Abstract
Purpose :
To understand the mechanisms underlying the scleral response to IOP elevation, we performed bulk RNA sequencing (RNA-seq) analysis of mouse sclera following bead-induced (BI) ocular hypertension (OHT). We identified and validated induction of cellular senescence by OHT.
Methods :
OHT was induced in adult C57BL6J mice (n=15) by anterior chamber BI and IOP was followed serially by rebound tonometry. Seven days after BI, eyes were harvested, axial length and width were measured, sclera were dissected, and RNA was isolated. Whole genome sequencing was performed on a NovaSeq 6000 at 2x100 bp read length and gene sequence enrichment analysis (GSEA) was used to identify regulated pathways. Transcripts of interest were validated using RT-qPCR of samples from independently conducted experiments. Beta-galactosidase (B-gal) assays were performed on scleral flatmounts from CD-1 mice and visualized under light microscopy to identify senescent scleral stromal cells. Human scleral fibroblasts were serially passaged or treated with bleomycin (50 mcg/ml) to induce senescence.
Results :
Gene expression profiles following OHT are distinct. After 7 days of OHT, 831 transcripts were upregulated and 595 were downregulated (2-fold, q-value 0.05) compared to control sclera from OHT mice. Signaling pathways regulated included epithelial to mesenchymal transition, inflammatory response, IL6-STAT3, hypoxia, and two senescence gene sets (SenMayo, q-value 9.1E-19; and Rat Aging, q-value 3.6E-21). Induction of senescence-associated secretory phenotype (SASP) transcripts (IL6, MMP13, CXCR2, CXCL2, IL1b, VEGF, uPAR, FN) and cell-cycle exit (CDKN1A, CDKN2A) markers was validated using RT-qPCR of independent experiments. OHT led to a significant increase in B-gal positive cells in scleral stroma (5.6 cells/field in OHT versus 0.25 cells/field in controls, p<0.001, n=4; 16 images). Serial passage of primary scleral fibroblasts led to widespread cell senescence as assessed by B-gal assay after 6-9 passages and as did treatment of early passage cells with bleomycin for 48 hours.
Conclusions :
We identified pathways enriched in OHT sclera and validated evidence of OHT-induced scleral cell senescence. These findings could aid in further understanding the mechanisms that underly IOP-induced scleral remodeling in glaucoma and pathologic myopia.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.