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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE312883 | GEO | 2026/01/26

REPOSITORIES: GEO

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			Action	DRS
		Other
	GSE312883_ISCH_Muscle_Processed_DATA.csv.gz	Csv
	GSE312883_lEVs_Processed_DATA.csv.gz	Csv
	GSE312883_sEVs_Processed_DATA.csv.gz	Csv

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Project description:Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these 4 were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions.

Project description:: Type 2 diabetes (T2D) is associated with increased morbidity, mortality, and substantial healthcare costs. Peripheral insulin resistance, involving interconnected dysregulation of multiple organs, is considered a major driver of T2D. Extracellular vesicles (EVs) are suggested as mediators of this dysregulation based on their properties in intercellular communication. Given the role of skeletal muscle in glucose metabolism, the content of skeletal muscle-derived EVs may provide insights into mechanisms of T2D. To examine this, myotubes from severely obese female T2D donors and matched women with normal glucose tolerance (NGT) were cultured. Small EVs (sEVs) were isolated by differential centrifugation and filter columns and characterized by nanoparticle tracking analysis, flow cytometry, and transmission electron microscopy. The micro-RNA (miR) content of sEV was analyzed via Affymetrix microarray, while proteins were detected by LC-MS/MS. No group differences were found in sEV concentration, size, or EV-marker levels. In total, 495 proteins were detected in the sEVs, of which 55 were unique to the T2D group and 2 to the NGT group. Principal component analysis showed distinct clustering, demonstrating clearly different protein profiles. Quantification of the protein cargo revealed 194 proteins with significantly higher levels and 21 with significantly lower levels in the T2D group. While 208 miRs were detected, no significant group differences were observed. However, 40 miRs were unique to the T2D group and 5 to the NGT group. Pathway analysis of protein and miR data revealed associations with EV-related mechanisms such as exocytosis and protein homeostasis, as well as T2D-relevant pathways including some involved in glucose metabolism, inositol metabolism, and extracellular matrix organization. In conclusion, myotube-derived sEVs from severely obese female donors with or without T2D showed distinct proteome-profiles, however, no differences were observed in the miR content. Other sEV characteristics were similar between the groups.

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

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