Abstract
Purpose :
The choroid is a vascularised and pigmented stroma that supplies the outer retina. In late age-related macular degeneration, the choroid is the site from which aberrant blood vessel growth occurs. The aim of this study was to investigate the stromal cells, including fibroblasts, and their gene expression under normal and disease conditions using a laser model of neovascular AMD
Methods :
Male mice aged 10-12 weeks (strains: C57/Bl6 and TCRd-/-) underwent laser photocoagulation (Micron III, 523 nm light, 350 mW, 70 ms) to introduce 4 lesions/eye a model of nAMD. Seven days later lasered mice (n = 6) and unlasered mice (n=6) were euthanized. Eyes were dissected and the anterior eye, lens and retina were discarded. The RPE/choroid dissociated into a single cell solution using an enzymatic dissociation per N. Cells were labelled with DAPI as live/dead marker, 10X CellPlex, TotalSeq A, an epitope label for 174 antibodies Cells were sorted on BD Aria Fusion and underwent single cell capture using the 10X Chromium 3’HT kit. Sequencing was undertaken using NextSeq 2000 (Illumina). Reads were aligned and quantified against the mm10 reference genome annotations using Cell Ranger (v7.1) with downstream analysis performed in R and Bioconductor.
Results :
There were approximately 170,000 RPE/choroid live cells sorted with 24,839 biologically relevant cells captured and sequenced. Unbiased clustering using UMAP resulted in 23 clusters. The bulk of cells identified were stromal cells (46%) for clusters (5-8). Cluster 8 is proposed as fibroblasts. Cluster 8 had high gene expression for extracellular matrix genes GSN, CD34, COL3A1, IGFBP6, MFAP5, the vascular endothelial growth factor receptor (FLT1), and high epitope expression of CD36.
Conclusions :
The current study revealed the bulk of choroidal cells were stomal cells. This study identified CD36 a glycoprotein receptor for thrombospondin on stomal cells. This marker, CD36, has the potential to identify pathological fibroblasts in choroidal neovascularisation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.