Mary Flokis

Transforming Growth Factor-beta (TGF-β) promotes lens epithelial–mesenchymal transition (EMT) and fibrosis, contributing to anterior subcapsular cataract (ASC) formation. Transgenic mice overexpressing TGF-β1 in the lens have been studied for over three decades, and yet the impact of active TGF-β1-overexpression on the lens epithelial transcriptome is undefined. We have addressed this knowledge gap by examining the gene expression landscape of these unique lens epithelia. High-throughput RNA-sequencing was performed on isolated lens epithelia from three-week-old TGF-β1-overexpression transgenic mice from two independent lines, OVE853 and OVE918, and wild-type mice. Downstream analyses included comparisons with lens datasets (e.g., cataract surgery model) and investigations using various resources/tools (e.g., Gene Ontology, CompBio, and iSyTE). Compared to wild-type murine lens epithelia, 384 differentially expressed genes (DEGs) were commonly identified in the lens of both transgenic lines. Candidates involved in EMT, inflammatory response, extracellular matrix organization, and mechano-sensation were elevated, while those involved in lipid metabolism, Wnt-suppression, Bmp- and Notch-activation were reduced. Comparative analyses with temporal transcriptomes on a mouse cataract surgery model identified overlapping pathological pathways, and some elevated genes, for example, endoplasmic reticulum stress genes, were consistent with human ASC data. This study provides the first comprehensive transcriptomic characterization of two independent TGF-β1 transgenic ASC models and identifies novel candidate downstream genes and pathways associated with TGF-β1 overexpression. All our data is made user-friendly and accessible through iSyTE.

Transforming Growth Factor-beta (TGF-β) promotes lens epithelial-mesenchymal transition (EMT) and fibrosis, contributing to anterior subcapsular cataract (ASC) formation. Transgenic mice overexpressing TGF-β1 in lens have been studied for over three decades, and yet the impact of active TGF-β1-overexpression on the lens epithelial transcriptome is undefined. We have addressed this knowledge gap by examining the gene expression landscape of these unique lens epithelia. High-throughput RNA-sequencing was performed on isolated lens epithelia from three-week-old TGF-β1-overexpression transgenic mice from two independent lines, OVE853 and OVE918, and wild-type mice. Downstream analyses included comparisons with lens datasets (e.g., cataract-surgery model) and investigations using various resources/tools (e.g., Gene Ontology, CompBio, iSyTE). Compared to wild-type murine lens epithelia, 384 differentially expressed genes (DEGs) were commonly identified in lens of both transgenic lines. Candidates involved in EMT, inflammatory response, extracellular matrix organization, and mechano-sensation were elevated, while those involved in lipid metabolism, Wnt-suppression, Bmp- and Notch-activation were reduced. Comparative analyses with temporal transcriptomes on a mouse cataract-surgery model identified overlapping pathological pathways, and some elevated genes, for example endoplasmic reticulum stress genes, were in agreement with human ASC data. A major discovery was the identification of several novel TGF-β1-targets. All our data is made user-friendly accessible through iSyTE.

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial–mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-β-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-β2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (β-crystallin), epithelial cell adhesion (β-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-β2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-β-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-β immunolabelled for α-SMA, with minimal β-crystallin, whereas the PLECs demonstrated strong β-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-β-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-β. Smad-dependent and independent signalling was increased in the FGF-2/TGF-β2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-β-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-β leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.