Proteomic Atlas of Notochordal Cell‐Derived Extracellular Vesicles Highlights EV‐Specific NF‐κB Modulation and Functional Implications of the Protein Corona

The notochord is a central signalling hub during embryonic development. After regression of the notochord, notochordal cells (NCs) reside within the developing intervertebral disc. The NC-secretome, including matrix, proteins and extracellular vesicles (NC-EVs), promotes regeneration of degenerate intervertebral discs and cartilage. This study aims to unravel the NC-EV-associated proteome and modulatory role in regenerative processes. NC-EVs were isolated from conditioned medium from porcine NC-rich tissue using differential centrifugation, size exclusion chromatography and density gradient centrifugation. Proteomic analysis of NC-EVs was performed using LC-MS/MS and identified 4042 unique proteins, including EV-associated markers, matrisome proteins and proteins associated with critical nodes in canonical signalling pathways. To test NC-EV bioactivity, functional analysis was performed using SBE, TCF/LEF and NF-κB luciferase reporters. NC-EVs activated SMAD3 and inhibited IL-1β-mediated NF-κB signalling. NC-EV depleted controls showed similar effects, indicating that co-isolated proteins played a major role in modulating signalling. Therefore, NC-EVs were further purified and the GAG-rich biomolecular corona was modified with hyaluronidase. Post-hyaluronidase treatment, SMAD3 activation was enhanced only in methodological controls, while inhibition of IL-1β-mediated NF-κB signalling was NC-EV specific. This study is the first to reveal the NC-EV proteome and illustrates that proteins absorbed on the NC-EVs attribute to EV-mediated functional effects.