Abstract 373: Senescence Drives Changes In Vesicle Secretion And Extracellular Matrix Organisation To Enhance Vascular Amyloidosis

Abstract

Background: Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid fragment of milk fat globule EGF-factor 8 (MFGE8). Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid metabolism and accumulation in the extracellular matrix (ECM). MFGE8 has been identified as EV cargo in several cell types, including vascular smooth muscle cells (VSMCs). To determine the mechanisms of AMA formation with age, we explored the impact of VSMC senescence, EV secretion and ECM remodelling on medin accumulation. Methods: Primary human VSMCs were used for in vitro experiments, including EV isolation and synthesis of decellularised ECM for Western blotting, immunofluorescence and mass spectrometry analysis. Results: Medin was detected in EVs secreted from VSMCs. Small, round medin aggregates colocalised with EV markers in the ECM and medin was shown on the surface of EVs deposited in the ECM. Decreasing VSMC EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age. VSMC senescence increased EV secretion, increased EV medin loading and triggered extracellular deposition of medin in fibrillar form. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition. These changes led to enhanced EV-ECM binding in the senescent ECM and accelerated aggregation of medin into amyloid fibrils. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalised with EVs and medin. Knock-down of HSPG2 decreased medin fibril formation in the senescent ECM. Conclusions: Medin is secreted into the ECM by VSMC-derived EVs, which enhance its aggregation. Senescence induces changes in EV secretion and cargo and ECM composition. Together, these changes trigger accumulation of fibrillar medin, contributing to the age-associated development of AMA.