Abstract
Purpose :
Damage to the trabecular meshwork (TM) is commonly thought to lead to elevated intraocular pressure (IOP) and glaucoma development, but the exact mechanisms that increase IOP remain unresolved. Prior studies indicate Dexamethasone(Dex)-induced glaucoma may involve increased Wnt signaling activity. This study employs RNA sequencing to investigate the specific changes in gene expression that can result from Dex treatment of human TM cells.
Methods :
The TM from 3 human corneal tissue (TM 3, TM 4, and TM 5) were isolated and plated with 20% fetal bovine serum (FBS) media. After reaching confluency, each TM cell line was treated with dexamethasone (D) or ethanol vehicle (V). RNA was isolated from each group, and the data was sequenced using Illumina HiSeq 3000. The data was demultiplexed using Illumina Bcl2fastq2 v. 2.17. Using Bowtie2 v. 2.1.0, the reads were mapped, and gene expression was determined using RSEM v. 1.2.15. The data was normalized using the trimmed mean of M-values. Data analysis was performed using Rstudio. Heatmaps were generated using the pheatmap R package and were scaled by row.
Results :
RNA sequencing data was converted into heatmaps on Rstudio. Both figures displayed similar relationships across genes and treatment groups. DKK1 had lower expression in Dex treated groups, particularly TM 5 cells. SFRP1 and SFRP 4 expression was lower in Dex treated groups. FZD4 showed a stark increased expression in Dex treated groups, especially TM 5 cells.
Conclusions :
Overall, Dex treatment caused wide-ranging changes in the expression of Wnt signaling genes, consistent with the idea that Wnt signaling plays a key role in the human TM’s function of regulating IOP. This is supported by a decreased expression of DKK1, SFRP1 and SFRP4, which are Wnt signaling inhibitors, in Dex-treated groups. Similarly, FZD4 is a receptor in the canonical Wnt pathway, where increased expression leads to greater Wnt signaling. Together, these results indicate a potential mechanism which involves the reduction of Wnt inhibition in glaucomatous TM cells.
This is a 2021 ARVO Annual Meeting abstract.