Abstract 28: p62-Enriched Inclusion Bodies in Macrophages Play a Protective Role in Atherosclerosis

Abstract

Polyubiquitinated protein aggregates have been classically associated with human neurodegenerative, liver, and muscle disorders, but not atherosclerosis. Since autophagy-deficient atherosclerotic macrophages have markedly increased levels of p62, a chaperone protein critical for the autophagic turnover of polyubiquitinated proteins, we aimed to evaluate the role of p62 and protein aggregation in atherosclerosis. We first tested the effects of atherogenic lipid loading in peritoneal macrophages. Incubation with either cholesterol crystals or oxidized LDL leads to dramatically increased p62 protein that completely co-localizes with polyubiquitinated proteins as cytoplasmic inclusions or as aggregates partially sequestered by lysosomes. Such accumulation was intensified in autophagy-null (ATG5-/-) macrophages, where massive cytoplasmic ubiquitin-positive p62 aggregates form. Our in vitro observations are recapitulated in vivo. Aortas from atherosclerotic (ApoE-/-) mice have marked elevations in p62 and polyubiquitinated proteins that predominantly co-localize with plaque macrophages, a process that is further exacerbated in the autophagy-deficient setting. Remarkably, we find nearly identical results in human carotid endarterectomy samples, suggesting that p62-enriched protein aggregates are a characteristic feature of atherosclerosis. The homeostasis of cytoplasmic inclusions is dependent on the presence of p62 since lipid-loaded p62-null macrophages accumulate polyubiquitinated proteins in a diffuse and disrupted cytoplasmic pattern. The disruption of these aggregates has functional consequences manifested as increased secretion of IL-1β and enhanced macrophage apoptosis. Consistent with our in vitro observations, p62-deficient mice have increased atherosclerotic plaque formation. Furthermore, p62-deficiency increases plaque burden even in the highly atherosclerosis-prone macrophage-specific ATG5-null mice. Taken together, these data suggest that by sequestering cytotoxic ubiquitinated proteins, p62-enriched inclusion body formation is a protective response during atherogenesis, a setting where the clearance of protein aggregates is disrupted.