Background Ageing is a major risk factor for several vascular pathologies. The age-related accumulation of amyloid is commonly associated with degenerative diseases, such as Alzheimer’s disease. Exosomes have recently been recognised as key mediators of amyloid formation, particularly in the brain. Exosomes have also been implicated in vascular smooth muscle cell (VSMC) calcification, another manifestation of ageing.
MFGE8 is an age-associated protein expressed by VSMCs and secreted by exosomes. MFGE8 is an amyloid precursor and can be cleaved into a 50-amino acid peptide called medin, which forms aortic medial amyloid (AMA) in ageing vessel walls. The mechanism of AMA formation and deposition is unknown.
The aims are to study 1) changes in exosome secretion and content with age, 2) amyloid protein loading in exosomes and 3) if MFGE8 and/or AMA can promote calcification.
Methods Western blotting, qPCR, and immunostaining were used to study medin and MFGE8 expression in VSMCs, at different ages and in calcifying conditions. FACS analysis was used for quantification of exosome secretion. Exosomes were isolated by differential ultracentrifugation. Extracellular matrix (ECM) was synthesised in vitro for immunofluorescent staining and Western blotting. Cresolphthalein assays were used to quantify calcification of VMSCs.
Results MFGE8, medin and medial calcification were present in the aortas of old, but not young subjects (table 1). MFGE8 was expressed by VSMCs and secreted by exosomes. Medin is deposited in the ECM and blocking exosome release decreased its deposition. The expression and secretion of MFGE8 increased in calcifying conditions and recombinant MFGE8 increases calcification while siRNA knockdown of MFGE8 decreased calcification.
Summary/conclusion Medin and MFGE8 are abundant in aged subjects and are secreted by exosomes into the ECM. Exosome release is increased with age, which could contribute to the deposition of medin in the ECM and the formation of amyloid. MFGE8 may play a role in accelerating calcification by inducing an osteogenic phenotype via the ERK pathway. Both MFGE8 and medin secretion by exosomes could contribute to the age-related development of vascular calcification.
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