Modulation of endothelial cell function and pathologic ocular angiogenesis by microRNA-150

Abstract

Purpose: Pathologic ocular angiogenesis is a major cause of blindness in several vascular eye diseases. In order to develop effective targeted therapeutics, it is critical to define factors dysregulated in pathologic neovessels. MicroRNAs are small non-coding RNAs regulating gene expression at the post-transcriptional level, and may mediate pathologic angiogenesis. Here we investigated the potential role of a specific microRNA, miR-150, in regulating ocular angiogenesis.

Methods: Two mouse models of pathologic ocular angiogenesis were used: the oxygen-induced retinopathy (OIR) and the laser-induced choroidal neovascularization (CNV). In OIR, neonatal mice were exposed to 75% O₂ from postnatal day 7 to 12. MicroRNA array was performed with OIR retinas compared with normoxic controls, followed by verification with RT-PCR and localization with laser capture microdissected (LCM) retinal neural and vascular layers. Pathologic retinal neovascularization (NV) in OIR was quantified in miR-150 knockout (miR-150^-/-) and wild type (WT) mice, and WT mice injected intravitreally with miR-150 mimic and negative control. For CNV model, adult miR-150^-/- and WT mice were treated with laser photocoagulation and CNV lesions were analyzed at 1 week post laser. Effects of miR-150 on endothelial cell function were analyzed in human retinal microvascular endothelial cells (HRMECs). Putative target genes of MiR-150 were validated in both retinas and HRMECs.

Results: Expression of miR-150 was found highly enriched in LCM retinal blood vessels isolated from normal retinas compared with retinal neurons, and significantly suppressed in pathologic NV isolated from OIR retinas. OIR-treated miR-150^-/- retinas revealed increased retinal NV compared with WT. Intravitreal injection of miR-150 mimic significantly decreased NV in OIR. In laser-induced CNV, miR150^-/- mice showed significantly larger CNV lesions compared with WT. HRMECs treated with miR-150 mimic revealed substantially decreased levels of cell proliferation, migration, and tubular formation. Loss of miR-150 led to strong upregulation of predicted target angiogenic genes (Cxcr4, Dll4, Fzd4, Plxnd1, and Kdr) in OIR retinas in vivo and in HRMECs in vitro.

Conclusions: Our findings indicate that vascular-enriched miR-150 is a novel endogenous inhibitor of pathologic ocular NV. MiR-150 may represent a potential therapeutic target to develop treatments for neovascular eye diseases.