Using an Accelerated Aging Model and an Optimised Proteomic Approach to Identify Extracellular Vesicle-Associated Proteins in Inflammaging

Abstract

Senescent cells contribute to inflammaging through the senescence-associated secretory phenotype (SASP). While the SASP has traditionally been identified by the release of soluble protein factors such as cytokines, extracellular vesicles (EVs) have recently emerged as important components of the SASP due to their cargo of diverse bioactive molecules. Individuals with early or accelerated aging often exhibit an increased burden of senescent cells. Hutchinson-Gilford Progeria Syndrome (HGPS), a rare and fatal genetic disorder, serves as a valuable model for studying accelerated biological aging and has played a pivotal role in elucidating SASP mechanisms. However, the composition and functional roles of EVs in HGPS remain poorly understood.

In this study, we optimized a workflow for isolating and characterizing EVs from HGPS fibroblasts cultured in vitro. Using liquid chromatography–tandem mass spectrometry (LC-MS/MS) in the data independent acquisition (DIA) mode, we identified over 1,500 unique proteins in EVs secreted by both HGPS and age-matched healthy control fibroblasts. Among these, we observed significant dysregulation of Major Histocompatibility Complex I (MHC-I) Human Leukocyte Antigen C (HLA-C) in EVs derived from mixed populations of replicating and senescent HGPS fibroblasts, compared to healthy controls. Notably, LNPEP (also known as IRAP), a protein involved in MHC-I antigen loading in endosomes, was upregulated in HGPS fibroblasts. CD9, a canonical EV marker implicated in inflammatory signaling was also upregulated in HGPS-derived EVs.

These findings suggest that EVs may participate in modulating immune responses to senescent cells, potentially contributing to the impaired clearance of senescent cells observed in age-related diseases. Our study provides novel insights into the EV-mediated component of the SASP in HGPS and underscores the broader relevance of EVs in diseases characterized by accelerated aging and cellular senescence.