Abstract
Purpose :
Sub-retinal fibrosis, observed at the end stage of neovascular age-related macular degeneration (AMD), is the leading cause of irreversible blindness in AMD. Although the pathogenesis of sub-retinal fibrosis is still unclear, excessive ECM appears to be a key event. Interestingly, the source of excessive ECM proteins, such as collagen, is unknown since fibroblasts are not resident in the healthy retina. Here, we create an atlas of collagen producing cells in the retina using Collagen-1 (Col1a1)-YFP reporter mice. We compare the dynamics of collagen production and cell types involved, in vivo over time, in mice receiving one- and two-stage laser-induced choroidal neovascularization (LiCNV) model to model subretinal wound healing and fibrosis.
Methods :
LiCNV was carried out using the Micron IV platform (532nm, 300mW, 100ms, 50um spot size, 3 spots per eye) in 12 week old Col1-YFP reporter mice. After 7 days, a cohort of the mice received a second laser burn directly over the initial lesion. On days 5 (1-stage) and 17 (2-stage), retinal tissue was harvested and retinal pigment epithelium (RPE)/choroid was flatmounted and regions around the CNV lesions were excised. Tissue was digested and live single cells were sorted into YFP+ and YFP- groups using fluorescence-activated cell sorting (FACS). YFP+ vs YFP- RPE/choroid tissue was further analysed by scRNA sequencing using 10x genomics and illumina NextSeq 500 platform.
Results :
FACS demonstrated negligible YFP+ cells in the retina compared to the RPE in both LiCNV models, indicating that cells involved in collagen production were not in the retina but in the RPE tissue. scRNAseq analysis revealed that fibroblasts, fibrocytes and pericytes all expressed transcript for Col1a1, however Col1a1 protein expression identified by Col1a1-YFP positivity clearly defined the fibroblast group into Col1a1 high-expressing subsets and Col1a1 low-expressing subsets. We further identified subclusters of disease-associated fibroblasts (DAFs) found only in injured tissue. We also found two well-defined Col1a1-YFP- cell clusters that appear only in the 2-stage fibrosis model.
Conclusions :
We have identified a gene signature for DAFs that are found in both 1- and 2-stage LiCNV injury models, and have also identified cell clusters that are found only in the 2-stage model of subretinal fibrosis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.