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.

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM’s structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

Elevated intraocular pressure (IOP) is a major risk factor for the development of primary open-angle glaucoma (POAG). This is from an increased aqueous humour (AH) outflow resistance through the trabecular meshwork (TM). The pathogenic mechanisms leading to the increase in TM outflow resistance are poorly understood but are thought to be from a dysregulation of the TM extracellular matrix (ECM) environment. ECM modification and turnover are crucial in regulating the resistance to aqueous outflow. ECM turnover is influenced by a complex interplay of growth factors such as transforming growth factors (TGFβ) family and matrix metalloproteinases (MMPs). Elevated TGFβ2 levels result in an increase in ECM deposition such as fibronectin leading to increased resistance. Fibronectin is a major component of TM ECM and plays a key role in its maintenance. Thrombospondins (TSP)-1 and -2 are important regulators of the ECM environment. TSP-1 has been implicated in the pathogenesis of POAG through activation of TGFβ2 within the TM. TSP-2 does not contain the catalytic domain to activate latent TGFβ, but is able to mediate the activities of MMP 2 and 9, thereby influencing ECM turnover. TSP-2 knock out mice show lower IOP levels compared to their wild type counterparts, suggesting the involvement of TSP-2 in the pathogenesis of POAG but its role in the pathogenesis of POAG remains unclear. The purpose of this study was to investigate the role of TSP-2 in trabecular meshwork ECM regulation and hence the pathogenesis of POAG. TSP-1 and TSP-2 expressions in immortalised glaucomatous TM cells (GTM3) and primary human non-glaucomatous (NTM) and glaucomatous cells (GTM) were determined by immunocytochemistry, immuno-blot analysis and qPCR following treatment with TGFβ2 and Dexamethasone. The level of ECM protein fibronectin was determined in TM cells using immuno-blot analysis following treatment with TSP-1 or -2. TM cells secrete TSP-1 and -2 under basal conditions at the protein level and TSP-2 mRNA and protein levels were increased in response to TGFβ2 three days post treatment. Exogenous treatment with TSP-2 up-regulated the expression of fibronectin protein in GTM3 cells, primary NTM and GTM cells. TSP-1 did not affect fibronectin protein levels in GTM3 cells. This suggests that the role of TSP-2 might be distinct from that of TSP-1 in the regulation of the TM cell ECM environment. TSP-2 may be involved in the pathogenesis of POAG and contribute to increased IOP levels by increasing the deposition of fibronectin within the ECM in response to TGFβ2.

The loss and dysfunction of trabecular meshwork (TM) cells are implicated in aging and primary open-angle glaucoma. TM progenitor cells (TMPCs) contribute to the population and function of the TM, but their identity is not well elucidated. This study aimed to identify the expression profile of differentially expressed genes (DEGs) in human TM cell cultures, TM-derived spheres, and their differentiated progeny. Primary normal human TM cells (PTM) from three donors were cultured, de-differentiated into spheres, and re-differentiated into TM cells (DTM). RNA-Seq was performed using Illumina NGS, and bioinformatics analysis was conducted with Tuxedo, Bowtie2, Tophat, Cufflinks, and Ingenuity Pathway Analysis (IPA). DEGs were validated via Nanostring, RT-qPCR (in five independent donors), immunocytochemistry, and western blotting. RNA-seq identified significant DEGs in PTM, TM progenitor cells (TMPCs), and DTM cells. Gene expression in TMPCs differed significantly from PTM and DTM cells. Nanostring and RT-qPCR confirmed 70 DEGs upregulated in TMPCs (P < 0.05). Immunocytochemistry highlighted distinct markers in TMPCs (SOX2, NOTCH1, ANKG, MGP) versus PTM and DTM cells (TAGLN, TEM7, SPARC). Western blotting further analyzed MGP, TAGLN, and SPARC proteins, revealing significant upregulation of MGP in TMPCs and downregulation of TAGLN and SPARC in spheres compared to PTM cells. Pathway analysis revealed activation of cell cycle checkpoint regulation, SUMOylation, and STAT3 pathways in TMPCs, with HGF, MMP9, KDR, IGF1, and FOS as key node genes in TMPC development. RNA-Seq identified novel expression profile of potential TM markers and activated pathways in TMPCs, providing insights into TMPC behaviours in physiological and pathological conditions. Graphical Abstract

Primary open-angle glaucoma (POAG) is a major cause of irreversible blindness, yet its underly mechanisms remain unclear. Elevated intraocular pressure (IOP), the only modifiable risk factor for POAG, arises from increased resistance to aqueous humour outflow within the conventional outflow pathway, which comprises the trabecular meshwork (TM) and the inner wall of Schlemm’s canal. Dysfunction and cellular loss within this pathway, particularly in the TM, are consistent features of the disease; however, the mechanisms responsible for impaired tissue maintenance and regenerative failure remain unclear. Trabecular meshwork progenitor cells (TMPCs) have the capacity to replace lost TM cells, suggesting that impaired progenitor function may contribute to disease progression. Transforming growth factor beta 2 (TGFβ2), which is consistently elevated in the aqueous humour of POAG patients, plays a key role in regulating stem cell differentiation. We proposed that excess TGFβ2 disrupts TMPC function, leading to progenitor depletion and TM dysfunction. Here, we show that TGFβ2 drives TMPCs toward a differentiated, fibrotic phenotype, increasing TM and profibrotic gene expression while reducing progenitor markers. These effects are mediated through TGFβ2–SMAD2/3 signalling, as inhibition of this pathway preserves TMPC characteristics and suppresses fibrotic gene induction. Our findings identify TGFβ2–SMAD2/3 signalling as a regulator of TMPC fate in vitro and suggest a potential mechanism by which elevated TGFβ2 may influence TM progenitor cell behaviour under pathological conditions.

The present study aimed to understand the role of miRNAs in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. For this, total RNA was extracted from cultured HTM cells with known GC responsiveness using Human organ-cultured anterior segment (HOCAS) (GC-responder GC-R; n=4) and GC-non-responder (GC-NR; n=4) after treatment with either 100nM dexamethasone (DEX) or ethanol (ETH) for 7 days. Differentially expressed miRNAs (DEMIRs) were compared among 5 groups and validated by RT-PCR. There were 13 and 21 DEMIRs identified in Group #1 (ETH vs DEX-treated GC-R) and Group #2 (ETH vs DEX-treated GC-NR) respectively. Seven miRNAs were found as common miRNAs dysregulated in both GC-R and GC-NR (Group #3). There were 6 and 14 unique DEMIRs were identified in GC-R (Gropu#4) and GC-NR (Group#5) HTM cells respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. Integrative analysis of miRNA-mRNA of the same set of HTM cells revealed several molecular regulators for GC non-responsiveness. This is the first study revealed a unique miRNA signature between GC-R and GC-NR HTM cells which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.