Identifying nuclear alterations in Schlemm’s canal cells interacting with glaucomatous trabecular meshwork cells using a 3D ECM hydrogel co-culture model

Abstract

Purpose : Schlemm’s canal (SC) inner wall endothelial cells closely interact with the trabecular meshwork (TM). Biomechanical alterations in this microenvironment are responsible for increased outflow resistance in ocular hypertensive glaucoma. However, there is a critical gap in our understanding of how glaucomatous TM cells and their pathologically altered/stiffened extracellular matrix (ECM) affect SC cell biology. Using our 3D ECM hydrogel co-culture model, we investigate SC cell nuclear and chromatin dynamics in response to glaucomatous TM substrate cues.

Methods : Human SC cells were grown on biomimetic ECM hydrogels encapsulated with normal TM (NTM) or glaucomatous TM (GTM) cells. Glaucomatous SC cells served as benchmark controls. SC cell nuclear architecture and chromatin condensation were assessed using DAPI-stained confocal Z-stacks. mRNA expression of histone deacetylases (HDACs) was quantified by qPCR. Genome-wide expression profiling was performed using 100 bp paired-end RNA sequencing; data was processed using established pipelines with Partek-Flow (i.e., alignment to hg38 human genome, two co-factor DESeq2 differential gene expression analysis with p-value cut-off of 0.05 and fold-change of ≥2.0). Gene ontology analysis was performed using WebGestalt.

Results : SC cells on GTM hydrogels exhibited decreased nuclear volume and increased chromatin condensation compared to NTM hydrogels (p<0.05), consistent with reduced chromatin accessibility, with nuclei displaying a flattened oblate shape (p<0.05). Concurrently, SC cells on GTM hydrogels showed increased HDAC2 mRNA expression (p<0.01) compared to NTM hydrogels, suggesting possible repressive epigenetic modifications. Overall patterns of induced nuclear changes were remarkably similar to glaucomatous SC cell controls. A total of 105 genes were differentially expressed in SC cells on GTM hydrogels compared to NTM hydrogels. We identified functional enrichment of genes involved in cytoskeletal protein (actin) binding, ECM, integrin family cell surface interactions, and syndecan-1-mediated signaling.

Conclusions : Our data suggest that SC cells respond to glaucomatous TM substrate cues by altering their nuclear phenotype and transcriptional activity, potentially involving direct mechanical cytoskeleton-nuclear membrane-chromatin linkage and indirect signaling pathways.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.