Abstract
Purpose :
Cold preservation of donor corneas is the standard practice in the USA and many other countries. It is known that cold storage of corneas causes cell death, damage to the actin cytoskeleton, and possibly prolonged stress signaling secondary to oxidative stress. To further explore damage to the endothelium, this study has investigated altered gene expression in the endothelium following cold storage of donor corneas.
Methods :
Total RNA was extracted from donor corneas held in cold storage (4° C) for 3 days (Short-term cold storage, SCS; n = 2), 7 days (Intermediate cold storage ICS; n = 2), and 14 days (Long-term cold storage; LCS; n = 2) using RNeasy kit. The samples were analyzed using Illumina NextSeq 500. Fastq data files were downloaded, and data was aligned using STAR (v2.7.10b), where Hg38.p13 was used as the reference genome. Read counts for each gene for all 6 samples were obtained using HTSeq (v2.0.4). Differentially expressed genes were obtained using DESeq2 implemented in R 4.2.2. The pathway analysis was performed using FunRich 3.0.
Results :
Bulk RNA sequencing showed the number of differentially expressed genes in short, intermediate, and long storage. Between SCS vs. ICS, 782 and 668 genes were upregulated (log2fold change > 1) and downregulated (log2fold change < -1), respectively. In contrast, between SCS vs. LCS, 405 and 414 genes were upregulated and downregulated, respectively, and between ICS vs. LCS, 1105 and 1213 genes were upregulated and downregulated, respectively. In independent experiments, we have observed CS to induce significant morphological changes and adversely affect the barrier integrity of the endothelium. Pathway analysis using the differential expressed genes showed a significant alteration in the nuclear transcriptional activity, exosomes, and ECM. Specifically, we noticed reduced Wnt transcripts concurrent with reduced HMGB2 expression, indicating Wnt-mediated alterations in ECM and cytoskeletal proteins during CS.
Conclusions :
Altered transcriptomics can be implicated in the acute and long-term damage to the corneal endothelium secondary to cold storage. We have observed CS-induced downregulation of Wnt signaling, indicating a potential role of supplementing Wnt proteins to the Optisol™ medium.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.