SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema

Abstract

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs (miRNAs) from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular miRNAs in concentrated conditioned medium (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEVmiRNA profiles-but not cCMmiRNAs-were impacted by lipedema. Seven sEVmiRNAs (miR-16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR-144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy individuals, whereas only one cCMmiRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema. Lipedema is a chronic, progressive disease characterized by bilateral, symmetrical, disproportional deposition of adipose tissue in the extremities and buttocks 1. Patients suffer from pain, reduced joint mobility, hematoma, edema and psychological impacts 2. It was first described in 1940 as a connective tissue disorder, characterized by fluid being collected in the interstitium instead of entering into lymphatics 3. This excess fluid in the interstitium potentially leads to growth of adipose tissue and hypoxia, which in turn might enhance angiogenesis of pathologic vessels 4,5. The area of lymphatic vessels and the number of blood vessels were found increased in non-obese lipedema patients compared to controls 6. Examination of adipose tissue from lipedema patients demonstrated hypertrophic adipocytes, crown-like structures and increased number of macrophages 6-8. Besides functioning as an energy storage, white adipose tissue (WAT) responds differentially to physiological and pathological metabolic changes by secreting a large diversity of proteins, hormones, lipids, non-coding ribonucleic acids (RNAs)-including microRNAs (miRNAs)-and extracellular vesicles (EVs) 9,10. Small EVs (sEVs) are a fraction of 70-150 nm sized, membrane-enclosed particles, which contain cell-type specific proteins, enzymes, growth factors, cytokines, lipids, as well as coding and non-coding RNAs. It has been repeatedly reported, that WAT-derived vesicular miRNAs are involved in metabolic regulations 11,12 and adipose tissue is considered a significant source of circulating sEV-miRNAs 11. By acting in an autocrine, paracrine as well as systemic manner, these factors can contribute to metabolic abnormalities, modulation of osteogenic differentiation, inhibition of...

MiRNA Profiling in Plasma Neural-Derived Small Extracellular Vesicles from Patients with Alzheimer’s Disease

“…Normalized delta Cq-values (dCq) were used for clustering analysis based on Euclidean distance and complete linkage. Unit variance row scaling was applied for hierarchical clustering and principal component analysis (PCA) [ 30 , 31 ].…”

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…This fact has popularized the use of SEC amongst its competitors for blood-based exosome-associated biomarker discovery research [52], [58]. SEC has been used successfully to isolate, purify and enrich exosomes from a variety of biological fluids including plasma [68]- [73], serum [74]- [76], bovine and human milk [77], [78], urine [79]- [82], saliva [83], [84], tears [83], cerebrospinal fluid [85], [86], synovial fluid [87], nasal lavage [89], seminal fluid [88] and stromal vascular fraction (SVF) from adipose tissue [63], [64] (Table 3). Recent findings reported at the ISEV Virtual Conference 2020 suggest SEC is superior to other techniques in isolation of pure exosomes from human body fluids (unpublished findings).…”

“…SEC coupled techniques are specifically useful within the context of adipose tissue-derived exosomes [61], [63], [64]. Flaherty et al [64] reported that standard isolation techniques are inefficient for extraction of exosomes from adipocytes given their high lipid content (which could affect vesicle density).…”

“…This is a crucial caveat to using density-based dUC for purification of adipose-derived exosomes. A combination technique of SEC and UF was effectively utilized to isolate adipocyte-exosomes from SVF and adipose tissue conditioned media [63], [64]. Another group combined SEC with UC to isolate adipocyte-exosomes (identified by presence of adipocyte markers) from platelet-free human plasma samples [61].…”

“…It is important to note that a small adipose-exosome fraction does not correlate with the effect or clinical relevance of these vesicles. Seven miRNAs contained within adipose-tissue derived EVs, involved in the regulation of adipogenesis, oxidative stress, inflammation, fibrosis and fat metabolism, were differentially expressed in lipedema patients compared to healthy controls, implicating the applicability of adipose-exosomes as biomarkers of disease [63]. Hence, using a purification technique that can use small starting volumes while maximizing yield of functionally intact exosomes free from non-vesicular protein contamination, renders SEC coupled approach to be the optimal method for adipose tissue-derived exosome isolation.…”