Abstract Rationale The aberrant accumulation of transitional epithelial cells after lung injury are thought to directly promote pulmonary fibrosis (PF). The mechanisms that might regulate their accumulation and their consequences for PF are unknown. Epithelial cell death has been noted in human and experimental fibrosis, but whether transitional cell death protects from or promotes fibrosis is unknown. Transitional cells highly express cellular communication network factor 1 (CCN1), a matricellular protein with diverse functions in other tissues, including limiting hepatic and cutaneous fibrosis and promoting apoptosis. Its role in PF and in transitional cells is unknown. Methods A transitional cell-specific Ccn1 conditional knockout mouse (Ccn1 CKO) was generated and subjected to bleomycin-induced lung injury. Severity of fibrosis was assessed by hydroxyproline assay on lung homogenate. Immunofluorescence for transitional state markers, CCN1, and apoptosis markers was performed on fixed lung tissues (human PF explants and bleomycin-induced lung injury). Ccn1 CKO and wild-type (WT) alveolar type 2 cells were isolated by flow sorting and cultured to induce the transitional state in vitro. Ccn1 CKO and WT transitional cells were treated with etoposide. Intrinsic apoptosis activation was assessed by qPCR and immunocytochemistry for cleaved caspase 3. Results Ccn1 CKO mice treated with bleomycin demonstrated significantly more severe lung fibrosis, associated with increased accumulation transitional cells in vivo. Apoptotic transitional cells were noted by immunofluorescence in human PF and in both Ccn1 CKO and WT mice. In vitro, murine transitional cells treated with recombinant CCN1 demonstrated marked cell death. Ccn1 CKO transitional cells were protected from cell death in an etoposide-induced killing assay as measured by cleaved caspase 3 staining and qPCR for transcriptionally-regulated intermediates of the p53-dependent intrinsic apoptosis cascade. Conclusions Transitional epithelial cell-produced CCN1 is protective in experimental lung fibrosis, likely by inducing apoptosis in pathologic transitional cells. These findings suggest that CCN1 via transitional cell apoptosis may act as a “brake” on fibrotic remodeling after injury. In the future, strategies to limit the accumulation of transitional epithelial cells may improve clinical outcomes in PF. This abstract is funded by: F32HL172379, T32HL007749
Ting et al. (Fri,) studied this question.