Pulmonary fibrosis is a life-threatening disease that has been attributed to several causes. Specifically, vascular injury is thought to be involved in the pathogenesis of fibrosis. The effects of the antifibrotic drug pirfenidone on angiogenesis have not been fully elucidated. This study aimed to investigate the effects of pirfenidone in human lung fibroblast-endothelial cell co-culture network formation and to analyze the underlying molecular mechanisms. Human lung fibroblasts were co-cultured with human umbilical vein endothelial cells to establish a co-culture network cell sheet. The influence of pirfenidone was evaluated for protective effect on the endothelial network in cell sheets stimulated with transforming growth factor β (TGF-β). Results indicated that TGF-β disrupted the network formation. Pirfenidone and Y27632 (Rho-associated coiled-coil containing protein kinase [Rho-kinase or ROCK] inhibitor) protected against the TGF-β-induced endothelial network disruption. TGF-β activated Rho-kinase signaling in cells composing the co-culture cell sheet, whereas pirfenidone and Y27632 inhibited these effects. In conclusion, TGF-β-induced Rho-kinase activation and disrupted endothelial network formation. Pirfenidone suppressed TGF-β-induced Rho-kinase activity in cell sheets, thereby enabling vascular endothelial cells networks to be preserved in the cell sheets. These findings suggest that pirfenidone has potential vascular network-preserving effect via inhibiting Rho-kinase activity in vascular injury, which is a precursor to pulmonary fibrosis.
Keywords: ROCK; Rho-kinase; angiogenesis; interstitial pneumonia; pirfenidone; pulmonary fibrosis; transforming growth factor β (TGF-β).