Targeting CCN Proteins in the Treatment of Dermal Fibrosis
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.