disorders, including the inflammatory connective tissue disease systemic sclerosis (SSc), contribute to 45% of deaths in the Western world. Currently there is no universally agreed-upon treatment for fibrosis. The CCN family of matricellular proteins are tightly spatiotemporally regulated proteins involved in development and wound healing, and are aberrantly expressed in fibrotic disease, including in SSc. CCN1 and CCN2 are overexpressed in SSc and contribute to the progression of animal models of fibrosis. CCN3 is reciprocally regulated to CCN1 and CCN2 and has been shown to suppress CCN2-mediated fibrogenic activity in kidney fibrosis. This led to the development of therapeutic peptides based on CCN3 (BLR-200), which have anti-fibrotic activity in vitro. The exact mechanisms of CCN protein activity in dermal fibrosis, including the anti-fibrotic ability of CCN3, have not yet been elucidated. Given this, I tested the hypothesis that CCN protein dysregulation permits a pro-fibrotic microenvironment, leading to increased myofibroblast activation and collagen deposition, that can be prevented by treatment with CCN3-based peptides. First, I used cell culture models to investigate CCN regulation in dermal fibroblasts and found that CCN1 and CCN2 were reciprocally expressed to CCN3 through divergent non-canonical TGFβ signalling pathways. Moreover, in cell culture, CCN3 overexpression inhibited CCN2 protein expression in dermal fibroblasts. Second, I used a murine model of bleomycin-induced SSc dermal fibrosis to assess the ability of BLR-200 to prevent fibrosis and impair early fibroblast response to a pro-inflammatory microenvironment. I found that BLR-200 prevented fibrosis in bleomycin-induced SSc fibrosis, as indicated by impairment of skin thickening, collagen deposition, and myofibroblast activation. Furthermore, BLR-200 treatment prevented bleomycin-induced CCN1 and CCN2 expression. Finally, through single-cell RNA-sequencing analysis, I found that BLR-200 impaired the ability of collagen-expressing fibroblasts to respond to bleomycin-induced inflammatory-driven fibrosis. BLR-200 prevented overexpression of pro-inflammatory genes including Il6, Cxcl2, and NLRP3 inflammasome markers. The results presented here suggest that CCN proteins play an important role in dermal fibrosis. Targeting the pro-fibrotic activity of CCN1 and CCN2 using endogenously derived CCN3-based peptides can prevent multiple pro-fibrotic changes and represents a novel therapeutic approach for treatment of SSc fibrosis.

Summary for Lay Audience

Fibrotic diseases such as systemic sclerosis (scleroderma; SSc) currently have no therapy. These diseases are characterized by extensive scar tissue deposition (largely comprised of collagen), resulting in tissue dysfunction, organ failure, and often death. In the scar tissue, excessive mechanical tension is responsible for the maintenance of the scar. The cells within scar tissue, known as fibroblasts, contribute to this mechanical tension and are essential for fibrosis. A group of proteins called Cellular Communication Network (CCN) proteins are also involved in maintenance of this scar tissue. Two members of this group, CCN1 and CCN2, are highly expressed in scar tissue and promote the mechanical tension that drives overaccumulation of scar tissue. A related member of the group, CCN3, is not found in scar tissue, and might act to inhibit the effects of CCN1 and CCN2. In my thesis, I investigate this group of proteins in skin fibrosis, and test whether a therapeutic treatment based on CCN3 can block skin fibrosis in mice. I found that CCN3 inhibited CCN2 in skin fibroblasts, indicating the potential of CCN3 to prevent the fibrotic activity of CCN2 in skin fibrosis. Second, I found that CCN3-based treatment prevented excessive scar tissue deposition and overall fibrotic changes in the skin of mice. Furthermore, CCN3-based treatment inhibited CCN1 and CCN2 in these mice. I also found that CCN3-based treatment impaired the ability of fibroblasts to contribute to the fibrosis. Overall, my thesis contributes to our knowledge of the role of CCN proteins in dermal fibrosis. These results also emphasize the therapeutic potential of CCN3 and CCN3-based therapies for the treatment of skin fibrosis in SSc and potentially other fibrotic diseases.

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

Supplemental File - PBS Clusters.xlsx (61 kB)
Representative genes for each PBS cluster

Supplemental File - BLM Clusters.xlsx (54 kB)
Representative genes for each BLM cluster

Supplemental File - BLR200 Clusters.xlsx (46 kB)
Representative genes for each BLR200 cluster

Available for download on Friday, December 15, 2023

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