Abstract
Purpose :
Subretinal fibrosis often develops in those with longstanding neovascular age-related macular degeneration (nAMD). While anti-VEGF therapy has proven effective in treating nAMD, fibrosis is correlated with poor vision in a majority of patients and remains untreatable. This study examined the immunological changes and efficacy of two antibody therapies on the development of subretinal fibrosis in a mouse model of choroidal neovascularisation (CNV).
Methods :
An image guided photocoagulation laser was used to induce CNV via applying 4 lesions (Micron III, 532nm, 350mW, 50mm, 70ms) in 8-week-old C57BL/6J mice (n=4-20). Mice were intravitreally injected with an antibody against CD47, CD74, or vehicle (PBS or saline). At 2 and 7 days post laser injury, eyes were enucleated for histology, and RPE-choroid isolated for flow cytometry (CytoFLEX S and LX) and RNA-seq (NovaSeq 6000). Differentially expressed genes (DEGs; FDR<0.05) and Gene Ontology (GO) enrichment were determined using EdgeR and clusterProfiler R packages respectively. CNV leakage area was assessed by fluorescein angiography and quantified using a customised ImageJ macro. Thickness of fibrotic lesions was measured using Masson trichrome-stained paraffin sections and ImageJ.
Results :
The percentage of myeloid leukocytes (CD45+CD11b+), neutrophils (Ly6G+) and inflammatory monocytes (Ly6C-Hi) increased significantly in RPE-choroid at 2 days post laser injury relative to untreated control mice (P<0.05) and returned to baseline at 7 days (P>0.05). These were also accompanied by a significant increase of pro-fibrotic CXCR4+ myeloid cells at 2 days (P<0.05). Moreover, GO analysis revealed activation of pathways associated with myeloid leukocyte chemotaxis, fibroblast proliferation or migration. Notably, DEGs involved in CXCR4 signalling, including Cd74 and CD47 ligand Thbs1, were significantly upregulated in CNV. However, treatment with anti-CD47 or anti-CD74 antibody failed to reduce CNV leakage or fibrosis 7 days post laser injury (P>0.05).
Conclusions :
We identified a recruitment of neutrophils and inflammatory monocytes to the subretinal space following induction of laser-induced CNV. Although our findings pointed a recruitment of pro-fibrotic myeloid cells during CNV and subretinal fibrosis, targeting chemotaxis via CD47 or CD74 did not attenuate pathology suggesting more work is needed to understand the mechanisms of subretinal fibrosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.