Cataracts are the leading cause of blindness worldwide. Although surgery is a successful method to restore vision loss due to cataracts, post-surgical complications can occur, such as secondary cataracts, also known as posterior capsular opacification (PCO). PCO arises when lens epithelial cells (LEC) are left behind in the capsular bag following surgery and are induced to undergo epithelial to mesenchymal transition (EMT). Following EMT, LEC morphology and phenotype are altered leading to a loss of transparency and vision. Transforming growth factor (TGF)-β-induced signaling through both canonical, TGF-β/Smad, and non-canonical, β-catenin/Wnt and Rho/ROCK/MRTF-A, pathways have been shown to be involved in lens EMT, and thus PCO. However, the interactions between these signaling pathways in the lens have not been thoroughly explored. In the current study we use rat LEC explants as an ex vivo model, to examine the interplay between three TGF-β-mediated pathways using α-smooth muscle actin (α-SMA) as a molecular marker for EMT. We show that Smad3 inhibition via SIS3 prevents nuclear translocation of β-catenin and MRTF-A, and α-SMA expression, suggesting a key role of Smad3 in regulation of MRTF-A and β-catenin nuclear transport in LECs. Further, we demonstrate that inhibition of β-catenin/CBP interaction by ICG-001 decreased the amount of phosphorylated Smad3 upon TGF-β stimulation in addition to significantly decreasing the expression levels of TGF-β receptors, TBRII and TBRI. Overall, our findings demonstrate interdependence between the canonical and non-canonical TGF-β-mediated signaling pathways controlling EMT in the lens.

Fibrotic cataracts, posterior capsular opacification (PCO), and anterior subcapsular cataracts (ASC) are mainly attributed to the transforming growth factor-β (TGFβ)-induced epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs). Previous investigations from our laboratory have shown the novel role of non-canonical TGFβ signaling in the progression of EMT in LECs. In this study, we have identified YAP as a critical signaling molecule involved in lens fibrosis. The observed increase in nuclear YAP in capsules of human ASC patients points toward the involvement of YAP in lens fibrosis. In addition, the immunohistochemical (IHC) analyses on ocular sections from mice that overexpress TGFβ in the lens (TGFβ^tg) showed a co-expression of YAP and α-SMA in the fibrotic plaques when compared to wild-type littermate lenses, which do not. The incubation of rat lens explants with verteporfin, a YAP inhibitor, prevented a TGFβ-induced fiber-like phenotype, α-SMA, and fibronectin expression, as well as delocalization of E-cadherin and β-catenin. Finally, LECs co-incubated with TGFβ and YAP inhibitor did not exhibit an induction in matrix metalloproteinase 2 compared to those LECs treated with TGFβ alone. In conclusion, these data demonstrate that YAP is required for TGFβ-mediated lens EMT and fibrosis.

Fibrotic cataracts have been attributed to transforming growth factor-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT). Using mouse knockout (KO) models, our laboratory has identified MMP9 as a crucial protein in the TGF-β-induced EMT process. In this study, we further revealed an absence of alpha-smooth muscle actin (αSMA) and filamentous-actin (F-actin) stress fibers in MMP9KO mouse lens epithelial cell explants (LECs). Expression analysis using NanoString revealed no marked differences in αSMA (ACTA2) and beta-actin (β-actin) (ACTB) mRNA between the lenses of TGF-β-overexpressing (TGF-βtg) mice and TGF-βtg mice on a MMP9KO background. We subsequently conducted a protein array that revealed differential regulation of proteins known to be involved in actin polymerization and cell migration in TGF-β-treated MMP9KO mouse LECs when compared to untreated controls. Immunofluorescence analyses using rat LECs and the novel MMP9-specific inhibitor, JNJ0966, revealed similar differential regulation of cortactin, FAK, LIMK1 and MLC2 as observed in the array. Finally, a reduction in the nuclear localization of MRTF-A, a master regulator of cytoskeletal remodeling during EMT, was observed in rat LECs co-treated with JNJ0966 and TGF-β. In conclusion, MMP9 deficiency results in differential regulation of proteins involved in actin polymerization and cell migration, and this in turn prevents TGF-β-induced EMT in the lens.

The cornea is an anterior eye structure specialized for vision. The corneal endothelium and stroma are derived from the periocular mesenchyme (POM), which originates from neural crest cells (NCCs), while the stratified corneal epithelium develops from the surface ectoderm. Activating protein-2β (AP-2β) is highly expressed in the POM and important for anterior segment development. Using a mouse model in which AP-2β is conditionally deleted in the NCCs (AP-2β NCC KO), we investigated resulting corneal epithelial abnormalities. Through PAS and IHC staining, we observed structural and phenotypic changes to the epithelium associated with AP-2β deletion. In addition to failure of the mutant epithelium to stratify, we also observed that Keratin-12, a marker of the differentiated epithelium, was absent, and Keratin-15, a limbal and conjunctival marker, was expanded across the central epithelium. Transcription factors PAX6 and P63 were not observed to be differentially expressed between WT and mutant. However, growth factor BMP4 was suppressed in the mutant epithelium. Given the non-NCC origin of the epithelium, we hypothesize that the abnormalities in the AP-2β NCC KO mouse result from changes to regulatory signaling from the POM-derived stroma. Our findings suggest that stromal pathways such as Wnt/β-Catenin signaling may regulate BMP4 expression, which influences cell fate and stratification.

Vascular leak is a key driver of organ injury in diseases such as Acute Respiratory Distress Syndrome caused by viruses, including COVID-19. Strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the Angiopoietin-1 (Angpt1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANGPT1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANGPT1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α (C4BP). We refer to this new fusion protein biologic as C4BP-ANG1, which forms a stable heptamer and induces TIE2 phosphorylation in cultured cells, and in the lung following i.v. injection of mice. Injection of C4BP-ANG1 ameliorates VEGF- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability, and therefore is a promising candidate treatment for inflammatory endothelial dysfunction.