Abstract
Purpose :
Pathological changes in the extracellular matrix (ECM) of the human Trabecular Meshwork (hTM) is associated with ocular hypertension. Whether cell-ECM interactions drive this pathology is poorly understood. Using steroid treated hTM cell-derived matrices, we document the effect of pathologic ECM on healthy hTM cells at a transcriptional and mechanistic level.
Methods :
Normal primary hTM cells from transplant rejected donors were validated and cultured in the presence of 100nM Dexamethasone (DEX) or ethanol vehicle control for four weeks on a coverslip. The matrices were then decellularized and primary hTM cells from the same donor were plated on these matrices and cultured for an additional 72 hours. Total RNA was then extracted and used for total RNA-Seq. Sequencing libraries were prepared using NovaSeq 6000. After quality control, sequence reads were aligned to the human genome build hg38. Differential expression (DE) analyses was conducted using paired multi-factorial ANOVA. The expression of several DE genes identified by cross-referencing Han et al.'s most recent list of glaucoma-associated genes (ANGPTL2, PDE7B, C22orf23, ABLIM2, COL4A1, ADAM12, IFT122, SEMA6C) was validated using EvaGreen-based ddPCR assays. Gene ontology and pathway analyses of the DE genes were performed using the PANTHER, NDEx IQuery, and QIAGEN IPA softwares.
Results :
Differential analysis identified 58 up-regulated genes and 211 down-regulated genes in cells plated on DEX-induced ECM. PANTHER and NDEx IQuery analysis identified critical pathways and genes to be differentially altered. Notably, cytokine and oxidative stress induced inflammation, integrin signaling, matrix remodeling, and angiogenesis pathways were found to be significantly activated across analysis methods as compared to controls. These findings were further supported by previously reported proteomic studies.
Conclusions :
These results support the notion that pathogenic ECM can further induce pathologic changes in cells at least at the transcriptional level. Further studies are needed with a larger and more diverse set of samples in order to confirm if these translate to proteins and in comparison with human patient data.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.