Fibrosis-Related Gene and Protein Expression in Normal and Glaucomatous Trabecular Meshwork Cells.

Abstract

Purpose: Glaucomatous trabecular meshwork (GTM) tissue is characterized by excess fibrotic-like extracellular matrices, which negatively impacts aqueous humor outflow. Endothelial-to-mesenchymal transition (EndMT) is the process by which tissues develop fibrosis. In this study, we investigated fibrotic-related gene and protein profiles of non-glaucomatous trabecular meshwork (NTM) and GTM cells.

Methods: Primary cells were cultured from NTM (n = 6) and GTM (n = 5) age-matched cadaver eyes. RNA was harvested and mRNA profiling of 750 genes was performed using the human fibrosis panel (NanoString). Quantitative PCR (qPCR), Western blotting, and immunofluorescence microscopy were performed. A matrix metalloproteinase (MMP) fluorogenic assay was used to quantitate enzyme activity.

Results: Classic EndMT biomarkers, α-SMA, SNAI2, TWIST1, TWIST2, and VIM, were upregulated in GTM cells, whereas increased phosphorylated SMAD2-3 indicated increased TGFβ signaling. GTM cells had increased deposition of FN-EDA fibronectin fibrils, but reduced amounts of FN-EDB fibrils, and altered immunostaining of active α5β1 and αvβ3 integrins. NanoString analysis showed that 2 genes were upregulated and 28 genes were downregulated in GTM cells compared with NTM cells. Western immunoblotting confirmed increased protein levels of N-cadherin and decreased MMP2, CHI3L1, COL6A3, and SERPINF1 proteins in GTM cells. Whereas MMP2 gene and protein levels were reduced, there was increased MMP activity.

Conclusions: Increased expression of α-SMA, FN-EDA, N-cadherin, SNAI2, TWISTs, VIM, TGFβ signaling, and MMP activity are consistent with GTM cells acquiring an EndMT phenotype. In combination with tissue studies, cultured GTM cells are a useful in vitro model for studying the fibrotic process in glaucoma.