Slow interstitial fluid flow induces fibrotic responses in human tenon fibroblasts by stimulating TGF-β-signaling

Abstract

Purpose : Scarring prevents sustained therapeutic success in about one-third of filtering glaucoma surgeries. In dermal fibroblasts, slow interstitial fluid flow promotes scar formation. Since interstitial fluid flow acts on human tenon fibroblasts (HTF) after filtering surgery, we developed experimental 2D and 3D models to investigate the hypotheses that scar formation in HTF is also stimulated by slow fluid flow and that the TGF-β pathway has a role in this.

Methods : HTF were cultured in a 2D or 3D model and continuously perfused at slow flow rates (150 µl/h in 2D or 180 µl/h in 3D) for 48 h or 72 h resulting in 0.0006 dyn/cm² or 0.003 dyn/cm² of shear stress, respectively. A small molecule inhibitor (E-616452 (RepSox), at 0.1 µM f.c.) of the TGF-β receptor ALK5 (ALK5-I.) was added to the perfusion medium. Static cell cultures served as controls. In the 2D model the cellular F-actin skeleton and cellular fibronectin 1 (FN1) were visualized by confocal immunofluorescence microscopy. In the 3D model, mRNA and whole cell lysates were extracted from HTF. qPCR and Western blot were used to analyze the expression of fibrosis-associated genes.

Results : In the 2D model, flow increased the density of intracellular F-actin stress fibers, led to loss of lamellipodia and increased deposition of FN1, which was more aligned and formed in thicker bundles. This effect was reduced by the addition of the ALK5-Inh. (n=3). In the 3D model (n=3), flow significantly increased expression of the scarring-associated genes ACTA2 (median fold change (FC) 5.3, p<0.001) , COL1A1 (FC 3.5, p<0.001), CTGF (FC 2.8, p<0.01) and showed a trend towards increased expression of FN1 (FC 1.6, p>0.05). With addition of the ALK5-Inh. to the medium, no increased expression of the investigated genes was observed (FC 0.9, 0.8, 0.2, 0.5, p>0.05). The amount of phosphorylated SMAD2, the main signal transducer of TGF-β receptor activation, was increased by flow and reduced by the addition of RepSox.

Conclusions : Our data are consistent with our hypothesis that slow fluid flow induces fibrotic changes in HTF by stimulating the TGF-β pathway. Inhibition of TGF-β receptor signaling reduces this flow response. In light of these data, inhibition of TGF-β signaling by small molecule inhibitors could be therapeutically useful.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.