EB3 inhibitor increases chromatin accessibility and reprograms neovascular endothelial cells.

In our previous studies, we described a critical role of microtubule-associated end binding protein 3 (EB3) in regulating pathological calcium release from endoplasmic reticulum (ER) stores through interaction with inositol 1,4,5-trisphosphate (IP3R3) within endothelial cells. The short cognate peptide we designed based on this interaction blocked VEGF-evoked ER calcium release and decreased laser-induced choroidal neovascularization (CNV) in both mouse and non-human primate models of age-related macular degeneration. Here we analyzed the global effects of this peptide on choroidal and retinal cells using single nuclei-RNA (snRNA) and single nuclei-assay for transposase-accessible chromatin (snATAC) sequencing of non-human primate eye tissue. Within the snRNA-seq transcriptome data, we detected 18 different cell types residing in the retina and choroid using well-established markers. For the characterization of snATAC-seq clusters, we performed Pearson correlation of the genes expressed in each cluster of our snRNA-seq data and the peaks found in each cluster of our snATAC-seq data. Our snATAC-seq analysis demonstrated a widespread increase in chromatin accessibility at the promoter region across different cell types. These changes in chromatin accessibility were associated with increased acetylation in lysine 27 of histone 3 (H3K27ac) but not lysine 8 of histone 4 (H4K8ac). Concurrent analysis between our snATAC-seq and snRNA-seq data further revealed increased chromatin opening of MEIS2 and PAX6 within our metabolic-active endothelial cells of neovascularized lesions, leading to activation of downstream transcription factors such as NCOR2, NR2C1, SREBF2, TCF4, TCF12, TEF, and THRA. The activation of this transcriptional program promoted neovascular regeneration and healing of ablated regions within CNV lesion of non-human primate eye in the treatment group. Taken together, we concluded that EB3 inhibition prevented aberrant angiogenesis through epigenetic reprogramming of injured endothelial cells and through activation of MEIS2 and PAX6's transcriptional program.