Abstract
Purpose :
Although healthy, non-glaucomatous Schlemm’s canal endothelial cells (SCECs) react to routine mechanical stress to maintain intraocular pressure (IOP) homeostasis, the direct effect of IOP on SCEC gene expression remains unclear. Using an experimental model of cyclic mechanical stretch (CMS), we sought to identify expression changes in mRNAs and lncRNAs in primary human SCECs in response to stretch.
Methods :
To mimic changes in IOP, SCECs from healthy human donors (n=3) were subjected to 15% CMS (1 cycle/sec) or without stretch for 24 hours using a Flexcell Tension 6000 system. Total RNA was isolated, and sequencing libraries were prepared using the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50bp sequencing with Illumina NextSeq 500. Sequencing reads were aligned to human genome hg38 and annotated/quantified against GENCODE release 38. After normalization, DESeq2 was used to identify differentially expressed mRNAs and lncRNAs. Transcripts with |fold change| (FC) > 1.5 and false discovery rate (FDR) < 0.1 were considered to be significant. WebGestalt and Ingenuity Pathway Analysis were used to perform gene ontology and pathway analyses. Reverse transcription and droplet digital PCR (ddPCR) were performed to validate differential expression of four select genes (DHCR7, PMEPA1, SCD, and FIBIN) in reference to GAPDH expression. Student’s t-tests with a significance level of 0.05 were used to compare normalized cDNA fold changes.
Results :
Through stranded total RNA-Seq, we identified 194 mRNAs and lncRNAs to be differentially expressed (|FC| > 1.5, FDR < 0.1) in stretched vs. non-stretched SCECs. These genes were particularly enriched in pathways involving sterol biosynthesis, extracellular matrix binding, and microtubule regulation. Our ddPCR assays successfully validated the up-regulation of DHCR7, PMEPA1 and SCD with FCs of 3.4, 2.2, and 3.1, respectively (t-test, P < 0.05), while FIBIN’s validation was borderline significant with a FC of 1.8 (t-test, P = 0.08).
Conclusions :
This was the first study to analyze genome-wide expression changes as a response to stretch in human SCECs. These results may help identify the pathways regulated by healthy SCECs in response to dynamic IOPs. Significant upregulation of IOP-associated cholesterol biosynthetic pathways may suggest a crucial role of membrane fluidity in healthy IOP homeostasis in SCECs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.