Primary Human Trabecular Meshwork Model for Pseudoexfoliation

Abstract

The lack of an animal model or an in vitro model limits experimental options for studying temporal molecular events in pseudoexfoliation syndrome (PXF), an age related fibrillopathy causing trabecular meshwork damage and glaucoma. Our goal was to create a workable in vitro model of PXF using primary human TM (HTM) cell lines simulating human disease. Primary HTM cells harvested from healthy donors (n = 3), were exposed to various concentrations (5 ng/mL, 10 ng/mL, 15 ng/mL) of transforming growth factor-beta1 (TGF-β1) for different time points. Morphological change of epithelial–mesenchymal transition (EMT) was analyzed by direct microscopic visualization and immunoblotting for EMT markers. Expression of pro-fibrotic markers were analyzed by quantitative RT-PCR and immunoblotting. Cell viability and death in treated cells was analyzed using FACS and MTT assay. Protein complex and amyloid aggregate formation was analyzed by Immunofluorescence of oligomer11 and amyloid beta fibrils. Effect of these changes with pharmacological inhibitors of canonical and non-canonical TGF pathway was done to analyze the pathway involved. The expression of pro-fibrotic markers was markedly upregulated at 10 ng/mL of TGF-β1 exposure at 48–72 h of exposure with associated EMT changes at the same time point. Protein aggregates were seen maximally at these time points that were found to be localized around the nucleus and in the extracellular matrix (ECM). EMT and pro-fibrotic expression was differentially regulated by different canonical and non-canonical pathways suggesting complex regulatory mechanisms. This in vitro model using HTM cells simulated the main characteristics of human disease in PXF like pro-fibrotic gene expression, EMT, and aggregate formation.

Alternate Causes for Pathogenesis of Exfoliation Glaucoma, a Multifactorial Elastotic Disorder: A Literature Review

“…We recently demonstrated a decreased UPR clearance in XFG compared to earlier forms of the disease associated with increased TGF levels in all disease stages, which suggest the potential regulation of the autophagy pathway and TGF-autophagy cross-talk involved in cell repair and aggregate clearance [50]. Autophagy is an intracellular trafficking system that conveys cytosolic constituents to the lysosome for ensuing degradation, which is crucial for misfolded protein clearance, cell homeostasis by ubiquitin-proteasomal degradation, and cell repair [51][52][53][54][55].…”

Role of Mitochondrial Mutations in Ocular Aggregopathy

“…Informed consent was obtained from all patients who underwent standardized ophthalmic examination including slit-lamp examination, gonioscopy, and fundus biomicroscopy, and IOP measurement by Goldman applanation. The definitions of XFS/XFG are detailed elsewhere [1][2][3].…”

“…Exfoliation glaucoma (XFG) is an age-related fibrillopathy characterized by protein deposits on various ocular surfaces. Transforming growth factor beta-1 (TGF-β1) has been shown to be increased in exfoliation syndrome (XFS) and XFG [ 1 , 2 ] eyes and is a key mediator for regulating extracellular matrix homeostasis [ 3 ], reactive oxygen species (ROS) production, and redox balance in the cell milieu [ 4 ]. ROS, in turn, induces/activates TGF-β1 and mediates many of the fibrogenic effects of TGF-β, forming a vicious cycle.…”

Magnesium and Its Role in Primary Open Angle Glaucoma; A Novel Therapeutic?

“…Studies have identified a link between elevated TGF-b2 levels in the AH and extracellular matrix (ECM) of patients with POAG (18)(19)(20)(21). It has been established that TGF-b isoforms regulate TM cellular fate due to their mesenchymal nature (22)(23)(24), with a heightened presence of TGF-b2 stimulating a myofibroblast phenotype, increasing contractile features, profibrotic protein expression (a-SMA, fibronectin) and ECM deposition (25,26). Overall, these factors contribute to a loss of cell-cell contact, increased stiffness, and fibrosis of the TM, leading to AH outflow resistance and increased IOP (26).…”