Free cholesterol loading of macrophages is associated with widespread mitochondrial dysfunction and activation of the mitochondrial apoptosis pathway.

Pin Mei Yao,Ira Tabas

doi:10.1074/jbc.m101419200

Abstract

Macrophage death in advanced atherosclerotic lesions leads to lesional necrosis and possibly plaque rupture and acute vascular occlusion. Among the likely causes of lesional macrophage death is intracellular accumulation of excess free cholesterol (FC), which is known to occur in vivo. We recently showed that FC loading of macrophages causes apoptosis, approximately 50% of which is mediated by activation of cell-surface FasL and triggering of the Fas pathway (Yao, P. M., and Tabas, I. (2000) J. Biol. Chem. 275, 23807-23813). To elucidate other pathways of death in FC-loaded macrophages, we investigated mitochondrial transmembrane potential (DeltaPsi(m)) and the mitochondrial apoptosis pathway in FC-loaded mouse peritoneal macrophages. Starting between 3 and 6 h of FC loading, DeltaPsi(m) was markedly decreased in the majority of macrophages and was independent of the Fas pathway. The decrease in DeltaPsi(m) by FC loading was not prevented by GSH, thus distinguishing it from 7-ketocholesterol-induced mitochondrial dysfunction. Cytochrome c release into the cytosol was noted by 4 h of FC loading, and activation of caspase-9 and effector caspases was observed at 6 h. Finally, we found that both cellular and mitochondrial levels of the pro-apoptotic protein Bax were increased severalfold as early as 4 h after FC loading. Thus, FC loading, perhaps via increased levels of Bax and/or cholesterol overloading of mitochondria, triggers cytochrome c release and activation of caspase-9 and the effector caspases, leading to macrophage apoptosis. These findings and our previous data support a model in which FC loading of macrophages promotes a dual program of caspase-mediated death.

Highlights

Macrophage death is a prominent feature of atherosclerotic lesions [1,2,3,4] and may affect lesion progression and/or complications
We recently showed that free cholesterol (FC) loading of macrophages causes apoptosis, ϳ50% of which is mediated by activation of cell-surface FasL and triggering of the Fas pathway
FC Loading of Macrophages Leads to Mitochondrial Dysfunction—As an initial assessment of mitochondrial function in FC-loaded macrophages, peritoneal macrophages from C57BL6 mice were incubated for 3, 6, or 9 h in medium alone or containing acetyl-LDL plus the acyl-CoA:cholesterol acyltransferase inhibitor compound 58035 to effect FC loading [13, 20]

Summary

Introduction

Macrophage death is a prominent feature of atherosclerotic lesions [1,2,3,4] and may affect lesion progression and/or complications. Macrophages in advanced lesions are known to accumulate excess FC (8 –11), and excess FC is a potent inducer of macrophage death [12, 13] In this regard, recent work from our laboratory has demonstrated that FC loading of macrophages leads to caspase-dependent externalization of phosphatidylserine and to DNA fragmentation, consistent with an apoptotic process [14]. Levels of cellular and mitochondrial Bax, which is known to induce cytochrome c release and mitochondrion-dependent caspase activation [15], are increased in FC-loaded macrophages. These findings and our previous data support a model in which FC loading of macrophages promotes a dual program of caspasemediated death

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Conclusion