Abstract Background/Aims Activated fibroblasts, called myofibroblasts, are responsible for the fibrosis seen in scleroderma. How to block myofibroblast activation and activity in scleroderma is unknown, but CCN3, a member of the CCN family of matricellular proteins, is anti-fibrotic and counteracts the profibrotic activity of the related protein CCN2. We have identified BLR-200 as a CCN3-derived peptide that retains CCN3’s antifibrotic properties in bleomycin-induced skin fibrosis. However, if CCN3’s and BLR-200’s antifibrotic properties translate to scleroderma patients is unknown. Methods To test the hypothesis that reduced CCN3 levels may exist in scleroderma patients, we used ELISAs to detect the amount of CCN2 and CCN3 in serum of healthy controls, early-onset diffuse (24 months duration) scleroderma patients and those of later (5 years) duration. To identify the fibroblast populations whose activation by bleomycin is suppressed by BLR-200, we conducted scRNAseq analysis of fibroblasts labelled postnatally with green fluorescent protein using mTmG mice, subjected or not to bleomycin-induced skin fibrosis in the presence or absence of BLR-200, that expressed a tamoxifen-dependent cre recombinase expressed under the control of a universal fibroblast-specific promoter/enhancer. Results CCN2 and CCN3 serum levels correlated well among healthy volunteers (controls) (r2=0.39, p 0.03, N = 20), whereas scleroderma patients of 2 years and of 5 years duration showed patient-specific dysregulation of CCN2 and CCN3 protein levels (r2=0.048 and r2=0.116, respectively, N = 20). CCN2 and CCN3 levels increased in serum with patients of early duration (p 0.05 vs p 0.0001, respectively); however, increases in CCN3, but not CCN2, levels were observed in patients with late duration (p 0.001 vs ns, respectively). In mice subjected to bleomycin-induced skin fibrosis, lineage tracing and scRNA-seq analyses revealed that myofibroblasts are derived from Pi16+/Col15+ve “universal” fibroblasts (N = 6, p 0.01). BLR-200 prevented myofibroblast differentiation of Pi16+/Col15+ve “universal” fibroblasts in response to bleomycin toward an engrailed-1/Col8A1-positive subset of “universal” fibroblasts, a pro-fibrotic cell type previously shown to specifically upregulated in skin derived from scleroderma patients with active disease. Conclusion Given what is known about the relative activities of CCN2 and CCN3 in vivo, it is likely that serum levels of CCN3 rise in scleroderma patients in the body’s attempt to suppress CCN2-dependent fibrosis. Why this dysregulation appears to be patient-specific is unclear and requires further investigation. BLR-200 specifically targets the prevention of formation of an engrailed-1/Col8A1-positive fibroblast subpopulation that is specifically upregulated in skin in scleroderma patients. Therefore, BLR-200 appears to be a novel anti-fibrotic agent of potential translational relevance, particularly to patients with low serum levels of CCN3. Disclosure A. Leask: None. R.J. Stratton: None. B.A. Abdi: None. Z.J. Kannan: None. J. Nguyen: None. B.L. Riser: Corporate appointments; CEO, BLR Bio, LLC.
Leask et al. (Wed,) studied this question.