Multiparametric Bulk and Single Extracellular Vesicle Pipeline for Identifying Adipose-Specific Signatures in Matched Human Biospecimens

Obesity remains a growing and global public health burden
across
a broad spectrum of metabolic, systemic, and neurodegenerative diseases.
Previously considered merely a fat storage depot, adipose tissue is
now recognized as an active endocrine organ crucial for the metabolic
and systemic regulation of local and distant organs. A burgeoning
line of investigation centers on adipose-derived extracellular vesicles
(ADEVs) and their pivotal role in obesity-associated pathobiology.
However, robust methodologies for specifically isolating and characterizing
human ADEVs are lacking. To bridge this gap, we developed a robust
multiparametric framework incorporating bulk and single EV characterization,
proteomics, and mRNA phenotyping. EVs from matched human visceral
adipose tissue, mature adipocyte-conditioned media, and plasma collected
from the same bariatric surgical patients were analyzed and subjected
to bottom-up proteomics analysis. This framework integrates bulk EV
proteomics for cell-specific marker identification and subsequent
single EV interrogation with single-particle interferometric reflectance
imaging (SP-IRIS) and total internal reflection fluorescence (TIRF)
microscopy. Our proteomics analysis revealed 76 unique proteins from
adipose tissue-derived EVs (ATEVs), 512 unique proteins from adipocyte
EVs (aEVs), and 1003 shared proteins. Prominent pathways enriched
in ATEVs included lipid metabolism, extracellular matrix organization,
and immune modulation, while aEVs were enriched in chromatin remodeling,
oxidative stress responses, and metabolic regulation pathways. Notably,
we identified obesity-associated and adipose-specific proteins, such
as TMEM120A and SACM1L, highly enriched in ATEVs, and histone variants,
including MACROH2A1, H3–3A, H2AC20, H2BC12, and H3–7,
uniquely upregulated in aEVs. Additionally, classical adipose-associated
proteins such as adiponectin and perilipin were highly enriched in
ADEVs and were confirmed in circulating plasma EVs. Colocalization
of key EV and adipocyte markers, including CD63 and PPARG, was validated
in circulating plasma EVs. In summary, our study paves the way toward
a tissue and cell-specific, multiparametric framework for an ‘adiposity
EV signature’ in obesity-driven diseases.