Encapsulated RUNX1 inhibitor (Ro24-7429) reduces angiogenesis in retinal endothelial cells

Abstract

Purpose :
Overexpression of runt-related transcription factor 1 (RUNX1) has been recently linked to aberrant angiogenesis in proliferative diabetic retinopathy and choroidal neovascularization. Topical delivery of small-molecule inhibitors of RUNX1 such as Ro24-7429 could lead to a novel and non-invasive treatment for a wide variety of angiogenic diseases of the eye. This study evaluated the in vitro effect of a nanoemulsion loaded with Ro24-7429 on migration and proliferation of human retinal endothelial cells (HRECs).

Methods : Ro24-7429 dispersed in the oil phase was homogenized and subsequently sonicated to fabricate an oil in water nano-emulsion. Dynamic light scattering (DLS) measured the drop size of the nanoemulsion. In vitro cytotoxicity and proliferation assays of unloaded (eNanoV), loaded (eNanoRo24) nanoemulsions, and Ro24-7429 in DMSO were used to test several concentrations. Scratch wound assay was performed for the evaluation of cell migration. Immunofluorescence staining (IF) for RUNX1 and delta-like four (DLL4) was carried on to assess the tip and stalk morphology of HRECs.

Results : We confirmed that the nano-emulsion constitutes lower than 200 nm in drop size. eNanoV and eNanoRo24 were not toxic for the HRECs even at the highest concentration tested of 8% v/v at 48h. A significant reduction (P-value<0.5) of proliferation and migration were observed after treatment. eNanoRo24 had higher reduction of proliferation than Ro24-7429 in DMSO. IF described a reduction of RUNX1 as well as DLL4 on treated HRECs. DLL4 is a protein-related to the tip phenotype of endothelial cells in sprouting angiogenesis.

Conclusions : eNanoRo24 reduced the proliferation and migration of HRECs in vitro. We did not observe any toxicity with application of eNanoRo24 in HRECs. Also, eNanoRo24 may improve the activity of the Ro24-7429. The inhibition of migration may be mediated by a change in the tip/stalk morphology. The reduction of both migration and proliferation strongly suggests that eNanoRo24 modulates the activity of endothelial cells and the angiogenic process, thus demonstrating the therapeutic potential of this drug delivery system.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.