Single-Cell atlas of aqueous humor outflow in glaucomatous Non-Human Primates identified MGP as a regulator of ECM remodeling.

PURPOSE: This study investigated the heterogeneity and molecular alterations of trabecular meshwork (TM) in glaucomatous non-human primates, aiming to identify potential pathogenic genes and signaling pathways involved in glaucoma. METHODS: TM tissues were analyzed using 10x Genomics single-cell RNA sequencing on the Illumina platform. After quality control, dimensionality reduction and clustering were performed. Differentially expressed genes (DEGs) were identified using Seurat and DESeq2. Functional enrichment analyses were conducted via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Protein expression was assessed by Western blotting, mRNA levels by RT-PCR, protein localization and fluorescence intensity by immunofluorescence, and mitochondrial morphology by transmission electron microscopy. RESULTS: We classified the outflow tissue into 10 cell subtypes, and TM cells were further classified into three subtypes: Beam A, Beam B, and JCT. Beam A cells exhibited the most significant proportional changes, and GO analysis indicated their strong association with extracellular matrix regulation. MGP emerged as the most significantly upregulated gene in both the overall TM population and the Beam A subcluster. Dexamethasone (DEX) treatment induced MGP expression in human primary TM cells, while MGP knockdown partially restored Wnt/β-catenin signaling, decreased Wnt5a levels, and reduced ECM deposition. CONCLUSION: This study generated a transcriptional atlas of TM in glaucoma. MGP may contribute to TM dysfunction via the Wnt/β-catenin-Wnt5a-ECM signaling pathway, offering novel insights into potential therapeutic targets for glaucoma intervention.