Abstract MP49: Macrophage-mediated Extracellular Digestive Exophagy Of Aggregated LDL Is Responsible For The Formation Of Cholesterol Crystals In Atherosclerotic Plaques

Abstract

Atherosclerosis is a disease that develops overtime through dyslipidemia and chronic inflammation. Formation of cholesterol crystals in the atherosclerotic plaques is a major contributor to the inflammatory response. However, how these crystals form in the atherosclerotic plaques remained poorly understood. We have observed that macrophage-mediated extracellular digestive exophagy of aggregated LDL releases a large amount of free cholesterol in the extracellular space. Therefore, we hypothesized that this release of cholesterol may play a major role in formation of cholesterol crystals in plaques. We have shown previously that TLR4 knockout in macrophages compromises digestive exophagy of aggregated LDL. Thus, to evaluate the role of digestive exophagy in cholesterol crystal formation, we examined cryosections of aortic plaques from Ldlr -/- mice that were gamma-irradiated and reconstituted with WT or Tlr4 -/- bone marrows. Interestingly, TLR4 KO in macrophages reduce both free cholesterol and cholesterol crystal in plaques compared to WT macrophages, suggesting a role of digestive exophagy in cholesterol crystal formation. To investigate whether digestive exophagy of aggregated LDL results in inflammatory response, we cultured bone-marrow derived macrophages expressing the inflammasome marker ASC-citrine with aggregated LDL over time. We observed a gradual increase in number of inflammasomes over a 24 h time period. We also observed extracellular cholesterol crystal formation as early as 2 h after the addition of aggregated LDL. Moreover, treating bone marrow-derived macrophages with acetylated LDL did not result in the formation of extracellular cholesterol crystals, suggesting this process is specific to a subset of modified LDL in the vessel wall. Overall, our data provide evidence that macrophage-mediated digestive exophagy of aggregated LDL is a major contributor to the formation of extracellular cholesterol crystal found in atherosclerotic lesions.