Hypercholesterolemia impairs the expression of angiogenic microRNAs in extracellular vesicles within ischemic skeletal muscles

Background/Objectives: 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. Methods: We used a mouse model of PAD and compared the response to hindlimb ischemia in hypercholesterolemic ApoE-/- vs. normocholesterolemic mice. Next generation sequencing (NGS) was used to perform a complete profiling of miR expression in ischemic skeletal muscles, and in EVs of varying sizes — large EVs (lEVs) and small EVs (sEVs) — within these muscles. Results: 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 show that 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 prediction of functional miR targets, pathway unions and gene unions, identified predictive resulting effects of HC-modulated miRs in EVs on processes having key roles in the modulation of angiogenesis and neovascularization, such as the regulation of actin cytoskeleton and focal adhesion, and HIF-1, MAPK, AMPK and PI3K-Akt signaling pathways. Conclusions: Our results constitute an important first step towards the identification of miRs that could be targeted to improve the angiogenic function of EVs in atherosclerotic conditions and reduce tissue damage in patients with severe PAD. Overall design: small RNAseq profiling of ischemic muscles and of sEVs and lEVs from ischemic muscles from ApoE ko mice on western diet chow for 4 weeks prior to ischemia induction and from control C57Bl/6 on regular chow.