Abstract
Purpose :
Metabolic adaptations and perfusion abnormalities lead to extracellular acidosis in atherosclerosis, neurodegeneration, and cancer. We speculate that this also occurs in Age Related Macular Degenration (AMD) and examined the role of acidosis on ARPE19 cells in culture. We previously observed that extracellular acidosis decreases ARPE19 cell glycolysis, mitochondrial respiration, ATP and NAD/NADH. These data suggest that metabolic/mitochondrial dysfunction could be an important component in AMD pathology. We examined the acute and chronic effects of extracellular acidosis on transcriptional changes in ARPE19 using RNA sequencing.
Methods :
Fully confluent 14 day cultures of ARPE19 cells were exposed to pH 7.4 or pH 6.5 for 12, 24 or 48 hours. Total RNA was extracted and analyzed on the Illumina NovaSeq 6000 platform with polyA selection library. The fastq files were aligned to human genome (GRCh38.p10) using STAR 2.4.1a. The transcript abundance was estimated using RSEM 1.2.22. The normalization and differential expression (DE) analysis were performed using edgeR and limma-voom R packages to identify the DE genes between pH 7.4 and pH 6.5 at different time points. The enriched pathways at each time point were obtained using GSEA.
Results :
We identified 326, 243 and 138 DE genes (absolute log2 fold change ≥ 1 and adjusted p-value < 0.05) at 12 hours, 24 hours and 48 hours, respectively, demonstrating the highest acidosis-induced gene expression changes occur at the earliest time point. The follow up gene set enrichment analysis showed ARF6 pathway, lysophospholipid pathway and TNF-Alpha signaling via NFκB were significantly altered in ARPE19 cell lines exposed to pH 6.5 at 12hrs (p-value < 0.05). Pathways associated with cell cycles were consistently down-regulated at all three time points.
Conclusions :
The results demonstrated that extracellular acidosis reprograms ARPE19 cell gene expression within 12 hours. The gene set enrichment analysis showed that the low pH environment interferes with multiple pathways related to AMD. Further study is required to determine how extracellular acidosis affect those pathways and to validate their potential role in macular degeneration.
This is a 2021 ARVO Annual Meeting abstract.