Free Cholesterol Loading of Macrophages Is Associated with Widespread Mitochondrial Dysfunction and Activation of the Mitochondrial Apoptosis Pathway

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, ∼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 (ΔΨm) and the mitochondrial apoptosis pathway in FC-loaded mouse peritoneal macrophages. Starting between 3 and 6 h of FC loading, ΔΨm was markedly decreased in the majority of macrophages and was independent of the Fas pathway. The decrease in ΔΨmby 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. 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, ∼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 (ΔΨm) and the mitochondrial apoptosis pathway in FC-loaded mouse peritoneal macrophages. Starting between 3 and 6 h of FC loading, ΔΨm was markedly decreased in the majority of macrophages and was independent of the Fas pathway. The decrease in ΔΨmby 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. free cholesterol fetal bovine serum 7-amino-4-trifluoromethylcoumarin benzyloxycarbonyl-Leu-Glu-His-Asp fluoromethyl ketone phosphate-buffered saline Dulbecco's modified Eagle's medium low density lipoprotein phosphatidylserine 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid Macrophage death is a prominent feature of atherosclerotic lesions (1Mitchinson M.J. Hardwick S.J. Bennett M.R. Curr. Opin. Lipidol. 1996; 7: 324-329Crossref PubMed Scopus (79) Google Scholar, 2Ball R.Y. Stowers E.C. Burton J.H. Cary N.R. Skepper J.N. Mitchinson M.J. Atherosclerosis. 1995; 114: 45-54Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 3Berberian P.A. Myers W. Tytell M. Challa V. Bond M.G. Am. J. Pathol. 1990; 136: 71-80PubMed Google Scholar, 4Bauriedel G. Schmucking I. Hutter R. Luchesi C. Welsch U. Kandolf R. Luderitz B. Z. Kardiol. 1997; 86: 902-910Crossref PubMed Scopus (14) Google Scholar) and may affect lesion progression and/or complications. For example, death of macrophages may contribute to the release of plaque-destabilizing and thrombogenic molecules in more advanced lesions. In support of this idea, “necrotic” cores of advanced atheromata, which contain the debris of dead macrophages (2Ball R.Y. Stowers E.C. Burton J.H. Cary N.R. Skepper J.N. Mitchinson M.J. Atherosclerosis. 1995; 114: 45-54Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 3Berberian P.A. Myers W. Tytell M. Challa V. Bond M.G. Am. J. Pathol. 1990; 136: 71-80PubMed Google Scholar), are located in areas predisposed to plaque rupture and acute thrombosis (5Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Crossref PubMed Scopus (2909) Google Scholar). Moreover, fragments of plasma membrane shed by apoptotic lesional cells are rich in thrombogenic tissue factor activity (6Mallat Z. Hugel B. Ohan J. Leseche G. Freyssinet J.M. Tedgui A. Circulation. 1999; 99: 348-353Crossref PubMed Scopus (595) Google Scholar). More directly, apoptotic macrophages, but not apoptotic smooth muscle cells or T cells, are greatly increased in ruptured plaques versusstable plaques (7Kolodgie F.D. Narula J. Burke A.P. Haider N. Farb A. Hui-Liang Y. Smialek J. Virmani R. Am. J. Pathol. 2000; 157: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), and atherectomy specimens from patients with unstable angina have approximately twice the number of dead intimal cells compared with specimens from patients with stable angina (4Bauriedel G. Schmucking I. Hutter R. Luchesi C. Welsch U. Kandolf R. Luderitz B. Z. Kardiol. 1997; 86: 902-910Crossref PubMed Scopus (14) Google Scholar). To elucidate the roles of macrophage death in atherosclerosis, it is necessary to gain a thorough understanding of the inducers and cellular death pathways involved. Although many molecules and processes have been proposed to cause macrophage death in lesions, intracellular accumulation of excess unesterified or free cholesterol (FC)1 has been the focus of several investigators. Macrophages in advanced lesions are known to accumulate excess FC (8Lundberg B. Atherosclerosis. 1985; 56: 93-110Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 9Small D.M. Bond M.G. Waugh D. Prack M. Sawyer J.K. J. Clin. Invest. 1984; 73: 1590-1605Crossref PubMed Scopus (144) Google Scholar, 10Rapp J.H. Connor W.E. Lin D.S. Inahara T. Porter J.M. J. Lipid Res. 1983; 24: 1329-1335Abstract Full Text PDF PubMed Google Scholar, 11Shio H. Haley N.J. Fowler S. Lab. Invest. 1979; 41: 160-167PubMed Google Scholar), and excess FC is a potent inducer of macrophage death (12Warner G.J. Stoudt G. Bamberger M. Johnson W.J. Rothblat G.H. J. Biol. Chem. 1995; 270: 5772-5778Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 13Tabas I. Marathe S. Keesler G.A. Beatini N. Shiratori Y. J. Biol. Chem. 1996; 271: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). 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 (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Most interestingly, approximately half of the apoptosis could be blocked by mutations in or inhibitors of the Fas pathway of cell death, implying a partial role for the Fas pathway in FC-induced apoptosis; the mechanism involves FC-induced activation of cell-surface FasL (Fasligand) (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). In this study, our goal was to determine the pathway(s) leading to caspase-dependent death in the substantial portion of FC-loaded macrophages that die independently of the Fas pathway. The data reported herein show that there is widespread, Fas-independent mitochondrial dysfunction in FC-loaded macrophages, as well as cytochrome c release, activation of caspase-9, and Fas-independent activation of effector caspases. Interestingly, levels of cellular and mitochondrial Bax, which is known to induce cytochrome c release and mitochondrion-dependent caspase activation (15Kroemer G. Reed J.C. Nat. Med. 2000; 6: PubMed Scopus Google Scholar), 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 caspase-mediated death. The tissue in was from and other tissue were from bovine serum was from and was for h at Rothblat G.H. J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar), an of was by of a was in and the in both and cells was and were from and and and were from c was from and was from and were from The protein was from was from other and and GSH, were from and were from Macrophages were from the of or 3 after the of of in of and as W. A. Tabas I. 1999; PubMed Scopus Google Scholar). the of the the cells were with and for the in and or as Y. Tabas I. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of the the cells were for the were by of and of cholesterol in The was by by of was to the To and acid were The was for at 4 a a For the in a of was as by G. C. G. S. S. D. P. Biol. Med. 2000; PubMed Scopus Google Scholar). of was in of and of was of this was to of medium to a of of was in and and in medium to a of Macrophages were or for and with or in at for The were with a with a and were to be in were with the and of an and was and with a To the of we the the cell in a with the from or FC-loaded macrophages were in and For the of were to and the protein were The were blocked in and and with the as Bax cytochrome c cytochrome and caspase-9 The was h at and the were at 4 The which was a or was for h at The were the from or FC-loaded macrophages were with for at to the cells were with for with and for at with which was bovine serum and and mouse The cells were with Bax for h in The was for h at in The cells were by as and FC-loaded macrophages were by at for at 4 The cell were with and with of and for the cells were with of a cell and the were at for at 4 to cells and The were and at for at 4 and the were severalfold the were in The and mitochondrial were to and as For the cell were in of and to the The was in the and at for Finally, the mitochondrial was in and for Bax by of caspases was in of or FC-loaded macrophages the caspase and In was the or the of the the macrophages were and in and in of and and The cellular was for and for at to the of protein the protein of the cell were at in of and or and of the in the of a that was in an and The were and were the of the Macrophages were or for with and by as (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). For to of cells were for the number of cells and are as 3 noted an of mitochondrial in FC-loaded macrophages, peritoneal macrophages from were for or h in medium or the to FC loading I. Marathe S. Keesler G.A. Beatini N. Shiratori Y. J. Biol. Chem. 1996; 271: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, G. D.M. G.J. J.K. Rothblat G.H. Biol. PubMed Scopus Google Scholar). The cells were with which is a that is in there is an transmembrane potential (ΔΨm) the mitochondrial membrane U. S. A. PubMed Scopus Google Scholar). in macrophages demonstrated consistent with a mitochondrial of The and of the were at Macrophages with FC for 3 h demonstrated a to that with cells In the of macrophages with FC for 6 or h was decreased The of macrophages with was from that of the macrophages of the data in of cells of is in G. More of mitochondrial with are in and The of macrophages is in A. In macrophages with FC for 6 h there were and the was with the that the FC-loaded cells not have a in the cellular of to a in the plasma membrane it is the of to the that is To macrophages or for 6 h were with that in ΔΨm is M. J. 1996; PubMed Scopus Google Scholar). in macrophages demonstrated the In macrophages with FC for 6 h showed markedly Finally, we investigated mitochondrial membrane potential with a that has been reported to be a of ΔΨm S. A. C. A. 1997; PubMed Scopus Google Scholar). in mitochondria, leading to and a there is and thus a decrease in the in FC loading was with a decrease in and an in of a decrease in In the data in and that by 6 h of FC loading, macrophages show markedly mitochondrial dysfunction. is that macrophages with an could be to or other in or that the cholesterol from could be to J. Lipid Res. 1996; Full Text PDF PubMed Google Scholar, G. S. C. L. V. S. L. P. Biol. 1999; PubMed Scopus Google Scholar). To we we mitochondrial dysfunction could be by macrophages with but not the other of To this we in the of macrophages by the H. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). Macrophages were with for 6 h with medium with or with cholesterol in 3 mitochondrial in macrophages with was markedly that observed in macrophages or macrophages with data are in D. Thus, a induce mitochondrial dysfunction in other are not necessary for mitochondrial dysfunction. the of apoptosis we could not the to the of in the cell with apoptosis in FC-loaded macrophages, and cell was the in macrophages with to the that be by we of the by G. C. G. S. S. D. P. Biol. Med. 2000; PubMed Scopus Google Scholar) that 7-ketocholesterol-induced death in is prevented by with the we could cause mitochondrial dysfunction in mouse peritoneal macrophages it could be prevented by in 4 macrophages to of and not of a mitochondrial that the which was in cells, was observed with and the mitochondrial was was the in In the decreased mitochondrial of macrophages with was not prevented at by with in Thus, mitochondrial dysfunction by be from that by a both by the of mitochondrial and by the of by with We the of apoptosis 7-ketocholesterol-induced apoptosis as well as mitochondrial dysfunction G. S. S. A. C. G. S. P. J. PubMed Scopus Google Scholar). In an the apoptotic cells in macrophages with in the of was compared with in the of and in macrophages that were not FC-loaded at Thus, consistent with the not macrophage apoptosis by an We have that a portion of macrophages with FC for h show of apoptosis, externalization of phosphatidylserine and DNA (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). These are decreased by in macrophages with Fas or FasL or in macrophages in the of an (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Thus, the Fas pathway of apoptosis is in a portion of FC-loaded macrophages. that activation of the Fas pathway to mitochondrial dysfunction in of cells C. S. A. C. J. PubMed Scopus Google Scholar), we FC-induced mitochondrial dysfunction was an Fas pathway. Macrophages from or from with the Fas S. T. 1995; Full Text PDF PubMed Scopus Google Scholar) were or for 6 h and with in the decrease in mitochondrial in FC-loaded macrophages was to that observed in macrophages were not Thus, in FC-loaded macrophages, mitochondrial dysfunction not Fas dysfunction be with release of cytochrome c and activation of the caspase by effector caspase activation and (15Kroemer G. Reed J.C. Nat. Med. 2000; 6: PubMed Scopus Google Scholar). To this we from and FC-loaded macrophages to cytochrome c in 6 and cytochrome c was decreased in macrophages with FC for 4 or h. To that data were not to mitochondrial protein in the FC we showed that the mitochondrial from and FC-loaded macrophages approximately of mitochondrial cytochrome and Finally, cytochrome c was in the cytosol of FC-loaded macrophages, but not in the cytosol of macrophages In FC loading of macrophages leads to a decrease in mitochondrial cytochrome c and an in cytochrome We to caspase-9 of and FC-loaded macrophages were with a caspase-9 that A. M.J. P.A. J. Med. 2000; PubMed Scopus Google Scholar). in activity was increased in FC-loaded macrophages. The of activity was to that observed in cells a in which caspase-9 activation is known to occur H. S. M. M. Y. N. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). it was necessary to show that the activity observed in FC-loaded macrophages was not to by effector caspases. To this we effector caspase activity the A. M.J. P.A. J. Med. 2000; PubMed Scopus Google Scholar). in activity was increased in FC-loaded macrophages, which effector caspase To determine the mitochondrial pathway be in this in we in which the other pathways of effector caspase the Fas and necrosis were In not we showed that activity was decreased by in FC-loaded macrophages from our previous study, we showed that of apoptosis at h of FC loading could be blocked by mutations in Fas or FasL or by FasL with an (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). that activity in of macrophages was that in macrophages is to between caspases in apoptotic in and the necrosis pathway with a necrosis factor at These data are consistent with a role for the mitochondrial pathway in effector caspase We the of M. J. L. Y. 2000; PubMed Scopus Google Scholar). in not effector caspase activity at the of our In the decreased activity to the in macrophages Although the for activity we found a substantial of activity in which activity was not by the For example, in of our the of activity in FC-loaded macrophages was and activity was the in A. In our data that or of the in activity in FC-loaded macrophages be by by effector caspases, but is of caspase-9 The to caspase-9 activation activation of caspase-9 P. D. I. M. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). an that caspase-9, we found that FC-loaded macrophages, but not macrophages, or in the and and macrophages and cells, the which as a at in the in These were to with cells In the data in 6 support the that FC loading of macrophages leads to cytochrome c release, caspase-9 and effector caspase We FC loading to the release of cytochrome the pro-apoptotic protein Bax is a known inducer of cytochrome c release, via the of Bax with mitochondrial (15Kroemer G. Reed J.C. Nat. Med. 2000; 6: PubMed Scopus Google Scholar), we investigated the of FC loading mitochondrial Thus, from macrophages or for 6 h were for Bax by in Bax was increased in the mitochondrial from FC-loaded macrophages. To between of Bax, which is to A. J.N. M. R. S.J. J. Biol. PubMed Scopus Google Scholar), mitochondrial we the in a to mitochondrial a in mitochondrial Bax in FC-loaded macrophages was To determine increased mitochondrial Bax an increased in cellular Bax or was to from the as has been reported in other A. J. S.J. J. PubMed Scopus Google Scholar, M. Johnson J. 1999; PubMed Google Scholar), we Bax in the from the as well as from macrophages or for 4 and h. Bax was increased Thus, FC loading of macrophages is with a in cellular Bax, and a portion of this increased Bax is with that mitochondrial Bax is increased in FC-loaded macrophages is by the in In macrophages, there was a of Bax that with but the of FC-loaded macrophages was more and More of FC-loaded macrophages with are in and The of Bax is demonstrated in and the in a The in partial of Bax and In the data in and that FC loading of macrophages is with a substantial in the of a between FC loading of macrophages and the release of cytochrome c and mitochondrion-dependent caspase Macrophage death and is likely to progression (2Ball R.Y. Stowers E.C. Burton J.H. Cary N.R. Skepper J.N. Mitchinson M.J. Atherosclerosis. 1995; 114: 45-54Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 3Berberian P.A. Myers W. Tytell M. Challa V. Bond M.G. Am. J. Pathol. 1990; 136: 71-80PubMed Google Scholar, 4Bauriedel G. Schmucking I. Hutter R. Luchesi C. Welsch U. Kandolf R. Luderitz B. Z. Kardiol. 1997; 86: 902-910Crossref PubMed Scopus (14) Google Scholar, V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Crossref PubMed Scopus (2909) Google Scholar, Z. Hugel B. Ohan J. Leseche G. Freyssinet J.M. Tedgui A. Circulation. 1999; 99: 348-353Crossref PubMed Scopus (595) Google Scholar, F.D. Narula J. Burke A.P. Haider N. Farb A. Hui-Liang Y. Smialek J. Virmani R. Am. J. Pathol. 2000; 157: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Among the inducers of macrophage death that are in lesions is excess intracellular FC accumulation (8Lundberg B. Atherosclerosis. 1985; 56: 93-110Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 9Small D.M. Bond M.G. Waugh D. Prack M. Sawyer J.K. J. Clin. Invest. 1984; 73: 1590-1605Crossref PubMed Scopus (144) Google Scholar, 10Rapp J.H. Connor W.E. Lin D.S. Inahara T. Porter J.M. J. Lipid Res. 1983; 24: 1329-1335Abstract Full Text PDF PubMed Google Scholar, 11Shio H. Haley N.J. Fowler S. Lab. Invest. 1979; 41: 160-167PubMed Google Scholar, G.J. Stoudt G. Bamberger M. Johnson W.J. Rothblat G.H. J. Biol. Chem. 1995; 270: 5772-5778Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 13Tabas I. Marathe S. Keesler G.A. Beatini N. Shiratori Y. J. Biol. Chem. 1996; 271: 22773-22781Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). For our laboratory has to understanding cellular pathways of FC-induced macrophage death. in this demonstrated that after h of FC loading in our cell of the cells externalization of phosphatidylserine and DNA fragmentation, and could be blocked by an of caspases and by Fas activation (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). The was to potential cellular pathways to Fas-independent death in We caspase activation and apoptosis of the of this pathway in cellular death (15Kroemer G. Reed J.C. Nat. Med. 2000; 6: PubMed Scopus Google Scholar). In this the data herein that FC loading of macrophages causes widespread, Fas-independent mitochondrial dysfunction between 3 and 6 h after the of FC an in cellular Bax at 4 and in activation of caspase-9 and effector caspases from our previous (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), externalization of phosphatidylserine and DNA we that at a portion of the Fas-independent apoptosis of FC-loaded macrophages is by a mitochondrial pathway release of cytochrome possibly by Bax, and caspase In the of this an the between increased levels of mitochondrial Bax, decreased mitochondrial and mitochondrial cytochrome c release in FC-loaded macrophages. to it is to that increased mitochondrial Bax a role in the release of cytochrome c and possibly in the in of this Bax FC loading of macrophages, which we have not been to cytochrome c release occur in the of mitochondrial R. B. A. S. C. R. G. A. J.C. J. Biol. PubMed Scopus Google Scholar), and mitochondrial occur in the of cytochrome c release J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). is that FC of mitochondrial leading to in a in ΔΨm and/or release of cytochrome is known to be to in macrophages A. S. A. U. I. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and mitochondrial has been to be by a cholesterol S. J. J.C. J. PubMed Scopus Google Scholar, A. G. A. PubMed Scopus Google Scholar, W. J. S. A. Am. J. Google Scholar, B. R. A. Res. PubMed Scopus Google Scholar). to this of mitochondrial cholesterol in and FC-loaded macrophages and to determine cholesterol of leads to the mitochondrial observed into the role of Bax in FC-induced mitochondrial dysfunction and cytochrome c release, the in this protein as a of cellular FC loading is a to from this Interestingly, the in mitochondrial Bax in other has been to occur via of Bax from the cytosol to the mitochondria, an in cellular Bax levels A. J. S.J. J. PubMed Scopus Google Scholar, M. Johnson J. 1999; PubMed Google Scholar). In the of FC-loaded macrophages, we observed an in cellular Bax, of which with work be to the mechanism of this increased of is it be to a role for the protein is a known inducer of Bax T. Reed J.C. 1995; Full Text PDF PubMed Scopus Google Scholar) and has recently been in cellular FC A. 2000; PubMed Scopus Google Scholar). Moreover, it to determine the of in Bax Although is not the of mitochondrial dysfunction or apoptosis in FC-loaded macrophages 4 and it is that other are to by may be involved. Moreover, there may be a role for from as and possibly A. S. A. U. I. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. J.C. J. PubMed Scopus Google Scholar, A. G. A. PubMed Scopus Google Scholar, W. J. S. A. Am. J. Google Scholar). Finally, of the of mitochondrial in to or of cytochrome is death (15Kroemer G. Reed J.C. Nat. Med. 2000; 6: PubMed Scopus Google Scholar). is mitochondrial dysfunction was in the of FC-loaded macrophages, caspase-dependent death in of cells (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Thus, the of cells that die with FC loading in a M. and I. Tabas, may be a of mitochondrial dysfunction. pathway of cell death may be in advanced atherosclerosis, in which macrophage necrosis is known to occur and may to the release of intracellular molecules that plaque rupture and thrombosis (1Mitchinson M.J. Hardwick S.J. Bennett M.R. Curr. Opin. Lipidol. 1996; 7: 324-329Crossref PubMed Scopus (79) Google Scholar, P. Curr. Opin. Lipidol. Scopus Google Scholar). In the data in this with in our previous (14Yao P.M. Tabas I. J. Biol. Chem. 2000; 275: 23807-23813Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) that FC loading of macrophages to caspase-dependent death by at an in cell-surface leading to activation of the Fas and activation of the mitochondrial death perhaps to an in mitochondrial Bax and/or cholesterol loading of Moreover, FC loading of macrophages leads to death, and FC-induced mitochondrial dysfunction may an role in this process we to the is to roles in caspase-dependent death may a of death, perhaps leading to the of macrophage in lesions, death may contribute to lesional plaque and acute vascular by macrophage death pathways and in and other be We for with the for the of this study, and for of the