Inhibition of LPA1 Signaling Impedes Conversion of Human Tenon's Fibroblasts into Myofibroblasts Via Suppressing TGF-ß/Smad2/3 Signaling.

ABSTRACT

Purpose: Lysophosphatidic acid (LPA) is a growth factor-like phospholipid that has been recognized as a profibrotic mediator in numerous tissues, yet, whether it plays a role in subconjunctival fibrosis remains to be investigated. Therefore, this study was designed to examine the effect of LPA1-3 signaling inhibitor, Ki16425 on the conversion of human Tenon's fibroblasts (HTFs) into myofibroblasts. Methods: Primary cultured HTFs were incubated with transforming growth factor-ß1 (TGF-ß1) alone or combined with Ki16425, the cell proliferation and migration were measured by Cell Counting Kit-8 and the scratch wound assay, respectively. HTFs contractility was evaluated with 3-dimensional (3D) Collagen Contraction assay. The mRNA and protein levels of α-smooth muscle actin (α-SMA), Snail and the phosphorylation levels of Smad2/3, p38MAPK, and ERK1/2 were determined by real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunofluorescence staining. Results: Ki16425 significantly prevent the proliferation and migration of Tenon's fibroblasts (HTFs) in a dose-dependent manner. Furthermore, Ki16425 blocked HTFs myofibroblast differentiation via downregulation of mRNA and protein expression of α-SMA. 3D collagen gel contraction assay demonstrated that Ki16425 effectively inhibits myofibroblast contraction induced by TGF-ß1. Mechanistically, we revealed that Ki16425 reduces Smad2/3 but not p38MAPK or ERK1/2 phosphorylation by TGF-ß1. By using an LPA1-specific inhibitor, AM095, we confirmed that LPA1 signaling but not LPA2 or LPA3 is involved in TGF-ß1 induced HTFs activation. Conclusions: Our results show that inhibition of LPA1 signaling presents potent antifibrotic effect in HTFs, which may serve as a promising intervention strategy for preventing subconjunctival fibrosis caused by glaucoma filtration surgery.