Slow Interstitial Fluid Flow Activates TGF-β Signaling and Drives Fibrotic Responses in Human Tenon Fibroblasts.

Abstract

Fibrosis limits the success of filtering glaucoma surgery. We employed 2D and 3D in vitro models to assess the effects of fluid flow on human tenon fibroblasts (HTF). HTF were exposed to continuous or pulsatile fluid flow for 48 or 72 h, at rates expected at the transscleral outflow site after filtering surgery. In the 2D model, the F-actin cytoskeleton and fibronectin 1 (FN1) were visualized by confocal immunofluorescence microscopy. In the 3D model, mRNA and whole cell lysates were extracted to analyze the expression of fibrosis-associated genes by qPCR and Western blot. The effects of a small-molecule inhibitor of the TGF-β receptor ALK5 were studied. Slow, continuous fluid flow induced fibrotic responses in the 2D and 3D models. It elicited changes in cell shape, the F-actin cytoskeleton, the deposition of FN1 and activated the intracellular TGF-β signaling pathway to induce expression of fibrosis-related genes, such as CTGF, FN1 and COL1A1. ALK5-inhibition reduced this effect. Intermittent fluid flow also induced fibrotic changes, which decreased with increasing pause duration. Slow interstitial fluid flow is sufficient to induce fibrosis, could underlie the intractable nature of fibrosis following filtering glaucoma surgery and might be a target for antifibrotic therapy.