RUNX1 mediates retinal angiofibrosis through endothelial-to-mesenchymal transition (Endo-MT)

Abstract

Purpose : Pathologic angiofibrosis is characterized by the formation of vision-disrupting fibrovascular membranes (FVMs) that grow along the surface of the retina. Found in diseases including proliferative diabetic retinopathy (PDR) and vein occlusions, these conditions are primarily thought to be a consequence of VEGF signaling. However, the fibrosis that often characterizes FVMs is not VEGF mediated. The purpose of this study was to assess the role of Runt-related transcription factor 1 (RUNX1) in the progression of angiofibrosis through the transition of endothelial cells to a mesenchymal phenotype (Endo-MT).

Methods : Expression of endothelial and mesenchymal biomarkers was identified in FVMs from donors with PDR via immunofluorescence (IF). Single cell RNA-seq (scRNA-seq) analysis was performed on patient-derived FVMs using the 10X genomics platform. Primary cultures of human microvascular retinal endothelial cells (HMRECs) were used to evaluate TNFα-induced upregulation of RUNX1 expression and transcriptional activity. Subseqeunt expression of endothelial and mesenchymal genes were determined using molecular biological techniques. The Kimba (TrVEGF029) mouse model of genetically induced VEGF overexpression was used to evaluate the effects of systemic RUNX1 inhibition. Animals were evaluated by fundoscopy and fluorescein angiography.

Results : IF staining revealed colocalization of RUNX1 and mesenchymal proteins in endothelial cells within FVMs. Gene expression profiling of FVM-derived endothelial cells by scRNA-seq supported a transition towards clusters of cells containing both endothelial and mesenchymal genes. In HMRECs, a TNFα-induced increase in RUNX1 expression was observed concomitant with a reduction in endothelial cell markers and an increase in mesenchymal markers. These observations were coincident with increased RUNX1 transcriptional activity. Pharmacological inhibition of RUNX1 was sufficient to prevent Endo-MT in HMRECs. In Kimba TrVEGF029 mice, systemic RUNX1 inhibition significantly improved retinal pathologies relative to untreated controls.

Conclusions : These findings provide insight into how RUNX1 may contribute to the progression of angiofibrosis via Endo-MT, and implicates RUNX1 as a putative angiofibrotic switch. Thus, therapeutics targeting RUNX1 may hinder the onset of vision threatening complications associated with angiofibrotic diseases of the eye.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.