Abstract 371: An Intact Autophagy-lysosomal System is Required for the Clearance of p62-enriched Inclusion Bodies in Macrophages and has Implications for Atherosclerosis
Autophagy is an essential catabolic cellular mechanism that involves degradation of dysfunctional proteins and organelles through the lysosomal machinery. Recent studies suggest that autophagy- deficiency induces atherosclerotic plaque formation in mouse models. Although the exact mechanisms are unknown, hyperactivation of the inflammasome, enhanced apoptosis/oxidative stress, and disrupted lipid efflux have been proposed. Herein, we describe an alternative mechanism involving the clearance of protein aggregates. It is known that autophagy-deficient macrophages have significantly increased levels of the chaperone protein p62. Since p62 binds polyubiquitinated proteins and facilitates their turnover via autophagy, disruptions in this process might be pathogenic in atherosclerosis. We first tested the effects of atherogenic lipid loading in peritoneal macrophages. Incubation with either cholesterol crystals or oxidized-LDL dramatically increased p62 protein levels with little effect on transcription, suggesting defects in the clearance of p62 protein. This coincided with the accumulation of polyubiquitinated proteins colocalizing with p62 present either as cytoplasmic inclusions or 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 progressive and marked elevations in p62 and polyubiquitinated protein levels that predominantly colocalize with plaque macrophages, a process that is further exacerbated in the autophagy-deficient setting. The homeostasis of cytoplasmic inclusions is dependent on the presence of p62 as lipid loaded p62-null macrophages showed polyubiquitinated protein accumulation present in a diffuse and disrupted cytoplasmic pattern. This appears to have functional consequences with p62-null macrophages displaying increased secretion of IL-1β likely through inflammasome activation. Taken together these data suggest that p62-positive inclusion body formation is a marker of atherosclerotic progression with potential pathophysiological consequences.
- © 2013 by American Heart Association, Inc.