Integrated single-cell RNA and ATAC sequencing of B-cell lymphoma-3(Bcl3) and endothelin-2(Edn2) proteins as targets to prevent glaucoma progression.

Background: Glaucoma is the leading cause of irreversible vision impairment worldwide. However, the mechanisms underlying primary open-angle glaucoma (POAG), the most common form of glaucoma, are not yet fully understood.

Methods: Single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing, and ATAC-seq datasets (GSE203499, GSE212186, GSE241782, GSE212188) containing data from glaucoma and control samples were analyzed to identify differentially expressed genes (DEGs) and chromatin accessibility changes. Functional enrichment, immune infiltration, and transcriptional regulatory networks were assessed using bioinformatics tools. Experimental validation was subsequently conducted using ELISA assays and RT-qPCR on human aqueous humor and serum, in vitro R28 cell excitotoxicity models, and in vivo retinal ischemia-reperfusion (I/R) and NMDA-induced glaucoma rat models. Pharmacological inhibitors (JSH-23 for NF-κB; TED-347 for Hippo) were finally tested for neuroprotective effects.

Results: Multi-omics integration identified Bcl3 and Edn2 as key proteins aberrantly upregulated in POAG, correlating with immune infiltration of M1 macrophages and CD4+ follicular T cells, and were enriched in the NF-κB and Hippo signaling pathways. Experimental validation confirmed elevation of Bcl3 and Edn2 expression in POAG patients' aqueous humor/serum, glaucoma animal models, and glutamate-injured R28 cells. Pharmacological inhibition of the NF-κB and Hippo pathways reduced Bcl3/Edn2 expression and mitigated retinal ganglion cell injury.

Conclusions: In summary, Bcl3 and Edn2 could be significant in understanding the molecular mechanisms underlying POAG.