Argonaute‐2 Promotes miR‐18a Entry in Human Brain Endothelial Cells

BACKGROUND Cerebral arteriovenous malformation (AVM) is a vascular disease exhibiting abnormal blood vessel morphology and function. miR-18a ameliorates the abnormal characteristics of AVM-derived brain endothelial cells (AVM-BEC) without the use of transfection reagents. Hence, our aim was to identify the mechanisms by which miR-18a is internalized by AVM-BEC. Since AVM-BEC overexpress RNA-binding protein Argonaute-2 (Ago-2) we explored the clinical potential of Ago-2 as a systemic miRNA carrier. METHODS AND RESULTS Primary cultures of AVM-BEC were isolated from surgical specimens and tested for endogenous miR-18a levels using qPCR. Conditioned media (CM) was derived from AVM-BEC cultures (AVM-BEC-CM). AVM-BEC-CM significantly enhanced miR-18a internalization. Ago-2 was detected using western blotting and immunostaining techniques. Ago-2 was highly expressed in AVM-BEC; and siAgo-2 decreased miR-18a entry into brain-derived endothelial cells. Only brain-derived endothelial cells were responsive to the Ago-2/miR-18a complex and not other cell types tested. Secreted products (eg, thrombospondin-1 [TSP-1]) were tested using ELISA. Brain endothelial cells treated with the Ago-2/miR-18a complex in vitro increased TSP-1 secretion. In the in vivo angiogenesis glioma model, animals were treated with miR-18a in combination with Ago-2. Plasma was obtained and tested for TSP-1 and vascular endothelial growth factor (VEGF)-A. In this angiogenesis model, the Ago-2/miR-18a complex caused a significant increase in TSP-1 and decrease in VEGF-A secretion in the plasma. CONCLUSIONS Ago-2 facilitates miR-18a entry into brain endothelial cells in vitro and in vivo. This study highlights the clinical potential of Ago-2 as a miRNA delivery platform for the treatment of brain vascular diseases.