RNA-Seq Transcriptomic Analysis of Human Trabecular Meshwork Explants Exposed to TGF-ß2: A Novel Approach Beyond Traditional Cell Culture Models

Glaucoma is a progressive optic neuropathy that can lead to irreversible blindness. Its main risk factor is elevated intraocular pressure (IOP). The trabecular meshwork (TM) acts as a filter between the anterior chamber of the eye and the aqueous humor collecting ducts, and dysfunction of this meshwork is responsible for the increased IOP in primary open-angle glaucoma (POAG). Considering that the culture conditions of human TM cells (HTMC) influence gene expression, we used human TM explants (HTMEx), which most closely mimic physiological conditions, to study the transcriptome of HTMC. The transforming growth factor-beta 2 (TGF-ß2) signaling pathway has been implicated in the pathophysiology of POAG. To better characterize the role of TGF-ß2 in this pathophysiology, we used bulk RNA sequencing and immunohistological analyses to establish gene signatures of TGF-ß2-exposed HTMEx and correlate them with morphological alterations. We identified differentially upregulated genes primarily involved in ECM regulation, as well as profibrotic TGF-ß signaling pathways, confirmed using confocal microscopy to highlight changes in trabecular architecture, TGFß2-induced F-actin rearrangements, and extracellular matrix (ECM) deposition. Enrichment analysis also revealed modulations of gene expression related to cytoskeletal organization, as well as activation of the bone morphogenic protein (BMP) and Wnt signaling pathways in response to TGF-ß2. Overall design: To gain a better understanding of the role of TGF-ß2 in the molecular pathology of glaucoma, we used bulk RNA-Seq to analyze changes in the transcriptome of HTMEx exposed to TGF-ß2.