Unraveling the web of nAMD: A transcriptomic analysis of age-related macular degeneration-associated cytokines in primary human retinal endothelial cells.

Neovascular age-related macular degeneration (nAMD) is a complex, blinding retinal disease underpinned by a dynamic and dysregulated interaction among pathogenic cytokines. The present study investigates the impact of six nAMD-associated cytokines, namely transforming growth factor-beta 1 and 2 (TGF-ß1/2), tumour necrosis factor-alpha (TNF-a), thrombin, interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), on the transcriptome of primary human retinal endothelial cells (HRECs), both individually and as a collective 'cocktail'. HRECS were treated for 24 hours before being harvested for bulk RNA-seq. In performing this study, we have described the different transcriptomic signatures of each cytokine in driving nAMD-associated inflammatory, angiogenic and fibrotic pathways when presented in isolation to primary HRECs. Additionally, this study dissects a novel 'cocktail' effect in the collective group characterised by further differential expression of genes, some of which were not induced by any of the individual cytokines. We explore the translatability of our study as an in vitro model of nAMD in endothelial cells by mapping the identified differential expression patterns to marker gene sets identified in previously performed scRNASeq of nAMD-affected human donor eyes. Overall design: n = 6 replicates of primary human retinal endothelial cells (HRECs) were treated with either no treatment (control), transforming growth factor-beta 1 or 2 (TGF-ß1/2), tumour necrosis factor-alpha (TNF-a), thrombin, interleukin-6 (IL-6) or vascular endothelial growth factor (VEGF), individually (all at 10 ng/ml) or as a combination of all 6 treatments with the same concentration as individual treatments (i.e. 10 ng/ml) for 24 hours, RNA extraction was performed at the same time point for all groups, followed by sequencing. We then performed gene expression profiling on each group with reference to the untreated control. Mapping of obtained differential expression profiles to open-access RNA-seq datasets was then performed to assess translatability of cytokine-treated HRECs as an in vitro model of retinal neovascularisation.