A Liquid Biopsy-Based Approach to Isolate and Characterize Adipose Tissue-Derived Extracellular Vesicles from Blood

Abstract

Obesity is a major risk factor for multiple chronic diseases. Anthropometric and imaging approaches are primarily used to assess adiposity, and there is a dearth of techniques to determine the changes in adipose tissue (AT) at the molecular level. Extracellular vesicles (EVs) have emerged as a novel and less invasive source of biomarkers for various pathologies. Furthermore, the possibility of enriching cell or tissue-specific EVs from the biofluids based on their unique surface markers has led to classifying these vesicles as “liquid biopsies”, offering valuable molecular information on hard-to-access tissues. Here, we isolated small EVs from AT (sEVAT) of lean and diet-induced obese (DIO) mice, identified unique surface proteins on sEVAT by surface shaving followed by mass spectrometry, and developed a signature of five unique proteins. Using this signature, we pulled out sEVAT from the blood of mice and validated the specificity of isolated sEVAT by measuring the expression of adiponectin, 38 adipokines on an array, and several adipose tissue-related miRNAs. Furthermore, we provided evidence of sEV applicability in disease prediction by characterizing sEVAT from the blood of lean and DIO mice. Interestingly, sEVAT‑DIO cargo showed a stronger pro-inflammatory effect on THP1 monocytes compared to sEVAT‑Lean and a significant increase in obesity-associated miRNA expression. Equally important, sEVAT cargo revealed an obesity-associated aberrant amino acid metabolism that was subsequently validated in the corresponding AT. Lastly, we show a significant increase in inflammation-related molecules in sEVAT isolated from the blood of nondiabetic obese (>30 kg/m2) individuals. Overall, the present study offers a less-invasive approach to characterize AT.

Chymase in Plasma and Urine Extracellular Vesicles: Novel Biomarkers for Primary Hypertension

“…Since an extensive characterization of plasma EVs had been performed by us in prior studies 8,10,32 , we limited the current analysis to confirm the expression of exosomal biomarkers CD63, TSG101, and Alix in urinary EVs by Western blotting ( Figure 2 ). Importantly, we did not observe any expression of endoplasmic reticulum protein Calnexin and Golgi protein GM130 in urinary EVs, confirming the purity of isolated EVs.…”

“…The cargo of these vesicles relates to the pathophysiological state of the parent cells. In support of the importance of characterizing EVs as a tissue-specific biomarker, we recently reported that EVs in the plasma could inform about corresponding molecular and metabolic alterations in the visceral adipose tissues of diet-induced obese mice 8 . The relevance of these experimental observations was further strengthened by the parallel finding of augmented inflammation-related molecules in adipose tissue-derived Evs isolated from the blood of obese patients 8 .…”

“…In support of the importance of characterizing EVs as a tissue-specific biomarker, we recently reported that EVs in the plasma could inform about corresponding molecular and metabolic alterations in the visceral adipose tissues of diet-induced obese mice 8 . The relevance of these experimental observations was further strengthened by the parallel finding of augmented inflammation-related molecules in adipose tissue-derived Evs isolated from the blood of obese patients 8 . Similarly, we recently reported the usefulness of tissue-specific sEV in plasma as usefulness biomarkers to assess neurocognitive decline as well as to predict treatment response in individuals with mild cognitive impairment [9][10][11] .…”

“…Flow cytometry was performed to assess the surface expression of exosomal tetraspanin markers (CD63, CD9 and CD81), as described by us previously 8,10 . Briefly, EVs were labeled with membrane labeling dye CellBrite 488 (Biotium, California, USA) with or without the CD63-PE (353004), CD81-PE (349506) (both from BioLegend, CA, USA), or CD9-APC (Invitrogen:17-0291-82) antibodies.…”

Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EVISF) reveals sex‐dependent changes in microglial EV proteome in response to Aβ pathology

“…Flow cytometry analysis was performed to evaluate the percentage of typical EV tetraspanin markers following methods and controls reported recently (Kumar et al, 2022(Kumar et al, , 2023Mishra et al, 2023)…”