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Primary open angle glaucoma is a leading cause of visual impairment and blindness which is commonly treated with drugs or laser but may require surgery. Tenon's ocular fibroblasts are involved in wound-healing after glaucoma filtration surgery and may compromise a favourable outcome of glaucoma surgery by contributing to fibrosis. To investigate changes in gene expression and key pathways contributing to the glaucomatous state we performed genome-wide RNA sequencing. Human Tenon's ocular fibroblasts were cultured from normal and glaucomatous human donors undergoing eye surgery (n = 12). mRNA was extracted and RNA-Seq performed on the Illumina platform. Differentially expressed genes were identified using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts and edgeR. Changes in biological functions and pathways were determined using Enrichr and clustered using Cytoscape. A total of 5817 genes were differentially expressed between Tenon's ocular fibroblasts from normal versus glaucomatous eyes. Enrichment analysis showed 787 significantly different biological functions and pathways which were clustered into 176 clusters. Tenon's ocular fibroblasts from glaucomatous eyes showed signs of fibrosis with fibroblast to myofibroblast transdifferentiation and associated changes in mitochondrial fission, remodeling of the extracellular matrix, proliferation, unfolded protein response, inflammation and apoptosis which may relate to the pathogenesis of glaucoma or the detrimental effects of topical glaucoma therapies. Altered gene expression in glaucomatous Tenon's ocular fibroblasts may contribute to an unfavourable outcome of glaucoma filtration surgery. This work presents a genome-wide transcriptome of glaucomatous versus normal Tenon's ocular fibroblasts which may identify genes or pathways of therapeutic value to improve surgical outcomes. [ABSTRACT FROM AUTHOR]

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with a complex, multifactorial aetiology. Raised intraocular pressure (IOP) is the most important clinically modifiable risk factor for POAG. All current pharmacological agents target aqueous humour dynamics to lower IOP. Newer therapeutic agents are required as some patients with POAG show a limited therapeutic response or develop ocular and systemic side effects to topical medication. Elevated IOP in POAG results from cellular and molecular changes in the trabecular meshwork driven by increased levels of transforming growth factor β (TGFβ) in the anterior segment of the eye. Understanding how TGFβ affects both the structural and functional changes in the outflow pathway and IOP is required to develop new glaucoma therapies that target the molecular pathology in the trabecular meshwork. In this study, we evaluated the effects of TGF-β1 and -β2 treatment on miRNA expression in cultured human primary trabecular meshwork cells. Our findings are presented in terms of specific miRNAs (miRNA-centric), but given miRNAs work in networks to control cellular pathways and processes, a pathway-centric view of miRNA action is also reported. Evaluating TGFβ-responsive miRNA expression in trabecular meshwork cells will further our understanding of the important pathways and changes involved in the pathogenesis of glaucoma and could lead to the development of miRNAs as new therapeutic modalities in glaucoma. [ABSTRACT FROM AUTHOR]

Aims/Purpose: Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, associated with activation of the TGF‐β pathway by TGF‐β1. MicroRNAs are small non‐coding genes that regulate mRNA expression. To investigate the role of TGFβ1 in glaucomatous changes in the trabecular meshwork (TM) and to determine how mRNA expression may be regulated by microRNAs in this condition, we performed a bioinformatics analysis to determine the microRNA‐mRNA interactome. Methods: Primary human trabecular meshwork cells were obtained from normal donors (n = 4) and treated with TGF‐β1 at 5 ng/mL for 24 h. RNA was extracted and miRNA‐Seq and mRNA‐Seq performed. Differential mRNA and miRNA expression was determined using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts, edgeR (miRNA) and DESeq2 (mRNA). Putative interactions between differentially expressed mRNAs and miRNAs, in which the direction of expression of the regulated mRNA was required to be opposite to that of the regulating miRNA, were determined using interaction databases miRTarBase, TarBase, TargetScan and miRWalk. To classify mRNA species by biological function and pathway, gene enrichment was performed using Enrichr. Results: The resulting microRNA‐mRNA interactome consisted of 1202 interactions. Some highly connected microRNAs were hsa‐let‐7e‐5p, hsa‐miR‐20a‐5p, hsa‐miR‐122‐5p, and hsa‐miR‐29c‐3p. Most genes in all enriched biological functions and pathways were indicated to be regulated by microRNAs. The sub‐interactomes of several specific XFG‐related enrichment terms such as oxidative stress, unfolded protein response, signal molecules and ECM remodelling were determined. Conclusions: This work presents a genome‐wide microRNA‐mRNA regulatory network for human TM cells treated with TGF‐β1 and may serve to generate unbiased hypotheses about regulatory functions of miRNAs found to be differentially expressed in XFG. [ABSTRACT FROM AUTHOR]