Genome-Wide Transcriptome Profiling of Human Trabecular Meshwork Cells Treated with TGF-β2.

Abstract

Glaucoma is a complex neurodegenerative disease resulting in progressive optic neuropathy and is a leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the predominant form affecting 65.5 million people globally. Despite the prevalence of POAG and the identification of over 120 glaucoma related genetic loci, the underlaying molecular mechanisms are still poorly understood. The transforming growth factor beta (TGF-β) signalling pathway is implicated in the molecular pathology of POAG. To gain a better understanding of the role TGF-β2 plays in the glaucomatous changes to the molecular pathology in the trabecular meshwork, we employed RNA-Seq to delineate the TGF-β2 induced changes in the transcriptome of normal primary human trabecular meshwork cells (HTM). We identified a significant number of differentially expressed genes and associated pathways that contribute to the pathogenesis of POAG. The differentially expressed genes were predominantly enriched in ECM regulation, TGF-β signalling, proliferation/apoptosis, inflammation/wound healing, MAPK signalling, oxidative stress and RHO signalling. Canonical pathway analysis confirmed the enrichment of RhoA signalling, inflammatory-related processes, ECM and cytoskeletal organisation in HTM cells in response to TGF-β2. We also identified novel genes and pathways that were affected after TGF-β2 treatment in the HTM, suggesting additional pathways are activated, including Nrf2, PI3K-Akt, MAPK and HIPPO signalling pathways. The identification and characterisation of TGF-β2 dependent differentially expressed genes and pathways in HTM cells is essential to understand the patho-physiology of glaucoma and to develop new therapeutic agents.

Temporal single cell transcriptome atlas of zebrafish anterior segment development reveals high degree of conservation between the trabecular meshwork and the annular ligament

“…To do so, we referenced genes in our data set to those that have been identified in mammalian, and particularly human AS tissues. Recent work examining adult cornea and the entire AS, also at single cell levels, was used to cross reference with our own (16–19, 29–32, 47). In doing so, we identified significant overlap in conservation of expression for CEp, CEn as well as the AL, compared to the cornea and trabecular meshwork, respectively.…”

“…In the absence of proper flow and drainage, the IOP can increase which in turn can lead to glaucoma, one of the leading causes of blindness in the US and worldwide (4,7,(13)(14)(15). Accordingly, an increasing amount of attention has been paid to understand the trabecular meshwork (TM) and the Schlemm's canal (SC), specifically the underlying genetics (4,(16)(17)(18)(19). These studies catalogued gene expression from adult tissues at the singe cell level and in doing so, uncovered numerous potential targets for the study of ICA function.…”

“…While recent studies have revealed some of the molecular composition of the adult cornea and ICA, there is still a lot more to uncover, particularly regarding the molecular mechanism regulating their development (16)(17)(18)(19)(29)(30)(31)(32). Work from various model systems, including zebrafish, chick, mouse, and human tissue, has firmly established the physiological landscape and developmental timing for the assembly of the AS (3,29,(33)(34)(35)(36)(37).…”

“…Furthermore, the current single cell transcriptomic data of the vertebrate eye includes tissue from a single, mostly adult time point. This includes recent work on human as well as monkey and pig AS samples (16)(17)(18)(19)(30)(31)(32). As such, in our current study we used developing zebrafish to systematically analyze transcriptomic changes within ASM isolated from the AS over the course of early development.…”

The TGFβ Induced MicroRNAome of the Trabecular Meshwork

“…Samples were incubated at 37 °C (5% CO2 and 95% humidity). Human TM characterisation was performed as previously described [39] including dexamethasone upregulation of myocilin protein expression (polyclonal rabbit anti-myocilin primary antibody was a kind gift from Dr. W. Daniel Stamer) as previously described by our group [40].…”