Single-Cell RNA Sequencing of Human Lamina Cribrosa Fibroblasts in Glaucoma
7 affiliations
- 1UCD Clinical Research Centre, Mater Misericordiae University Hospital, D07 KX5K Dublin, Ireland
- 2Department of Ophthalmology, Mater Misericordiae University Hospital, D07 R2WY Dublin, Ireland
- 3The Wellcome–Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast BT9 7BL, UK
- 4Systems Biology Ireland, School of Medicine, University College Dublin, D04 V1W8 Dublin, Ireland
- 5Department of Genetics, Trinity College Dublin, D02 PN40 Dublin, Ireland
- 6St Vincent’s University Hospital, D04 T6F4 Dublin, Ireland
- 7Department of Pharmacology & Neuroscience and the North Texas Eye Research Institute, University of North Texas Health Science Center, Fort Worth, TX 76107, USA
Abstract
The molecular mechanisms that underlie the extracellular matrix (ECM) remodelling of the human lamina cribrosa (LC) in glaucoma remain obscure. This is the first study to characterise human glaucomatous LC cells using single-cell RNA sequencing (scRNA-seq). Our aim is to characterise transcriptional changes in human LC cells from glaucomatous and normal eyes and identify signalling pathways contributing to optic nerve head remodelling. scRNA-seq was performed on LC cells from glaucomatous and normal donor eyes. Differential gene expression was analysed using Seurat. Cell identity was assessed using SingleR and GTEx reference datasets. Pathway enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA), and transcription factor (TF) motif enrichment was evaluated using i-cisTarget. Validation was performed using quantitative PCR, Western blotting, immunohistochemistry, and immunofluorescence. LC cells were treated with ISRIB and 4-PBA. Analysis of 6131 LC cells using shared nearest-neighbour clustering and UMAP visualisation identified four broad transcriptional groups, which were used for subsequent transcriptional characterisation. Cell type mapping identified a fibroblast/mesenchymal cell origin. Glaucomatous LC fibroblasts demonstrated a pro-fibrotic transcriptional profile, with overexpression of multiple pro-fibrotic genes, master TF regulons involved in cell fate decisions, and TF motifs including SRF, as well as key integrated stress response (ISR) genes such as ATF4, ATF3 and CREB3L1. Enriched pathways included E2F, G2M, the Hallmark EMT gene set and MYC, while the p53 pathway was downregulated. Pharmacologic treatment with ISRIB or 4-PBA reduced expression of stress-response and ECM genes, while ISRIB attenuated the increased cellular metabolic activity observed in glaucomatous LC cells. Bioinformatic analysis of scRNA-seq of human glaucoma LC cells identified increased expression of myofibroblast genes, an EMT-associated/profibrotic transcriptional programme, and cell cycle signalling pathways, TF regulatory elements and stress response genes.
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BibTeX
@article{normile2026singlecell,
title = {{Single-Cell RNA Sequencing of Human Lamina Cribrosa Fibroblasts in Glaucoma}},
author = {Caoimhe Normile and Mustapha Irnaten and Oisin Cappa and Vadim Zhernovkov and Jeffrey O’Callaghan and Bruce Moran and Eoghan Culligan and Sherri Feris and Stacy Curry and Abbot F. Clark and David Simpson and Colm O’Brien},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = nov,
doi = {10.3390/ijms27219892},
url = {https://www.mdpi.com/1422-0067/27/21/9892},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Single-Cell RNA Sequencing of Human Lamina Cribrosa Fibroblasts in Glaucoma
AU - Caoimhe Normile
AU - Mustapha Irnaten
AU - Oisin Cappa
AU - Vadim Zhernovkov
AU - Jeffrey O’Callaghan
AU - Bruce Moran
AU - Eoghan Culligan
AU - Sherri Feris
AU - Stacy Curry
AU - Abbot F. Clark
AU - David Simpson
AU - Colm O’Brien
PY - 2026
DA - 2026/11/04
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27219892
UR - https://www.mdpi.com/1422-0067/27/21/9892
AB - The molecular mechanisms that underlie the extracellular matrix (ECM) remodelling of the human lamina cribrosa (LC) in glaucoma remain obscure. This is the first study to characterise human glaucomatous LC cells using single-cell RNA sequencing (scRNA-seq). Our aim is to characterise transcriptional changes in human LC cells from glaucomatous and normal eyes and identify signalling pathways contributing to optic nerve head remodelling. scRNA-seq was performed on LC cells from glaucomatous and normal donor eyes. Differential gene expression was analysed using Seurat. Cell identity was assessed using SingleR and GTEx reference datasets. Pathway enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA), and transcription factor (TF) motif enrichment was evaluated using i-cisTarget. Validation was performed using quantitative PCR, Western blotting, immunohistochemistry, and immunofluorescence. LC cells were treated with ISRIB and 4-PBA. Analysis of 6131 LC cells using shared nearest-neighbour clustering and UMAP visualisation identified four broad transcriptional groups, which were used for subsequent transcriptional characterisation. Cell type mapping identified a fibroblast/mesenchymal cell origin. Glaucomatous LC fibroblasts demonstrated a pro-fibrotic transcriptional profile, with overexpression of multiple pro-fibrotic genes, master TF regulons involved in cell fate decisions, and TF motifs including SRF, as well as key integrated stress response (ISR) genes such as ATF4, ATF3 and CREB3L1. Enriched pathways included E2F, G2M, the Hallmark EMT gene set and MYC, while the p53 pathway was downregulated. Pharmacologic treatment with ISRIB or 4-PBA reduced expression of stress-response and ECM genes, while ISRIB attenuated the increased cellular metabolic activity observed in glaucomatous LC cells. Bioinformatic analysis of scRNA-seq of human glaucoma LC cells identified increased expression of myofibroblast genes, an EMT-associated/profibrotic transcriptional programme, and cell cycle signalling pathways, TF regulatory elements and stress response genes.
SN - 1422-0067
ER -