Identification of putative apoptomiRs that prevent endothelial cell proliferation

Abstract

Purpose: Ectopic proliferation and sprouting of endothelial cells are hallmark characteristics of pathological angiogenesis. Inhibiting various pathways in this process may allow the development of more effective treatments for patients suffering from neovascular retinal disease. We have previously reported that anti-mir-132 oligos inhibit neovascularization by inducing quiescence in sprouting endothelial cells. In this study, we introduce the exciting concept of preventing pathological angiogenesis by delivering a microRNA (miR) that promotes apoptosis (apoptomiR) in ectopically sprouting endothelial cells.

Methods: A high-throughput screen to identify dysregulated miRs was performed by exposing cultured human endothelial cells (HUVECs) to ionizing radiation to induce DNA damage and apoptosis. MTT viability assays were performed following transfection of the candidate miR oligos to examine their viability and proliferation capacities. Treated HUVECs were cultured in 3D-collagen matrices to analyze their ability to sprout in a tube-forming assay. Candidate apoptomiRs were also injected in OIR mice at the onset of hypoxia to determine their anti-angiogenic effects in vivo.

Results: Of five candidates identified from the high-throughput screen, miR-34a stood out as a strong apoptomiR candidate due to (1) its dramatic upregulation in HUVECs following ionizing radiation, and (2) its ability to decrease proliferating HUVEC viability upon transfection. Functionally, miR-34a was anti-angiogenic both in vitro (tube forming assay) and in vivo (OIR model). Importantly, miR-34a does not induce gross defects on existing vascular networks.

Conclusions: miR-induced apoptosis in proliferating vascular cells suppresses neovascularization. In this study, we identified miR-34a as a candidate apoptomiR that effectively prevents abnormal endothelial cell sprouting in vitro and in vivo. Mechanistic studies will help further elucidate miR-34a’s potential as a therapeutic agent. miR-34a may be an effective mono-therapy or could be used in combination with other anti-angiogenic compounds to treat a broad host of human ocular neovascular diseases.