Hypercholesterolemia Impairs the Expression of Angiogenic MicroRNAs in Extracellular Vesicles Within Ischemic Skeletal Muscles

<b>Background/Objectives</b>: In severe peripheral artery disease (PAD) with limb ischemia, hypercholesterolemia (HC) is associated with impaired neovascularization. Extracellular vesicles (EVs) are present within ischemic muscles, and they contain microRNAs (miRs) involved in several biological functions, including angiogenesis and neovascularization. <b>Methods</b>: We used a mouse model of PAD and compared the response to hindlimb ischemia in hypercholesterolemic ApoE<i>^-/-</i> vs. normocholesterolemic mice. Next-generation sequencing (NGS) was used to perform full miR expression profiling in ischemic skeletal muscles and in EVs of varying sizes-large EVs (lEVs) and small EVs (sEVs)-within these muscles. <b>Results</b>: We identified several miRs with potential pro-angiogenic effects (angiomiRs) that are reduced by HC in lEVs (Let-7b-5p, miR-151-3p, Let-7c-5p) or sEVs (miR-21a-5p, miR-196b-5p, miR-340-5p). As proof of principle, we showed that the overexpression of Let-7b-5p in lEVs, or miR-21a-5p in sEVs, can significantly increase the angiogenic capacity of these EVs in vitro. HC also impaired the enrichment of specific angiomiRs in lEVs (miR-100-5p), sEVs (miR-142a-3p), or in both lEVs and sEVs (miR-146b-5p). In silico approaches, including the prediction of miR targets, pathway unions, and gene unions, identified the resulting predictive effects of HC-modulated miRs in EVs on processes with key roles in the modulation of angiogenesis and neovascularization, such as the regulation of the actin cytoskeleton and focal adhesion and the HIF-1, MAPK, AMPK, and PI3K-Akt signaling pathways. <b>Conclusions</b>: Our results constitute an important first step towards the identification of specific miRs that could be targeted to improve EV angiogenic function in hypercholesterolemic conditions and reduce tissue ischemia in patients with severe PAD.