5224Endothelial autophagy triggers nuclear enrichment of miR-126-5p via a Mex3a-dependent pathway to confers endothelial protection and prevent atherosclerosis
Authors: Santovito D , Natarelli L , Egea V , Bidzhekov K , Blanchet X , Mourao A , Wichapong K , Aslani M , Horckmans M , Lutgens E , Von Hundelshausen P , Duchene J , Steffens S , Sattler M , Weber C 
Published: October 2019

Abstract

Background
MicroRNAs are versatile regulators of gene expression with profound implications for cardiovascular diseases and atherosclerosis. Among the most expressed in endothelial cells (ECs), the miR-126 duplex is crucial for angiogenesis and vascular protection, featuring different functional roles and homeostasis of the two strands. In line with separate activities, miR-126-3p and -5p undergo differential regulation in response to shear-stress in a KLF2-dependent manner. As both strands derive from a common precursor, the mechanism for strand specificity is unrelated to miRNA transcription and remains largely elusive as well as unknown is its relevance for vascular biology.

Methods and results
In human ECs (HUVECs) overexpression of KLF2 produced an up-regulation of the miR-126 precursor and miR-126-5p, but not miR-126-3p. Analysis of ECs overexpressing KLF2 or exposed to high-shear stress at transcriptional and protein level revealed the activation of the autophagic flux. Moreover, stimulation of autophagy by rapamycin replicated strand-specific regulation of miR-126. In particular, rapamycin promoted miR-126-3p degradation, while miR-126-5p was preserved and translocated to a nuclear reservoir complexed with the protein argonaute-2 (Ago2). Mutational scanning of fluorescently-labelled miR-126-5p revealed that nuclear shuttling required motifs distinct from the seed sequence. Size exclusion chromatography and Ago2-immunoprecipitation in autophagic ECs, as well as surface plasmon resonance (SPR) and electrophoretic mobility shift assays in vitro showed the formation of a ternary complex with the RNA-binding protein Mex3a. Preferential binding of miR-126-5p to Mex3a was confirmed by SPR, isothermal calorimetry, and nuclear magnetic resonance spectroscopy. Super-resolution microscopy by stimulated emission depletion (STED) visualized the Mex3a/Ago2 interaction on autophagosomal surfaces and silencing of Mex3a dampened rapamycin-induced nuclear miR-126-5p enrichment. Gain- and loss-of-function studies for miR-126-5p and Mex3a showed that Mex3a-guided nuclear miR-126-5p affect apoptosis mediators (e.g. caspase-3) at RNA and protein level, thus reducing the apoptotic rate. High-shear flow at non-predilection sites for atherosclerosis induced endothelial autophagy to promote nuclear enrichment of Ago2 and miR-126-5p in vivo in mice. Consistently, fewer nuclear Ago2 and miR-126-5p were observed in the endothelium of atheromatous area at bifurcations of human carotid arteries. These effects were abrogated in vivo by endothelial-specific deficiency in autophagy, thereby exacerbating atherosclerosis, and by Mex3a knock-out favouring apoptosis.

Conclusion
The autophagy-activated Mex3a-driven nuclear translocation represents a non-canonical mechanism by which miR-126-5p confer endothelial protection. Modulation of this pathway may open new opportunities for prevention and treatment of atherosclerosis.