C57BL/6 Mouse Primary Lung Fibroblasts

Specific Knockout of VCAN in Fibroblasts Attenuates Lung Fibrosis

This study aimed to elucidate the role and mechanism of versican (VCAN) in promoting pulmonary fibrosis (PF) and to identify therapeutic targets for PF. Given that fibroblasts are the main effector cells in the process of lung fibrosis, we investigated whether changes in VCAN expression during lung fibrosis were associated with fibroblast activation.

Primary lung fibroblasts were isolated from wild-type C57BL/6 mice along with VCAN^fl/fl (control) and VCAN^-/- (fibroblast-specific knockout) mice, and their activation levels were examined. The results of the WB experiments showed that bleomycin (BLM) significantly induced fibroblasts to express α-SMA and Collagen. However, in mice with specific VCAN knockout in fibroblasts, the ability of BLM to induce α-SMA and Collagen expression was significantly decreased (Fig. 1A). Immunofluorescence experiments for α-SMA also showed that BLM-induced α-SMA expression in fibroblasts was significantly lower in mice with specific knockout of VCAN than in mice treated with BLM alone (Fig. 1B). Activated fibroblasts showed significant migration capacity. Transwell and scratch assays demonstrated that BLM significantly increased the migratory capacity of fibroblasts. However, the fibroblast migration ability significantly decreased in the VCAN knockout group compared to that in the BLM group (Fig. 1C-D).

To further validate our experimental results, we used human and mouse-derived fibroblast cell lines to verify the effect of VCAN on fibroblast activation, the knockdown efficiency was validated, and those with high efficiency were selected for subsequent experiments (Fig. 1E). WB results showed that VCAN knockdown significantly inhibited BLM-induced fibroblast expression of α-SMA and Collagen (Fig. 1F, G). These results indicated that VCAN plays an important role in the activation of lung fibroblasts.