Abstract
Purpose :
Monocytes play a major role in the pathogenesis of age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). This study aimed to gain additional insight into the role of each monocytes subtype in the pathogenesis.
Methods :
PBMCs from AMD/PCV patients (n=10), and healthy control (n=9) were collected. Ficoll separations and FACS sortings of monocytes were carried out followed by loading of five thousand monocytes per well per sample into the chip for generation of barcoded cDNA libraries using chromium single cells 3’ reagent kits v3 of 10X Genomics. Libraries were sequenced on Illumina HiSeq X. Data were aligned to GRCh38 using CellRanger v.4.0.0. Potentially empty or multiple cells droplets were removed and the cells with 300~7,000 genes, UMI count 500~40,000 and mitochondrial reads ≤15% were kept. Residual multiplets were removed using DoubletFinder v.2.0.3. UMI counts were normalized using RCAv2. Identification of major cell type plus quality control was done using GlobalPanel in the RCAv2 followed by graph-based Louvain clustering from Seurat v.4.0.5. Finally, a total of 22,593 high quality monocytes were analyzed. Harmony v.1 was used for batch correction. To compare the diseases versus normal in each monocyte subtype for differentially expressed genes (DEG), FindMarkers function was used. Gene Set Enrichment Analysis (GSEA) was done using fgsea v.3.13 and molecular signature database datasets for Biocarta, KEGG, Reactome, WikiPathways and Gene Ontology at the website; https://www.gsea-msigdb.org/gsea/msigdb/.
Results :
A total of 22 genes were commonly upregulated while 61 genes were commonly downregulated in both AMD and PCV. GSEA showed a significant negative enrichment of caveola in classical monocytes of PCV (NES: -1.769, p=0.000, q= 0.026). Furthermore, a significant slit-robo signaling pathway was positively enriched in classical and intermediate monocytes of PCV (NES: 2.264, p=0.000, q=0.000 & NES: 2.446, p=0.000, q=0.000) and classical monocyte of AMD (NES: 1.994, p=0.000, q=0.001).
Conclusions :
Deficiency of caveola, especially of its structural core proteins, caveolins, and increasing of slit-robo signaling pathway in monocytes may contribute to the pathogenesis of AMD/PCV. Functional analysis of caveolins and slit-robo signaling pathway are recommended to obtain additional novel findings.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.