- Email: [email protected]
Cholesterol and phospholipid metabolism in macrophages
Biochimica et Biophysica Acta 1529 (2000) 164^174 www.elsevier.com/locate/bba
Review
Cholesterol and phospholipid metabolism in macrophages Ira Tabas * Departments of Medicine and Anatomy and Cell Biology, Columbia University, 630 West 168th Street, New York, NY 10032, USA Received 20 April 2000; accepted 16 August 2000
Abstract Cholesterol-loaded macrophages are present at all stages of atherogenesis, and recent in vivo data indicate that these cells play important roles in both early lesion development and late lesion complications. To understand how these cells promote atherogenesis, it is critical that we understand how lesional macrophages interact with subendothelial lipoproteins, the consequences of this interaction, and the impact of subsequent intracellular metabolic events. In the arterial wall, macrophages likely interact with both soluble and matrix-retained lipoproteins, and a new challenge is to understand how certain consequences of these two processes might differ. Initially, the major intracellular metabolic route of the lipoproteinderived cholesterol is esterification to fatty acids, but macrophages in advanced atherosclerotic lesions progressively accumulate large amounts of unesterified, or free, cholesterol (FC). In cultured macrophages, excess FC accumulation stimulates phospholipid biosynthesis, which is an adaptive response to protect the macrophage from FC-induced cytotoxicity. This phospholipid response eventually decreases with continued FC loading, leading to a series of cellular death reactions involving both death receptor-induced signaling and mitochondrial dysfunction. Because macrophage death in advanced lesions is thought to promote plaque instability, these intracellular processes involving cholesterol, phospholipid, and death pathways may play a critical role in the acute clinical manifestations of advanced atherosclerotic lesions. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Foam cell; Atherosclerosis ; Lipoprotein; Acyl-coenzyme A:cholesterol acyltransferase; Phosphatidylcholine; Cytidine 5P-triphosphate:phosphocholine cytidylyltransferase ; Apoptosis
1. Introduction A critical event in the developing atherosclerotic lesion is the entry of monocytes into the subendothelium of focal areas of the arterial wall [1]. It is likely Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; CE, cholesteryl ester; CT, CTP:phosphocholine cytidylyltransferase; ER, endoplasmic reticulum; FC, free cholesterol; HSL, hormone-sensitive lipase; PC, phosphatidylcholine; LDL, low-density lipoprotein; M-CSF, macrophage-colony stimulating factor; PL, phospholipid * Fax: +1-212-305-4834; E-mail: [email protected]
that this event is a biologic response to the focal subendothelial retention of lipoproteins [2,3]. After entry, the monocytes are exposed to endotheliumderived growth and di¡erentiation factors, such as macrophage-colony stimulating factor (M-CSF), leading to their di¡erentiation into macrophages [1]. Therefore, these focal areas of the arterial tree, like areas in the liver, lung, spleen, and in£ammatory lesions, become populated with di¡erentiated tissue macrophages. Arterial wall macrophages in developing atherosclerotic lesions, however, face a rather unique environment that distinguishes their fate from other tissue macrophages: in the subendothelial
1388-1981 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 0 ) 0 0 1 4 6 - 3
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
matrix, they encounter a virtual forest of matrixbound lipoprotein particles [2,3]. The macrophages react to this environment by internalizing and metabolizing these subendothelial lipoproteins, leading to the massive intracellular accumulation of lipoprotein-derived cholesterol [1,4]. Initially, the major storage form of cholesterol in these macrophages is cholesteryl fatty acid esters (CE), which are sequestered into membrane-bound cytoplasmic lipid droplets; these macrophages are often referred to as `foam cells' because of the `foamy' appearance of the cytoplasmic CE inclusions [1,4]. Other important metabolic events in these macrophages, particularly in advanced atherosclerotic lesions, is the accumulation of unesteri¢ed, or `free', cholesterol (FC) and increased rates of phospholipid biosynthesis [5,6]. Speci¢c consequences of atherosclerotic macrophages include both physical e¡ects, such as intimal thickening, and biological e¡ects, such as internalization of lipoproteins and secretion of biologically active molecules [1,7]. Examples of molecules secreted by macrophages include cytokines, growth factors, and pro-oxidants, which have been implicated in amplifying early atherogenic events, and proteases, which are thought to contribute to plaque instability, acute thrombosis, and acute clinical events [1,7]. Indeed, many studies, including recent in vivo investigations, have provided evidence that macrophage foam cells play roles both in early atherogenesis and in late lesional events [7^10]. In particular, mice with absent M-CSF and thus decreased tissue macrophages, including decreased arterial wall macrophages, have markedly decreased atherosclerotic lesion size [8]. Similarly, mice with genetic mutations in a chemokine for monocytes, monocyte chemotactic protein-1, or its monocyte receptor, CCR2, have less arterial wall macrophages and less atherosclerosis [9,10]. The role of lesional macrophages in these atherogenic events is critically in£uenced by processes related to intracellular cholesterol and phospholipid metabolism. For example, many of the critical physical properties of foam cell-rich lesions are due to the physicochemical properties of the intracellular CE droplets [11], and CE loading of macrophages may be an important stimulus for the secretion of matrix metalloproteinases in advanced lesions [12]. The accumulation of excess FC by macrophages leads to
165
important consequences related to cellular phospholipid metabolism and cell viability [5,6]. Thus, a clear understanding of these intracellular metabolic processes is essential to our elucidating how macrophages in£uence atherogenesis and to the development of novel anti-atherogenic therapeutic strategies directed at the lesional macrophage. This review will describe our current understanding of cholesterol and phospholipid metabolism in lesional macrophages and how these metabolic events might a¡ect speci¢c atherogenic processes. 2. The interaction of macrophages with atherogenic lipoproteins Cholesterol accumulation in lesional macrophages begins with the internalization of subendothelial lipoproteins. These lipoproteins enter the subendothelium from the plasma by either transcytosis through endothelial cells or possibly via `leakage' through transient gaps between endothelial cells [13^16]. Once in the subendothelial space, the lipoproteins can either egress back into the circulation or be retained on subendothelial matrix, principally by binding to proteoglycans and collagen [2,3]. As alluded to above, an increased tendency of lipoproteins to be focally retained by these subendothelial molecules, followed by biological responses to this retained material, appears to be a key event in the initiation of atherogenesis [2,3]. A major area of investigation in the cell biology of atherosclerosis is the exploration of how macrophages interact with lipoproteins. Most studies have explored this interaction by incubating soluble, monomeric lipoproteins with monolayers of macrophages plated on tissue culture plastic. This model, which emphasizes receptor-mediated endocytosis, may re£ect some aspects of lesional foam cell formation in early atherosclerotic lesions, where a portion of subendothelial lipoproteins are probably monomeric and in solution in the interstitial £uid [17]. In addition, the model can help us understand some post-uptake events, such as lipoprotein-CE hydrolysis in lysosomes and the threshold phenomenon of cholesterol esteri¢cation (see below). Based on this model, investigators have shown that native low-density lipoprotein (LDL) is a poor inducer of CE ac-
BBAMCB 55719 28-11-00
166
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
cumulation in cultured macrophages, which re£ects both the downregulation of the LDL receptor and peculiarities of intracellular metabolism of LDL-derived cholesterol [4,18]. It is possible, however, that the LDL receptor itself can contribute to foam cell formation, perhaps by mediating the uptake of atherogenic chylomicron remnant-like lipoproteins [4,19]. In this regard, Fazio and colleagues [20] have shown that LDL receptor knockout mice transplanted with LDL receptor-positive macrophages have larger lesion size than similar mice transplanted with LDL receptor-negative macrophages. The receptor-mediated endocytosis model has also revealed that macrophages can internalize large amounts of oxidatively modi¢ed LDL via a variety of receptors including type A and B scavenger receptors [18,21,22]. Interestingly, most forms of in vitro oxidized LDL are relatively weak inducers of macrophage CE accumulation [23], but studies with type A scavenger receptor knockout mice have suggested a role for this receptor in foam cell formation in vivo. It is possible, therefore, that this receptor and perhaps other oxidized LDL receptors mediate the uptake of other forms of atherogenic lipoproteins in the subendothelium (see below). Once lesions becomes established, the vast majority of lesional lipoproteins are avidly bound to matrix [17]. Moreover, both biochemical and morphological studies of human and animal lesions have shown that many of the matrix lipoproteins are in a fused or aggregated state [23^25]. This point is crucial, because cell culture studies have shown that aggregated and fused lipoproteins, unlike oxidized LDL, are among the most potent inducers of massive CE loading of macrophages [23,26^28]. Thus, the typical cell culture model described above may not accurately re£ect the lipoprotein engagement and uptake process ^ and unique consequences of this process ^ that probably occur in the majority of lesions. For example, using a new experimental system to better re£ect the interaction of macrophages with matrix-retained and aggregated LDL, we have shown that a unique event occurs during the initial engagement process. This event is characterized by prolonged contact between macrophage surface invaginations and the matrix-retained lipoproteins during which LDL-CE hydrolysis exceeds LDL-protein degradation [29]. This process, which does not require
the LDL receptor, is clearly di¡erent from events occurring during typical receptor-mediated endocytosis, where there is rapid uptake of ligand and parallel degradation of the CE and protein moieties of lipoproteins [30]. The receptor or receptors mediating this process and its possible physiologic signi¢cance remain to be determined. Similarities between the engagement of matrix-retained and aggregated lipoproteins and macrophage phagocytosis of immobilized ligand (often called `frustrated phagocytosis') may provide other examples of processes that distinguish macrophage-lipoprotein interactions in vivo from the uptake of monomeric, soluble lipoproteins. For example, speci¢c events related to reorganization of the actin cytoskeleton, mediated by speci¢c cell signaling pathways, can distinguish phagocytosis from endocytosis [31,32] and are thus likely involved when lesional macrophages engage subendothelial lipoproteins. Similarly, a feature that distinguishes phagocytosis, and particularly frustrated phagocytosis (above), from receptor-mediated endocytosis is a secretory response leading to the release of a number of bioactive molecules. The list includes potentially atherogenic molecules such as in£ammatory cytokines, reactive oxygen intermediates, lysosomal enzymes, neutral proteases, and even an apolipoprotein [33^ 39]. Thus, new models designed to study the interaction of macrophages with matrix-retained and aggregated lipoproteins are likely to reveal the role of speci¢c cell surface molecules, cell signaling reactions, and secretory events that are involved in macrophage foam cell formation in vivo. 3. Intracellular cholesterol metabolism in macrophages following the internalization of atherogenic lipoproteins The major pathway by which atherogenic lipoproteins deliver their cholesterol to cells is via endocytic or phagocytic delivery of the lipoproteins to degradative organelles (late endosomes, lysosomes, or phagolysosomes), followed by hydrolysis of the of the CE moiety to FC. This newly hydrolyzed FC, along with the original FC component of the lipoproteins, is then distributed to peripheral organelles. Most studies suggest that the initial transport of this
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
FC is to the plasma membrane [4,40], with subsequent transport to other organelles. The molecular basis of these transport processes is not fully known, but the protein that is defective in Niemann-Pick C disease, called npc1, clearly plays a role [41^44]. Two recent studies have suggested that FC is ¢rst transported to the plasma membrane and then to a type of endocytic vesicle in an npc1-independent process, followed by npc1-dependent tra¤cking either back to the plasma membrane or to other peripheral organelles [44,45]. Other molecules that may facilitate FC tra¤cking include lysophosphatidic acid [43], a protein called npc2 that has not yet been cloned but is defective in a rare type of Niemann-Pick C disease [41,42], and VPS4, which is a mammalian homologue of a yeast vacuolar sorting protein [46]. Several sites of post-plasma membrane FC transport are of particular importance in atherosclerotic macrophage biology. Recycling back to the plasma membrane may be critical in FC e¥ux from cells [47]. Transport to the mitochondria is necessary for conversion to 27-hydroxycholesterol by the mitochondrial enzyme sterol 27-hydroxylase [48]; Bjo«rkhem and colleagues [48,49] propose that this pathway is important for sterol e¥ux from macrophages. Transport to the endoplasmic reticulum (ER) is necessary for important negative feedback pathways [50] and for cholesterol esteri¢cation [51]. One feedback pathway occurs when FC interacts directly with the sterol sensing domain of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, a rate limiting enzyme in cholesterol biosynthesis, leading to the enzyme's degradation [52,53]. In another pathway, interaction of FC with a homologous sterol sensing domain in an ER protein called SCAP (sterol-response element binding protein (SREBP) cleavage activating protein) prevents a series of proteolytic cleavages that, in the absence of sterol, leads to activation of SREBP; proteolytically activated SREBP is a transcription factor for the LDL receptor gene as well as genes involved in endogenous cholesterol biosynthesis [50]. The cholesterol esteri¢cation reaction is particularly important in the atherosclerotic macrophage, because the CE resulting from this reaction is the major lipid that accumulates in lesional foam cells [4,51,54]. Via the plasma membrane pathway mentioned above, and perhaps also via another pathway
167
involving more direct transport of FC from the lysosomes to the ER [55,56], FC comes into contact with acyl-CoA:cholesterol acyltransferase (ACAT) [4,40, 51,57]; the form of ACAT in macrophages is called ACAT1, which distinguishes it from another form of ACAT in liver and intestine called ACAT2 [58]. Upon contacting ACAT, the activity of the enzyme is increased, most likely by a combination of substrate provision and allosteric activation [59]. An increase in the expression of ACAT in response to cholesterol is not likely to be a major factor in the increased activity in macrophages because there are only small changes in ACAT mRNA in response to elevated cholesterol [51], and cycloheximide actually stimulates overall ACAT activity [60,61]. This latter observation may indicate that a short-lived protein in cells acts to limit the cholesterol esteri¢cation pathway, but there has been no direct proof of this hypothesis. Tra¤cking of FC to ACAT is energy-dependent, requires an intact actin cytoskeleton, and probably involves vesicular transport [4,40]. Importantly, ACAT is stimulated when cellular FC levels reach a particular threshold level above the ambient cellular cholesterol concentration [62,63]. The Tabas and Lange laboratories have proposed that threshold phenomenon may involve the induction of the above-mentioned cholesterol transport pathway [4,64], and Lange and colleagues have recently shown that there is a dramatic rise in ER cholesterol content when threshold is reached [64]. The exact mechanism whereby a rise in cellular cholesterol triggers this putative vesicular transport pathway remains to be determined. Of potential interest in this regard, Rozelle et al. [65] have recently shown that decreasing cellular cholesterol content, perhaps by in£uencing the behavior of lipid-rich domains in the plasma membrane, can inhibit actin-propelled vesicular transport in cells. An important issue regarding the regulation and function of ACAT is its cellular location. Most investigators agree that the majority of the enzyme resides in the ER, perhaps concentrated in the rough ER, where it functions to prevent excess FC accumulation in the cell in general and ER in particular [51]. Two studies, however, have presented some data suggesting that a small portion of ACAT may be localized to the cell surface, where it may more directly
BBAMCB 55719 28-11-00
168
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
limit the FC content of the plasma membrane [66,67]. Curiously, cell surface ACAT in macrophages was found only when the cells were not attached to a substratum [67]. Finally, Tabas, Max¢eld, and colleagues [67,68] have demonstrated that another portion of ACAT in macrophages is localized to a paranuclear site in the vicinity of the transGolgi network and endocytic recycling compartment, which are both cholesterol-rich membranes. In a recent electron microscopic three-dimensional analysis of the Golgi apparatus [69], it was shown that elements of the ER enter into close apposition with trans elements of the Golgi. In fact, portions of the ER become morphologically indistinguishable from stacked Golgi cisternae, and this would provide an ideal site for transfer of cholesterol from a cholesterol-rich Golgi membrane to the ER. In terms of a potential function of ACAT in this paranuclear site, recent work has shown that cellular cholesterol content can in£uence endocytic tra¤cking [70^72], perhaps by in£uencing the FC content of the endocytic recycling compartment, trans-Golgi network, or both. Therefore, it is possible that paranuclear ACAT, by regulating the FC content in these organelles (i.e., converting FC into a neutral lipid), can directly in£uence endocytic tra¤cking. This section has dealt thus far with lipoproteincholesterol entering macrophages via endocytic or phagocytic pathways, where lipoprotein-protein degradation and CE hydrolysis occur in parallel. As mentioned in the previous section, however, macrophages interacting with matrix-retained and aggregated LDL demonstrate an initial period when LDL-CE uptake and hydrolysis exceeds LDL-protein degradation [29]. LDL-CE internalization and hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis [29]. The tra¤cking and metabolic fate of the cholesterol internalized by this process is not yet known. The LDL-CE is hydrolyzed by lysosomal acid lipase [29], suggesting initial tra¤cking to lysosomes. Thus, one would presume the cholesterol enters the same postlysosomal pathways as lipoprotein-cholesterol acquired by endocytosis or phagocytosis, but this remains to be experimentally proven. Finally, the CE from by the ACAT pathway can be rehydrolyzed to FC by a cytoplasmic neutral CE
hydrolase [73]. This `cholesteryl ester cycle' goes on continuously, but the ratio of CE hydrolysis to cholesterol esteri¢cation is increased during cholesterol e¥ux from cells, and the FC resulting from this hydrolysis is available for e¥ux [47,73]. The identity of this hydrolase enzyme is uncertain; previous evidence suggested that it was identical to hormone-sensitive lipase (HSL) [74], but a recent report showed that macrophages from HSL knockout mice are able to hydrolyze stored CE normally [75]. De¢nitive identi¢cation of neutral CE hydrolase in macrophages is important because its stimulation may be a way to accelerate reversal of foam cell formation in the presence of extracellular cholesterol acceptors like highdensity lipoprotein (HDL). 4. Free cholesterol accumulation and phospholipid biosynthesis in macrophages: possible relevance to foam cells in advanced atherosclerotic lesions As described above, a major metabolic pathway in lesional macrophages is the esteri¢cation of cholesterol. Macrophages in advanced lesions, however, have been shown to accumulate large amounts of FC [76^79], probably due to a combination of decreased FC e¥ux, decreased ACAT-mediated cholesterol esteri¢cation, and perhaps increased CE hydrolysis [5]. The potential relevance of this observation is that FC loading of cells in general and macrophages in particular is cytotoxic [80^82], and thus FC-induced toxicity may be an important cause of lesional macrophage death (below). The causes of cytotoxicity are almost certainly related to the inhibition of certain critical plasma membrane enzymes by a high FC:phospholipid (PL) ratio in the vicinity of these molecules [5,80,83]. At a normal FC:PL ratio, membranes contain areas of PL `packing defects', which provide `space' for integral membrane proteins to undergo conformational changes. In the presence of excess FC, however, these packing defects diminish, which restricts the conformational freedom of membrane proteins and inhibits their ability to function properly [80]. Other peripheral membrane proteins, particular those in the mitochondria, may also be adversely a¡ected by a high FC:PL ratio. In addition, another possible consequence of excess FC accumulation in macrophages is the precipitation of
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
intracellular cholesterol crystals [84^87], which may cause physical damage to cellular organelles. KellnerWeibel et al. [88] provided data to support the hypothesis that FC loading damages cells by a¡ecting proteins in peripheral organelles by showing that drugs that partially block FC transport out of lysosomes prevent FC-mediated toxicity in macrophages. Consistent with this ¢nding, we have recently shown that peritoneal macrophages from mice with the Niemann-Pick C mutation, which results in a similar block of FC transport (see above and [42,89^91]), are also protected from death by FC loading (D. Zhang, P.M. Yao and I. Tabas, manuscript in preparation). Another interesting observation related to the macrophages of advanced lesions is their accumulation of intracellular membrane whorls [92], and in situ labeling studies in experimental animals have demonstrated increased PL biosynthesis in lesional cells [93]. Based on these observations and those described above, we hypothesized that increased PL biosynthesis, by preventing the FC:PL ratio from reaching toxic levels, was an initial, transient adaptive response to intracellular FC accumulation. Indeed, we showed that cultured macrophages respond to FC loading by increasing their biosynthesis and mass of phosphatidylcholine (PC) [82,94,95]. The mechanism is activation of the rate limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT) [94]. FC activates CT by an incompletely characterized signaling pathway that requires dephosphorylation of CT itself and possibly other cellular proteins [95]. Macrophage sphingomyelin biosynthesis is also increased in FC-loaded macrophages [63], but the cellular pathways have not yet been de¢ned. If this response is adaptive, why does prolonged FC loading lead to macrophage death? Our studies have shown that shortly before the onset of toxicity in FC-loaded macrophages, CT activity begins to decrease [82]. Thus, the adaptive mechanism is not maintained with continuous FC loading. In fact, we can hasten the onset of toxicity by incubating the cells in choline-de¢cient medium, which inhibits PC biosynthesis at an early time point. To further test this point, we have used cell-speci¢c gene targeting to create mice whose major CT isoform, CTK, is absent in macrophages. Under normal growth conditions,
169
these macrophages are healthy due to a low level of PC biosynthesis e¡ected by the other CT isoform, CTL. With FC loading, however, these macrophages show markedly accelerated cell death [106]. These data indicate CT activity and a normal level of PC biosynthesis is necessary for macrophages to survive the initial period of FC loading. As alluded to above, the physiologic relevance of these studies may be related to an important event in advanced atherosclerotic lesions, namely, macrophage death (Fig. 1). Many studies have shown the presence of dead or dying macrophages in human and animal atherosclerotic lesions, particularly in advanced lesions [96^101]. On one hand, lesional macrophage death may limit the number of macrophages that accumulate in atheromata. On the other hand, macrophage death may contribute to lesion pathology by releasing plaque destabilizing enzymes (e.g., lysosomal proteases and matrix metalloproteinases) and pro-coagulant/thrombogenic molecules (e.g., tissue factor and phosphatidylserine). These events would be expected to contribute to plaque rupture and acute thrombosis, which precipitate most acute ischemic events [102]. In fact, plaque rupture often occurs in the vicinity of `necrotic' areas in lesions [102], and these areas have been shown to contain the debris of dead macrophages [86,103]. In this context, FC-induced toxicity may be an important cause of macrophage death in advanced atherosclerotic lesions [104]. Accad et al. [54] have recently studied hypercholesterolemic mice whose macrophage ACAT has been knocked out by gene targeting. The early atherosclerotic lesions in these mice are populated by CE-depleted macrophages, but the later lesions are mostly devoid of macrophages altogether. The authors speculated that there was progressive loss of lesional macrophages due to FCinduced toxicity [54]. Dying macrophages in lesions show morphologic and biochemical signs of both `necrosis' and `apoptosis' [97]. The potential physiologic implications of this distinction may relate to the fate of the dying cells: necrotic death is likely to result in the release of harmful cellular contents (above), whereas it is possible, though far from certain, that death by apoptosis may lead to `safe' disposal of the toxic cells via phagocytosis of apoptotic bodies [105]. In the case of FC-induced death, Rothblat and colleagues [88]
BBAMCB 55719 28-11-00
170
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
Fig. 1. Working hypothesis of how macrophage cholesterol and phospholipid metabolism may be related to macrophage death in atherosclerotic lesions. See text for details. MPs, macrophages.
showed morphologic features suggestive of apoptosis, and we recently extended these ¢ndings by showing the presence of biochemical signs of apoptosis, such as externalization of phosphatidylserine, DNA fragmentation, and caspase involvement [107]. Most remarkably, a portion of FC-induced death requires the Fas pathway, and FC loading induces the expression of cell surface Fas ligand [107]. Moreover, another pathway of apoptosis ^ mitochondrial dysfunction ^ is induced by FC loading [107]. Thus, two novel cellular e¡ects of FC loading in macrophages ^ induction of cell surface Fas ligand and mitochondrial dysfunction ^ appear to be responsible for at least a portion of the death that is induced by FC loading (Fig. 1). 5. Conclusion The prominence of cholesterol-loaded macrophages in all stages of atherosclerotic lesions has been appreciated for several decades, but only recently have in vivo models of genetically altered macrophage physiology taught us that these cells play critical roles in atherogenesis. This insight highlights the importance of investigating the biology of the cholesterol-loaded macrophage, with an emphasis on understanding cellular and molecular mechanisms that account for the atherogenicity of these cells. There can be no doubt that many important aspects related to the role of macrophages in atherosclerosis
are directly linked to the accumulation and metabolism of cholesterol by these cells. One particularly important example of this principle may be the cell biologic events that lead to excess free cholesterol accumulation, which in turn in£uence critical aspects of macrophage phospholipid metabolism and susceptibility to cell death. Circumstantial evidence linking macrophage death to necrotic core formation, plaque rupture, and acute thrombosis provides a potential link between the basic cellular processes of macrophage cholesterol and phospholipid metabolism and the most important clinical manifestation of atherosclerotic vascular disease. For the future, our increasing knowledge of lesional macrophage biology is likely to lead to novel therapeutic strategies that complement the lipid lowering strategies that are currently available. Moreover, if investigators' speculations about the role of macrophages in plaque rupture prove correct, work in this area may lead to novel therapies that speci¢cally aim to prevent the formation of rupture-prone lesions and acute vascular events. Acknowledgements The author expresses gratitude to colleagues in his laboratory who have worked on various aspects of the work reviewed herein, including Drs. Xiangxi Xu, Paul Skiba, Yoshimune Shiratori, Wei Tang, Xavier Buton, Pin Mei Yao, Dajun Zhang, and
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
Sana Sakr. The author's research has been supported in part by NIH Grants HL 57560, HL 54591, and HL 56984. References [1] R. Ross, Cell biology of atherosclerosis, Annu. Rev. Physiol. 57 (1995) 791^804. [2] K.J. Williams, I. Tabas, The response-to-retention hypothesis of early atherogenesis, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 551^561. [3] K.J. Williams, I. Tabas, The response-to-retention hypothesis of atherogenesis, reinforced, Curr. Opin. Lipidol. 9 (1998) 471^474. [4] I. Tabas, in: D. Freeman, T.Y. Chang (Eds.), Intracellular Cholesterol Tra¤cking, Kluwer, Boston, MA, 1998, pp. 183^196. [5] I. Tabas, Free cholesterol-induced cytotoxicity. A possible contributing factor to macrophage foam cell necrosis in advanced atherosclerotic lesions, Trends Cardiovasc. Med. 7 (1997) 256^263. [6] I. Tabas, Phospholipid metabolism in cholesterol-loaded macrophages, Curr. Opin. Lipidol. 8 (1997) 263^267. [7] P. Libby, S.K. Clinton, The role of macrophages in atherogenesis, Curr. Opin. Lipidol. 4 (1993) 355^363. [8] J.D. Smith, E. Trogan, M. Ginsberg, C. Grigaux, J. Tian, M. Miyata, Decreased atherosclerosis in mice de¢cient in both macrophage colony-stimulating factor (op) and apolipoprotein E, Proc. Natl. Acad. Sci. USA 92 (1995) 8264^ 8268. [9] L. Boring, J. Gosling, M. Cleary, I.F. Charo, Decreased lesion formation in CCR23=3 mice reveals a role for chemokines in the initiation of atherosclerosis, Nature 394 (1998) 894^897. [10] L. Gu, Y. Okada, S.K. Clinton, C. Gerard, G.K. Sukhova, P. Libby, B.J. Rollins, Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-de¢cient mice, Mol. Cell 2 (1998) 275^281. [11] C.L. Lendon, M.J. Davies, G.V.R. Born, P.D. Richardson, Atherosclerotic plaque caps are locally weakened when macrophage density is increased, Atherosclerosis 87 (1991) 87^ 90. [12] Z.S. Galis, G.K. Sukhova, R. Kranzho«fer, S. Clark, P. Libby, Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading enzymes, Proc. Natl. Acad. Sci. USA 92 (1995) 402^406. [13] G.V. Born, New determinants of the uptake of atherogenic plasma proteins by arteries, Basic Res. Cardiol. 89 (1994) 103^106. [14] I. Snelting-Havinga, M. Mommaas, V.W. Van Hinsbergh, M.R. Daha, W.T. Daems, B.J. Vermeer, Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries, Eur. J. Cell Biol. 48 (1989) 27^36.
171
[15] E. Vasile, M. Simionescu, N. Simionescu, Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ, J. Cell Biol. 96 (1983) 1677^1689. [16] S. Weinbaum, S. Chien, Lipid transport aspects of atherogenesis, J. Biomech. Eng. 115 (1993) 602^610. [17] E.B. Smith, I.B. Massie, K.M. Alexander, The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin, Atherosclerosis 25 (1976) 71^84. [18] M.S. Brown, J.L. Goldstein, Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis, Annu. Rev. Biochem. 52 (1983) 223^261. [19] C. Koo, M.E. Wernette-Hammond, T.L. Innerarity, Uptake of canine-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor, J. Biol. Chem. 261 (1986) 11194^11201. [20] M.F. Linton, V.R. Babaev, L.A. Gleaves, S. Fazio, A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation, J. Biol. Chem. 274 (1999) 19204^19210. [21] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Witztum, Beyond cholesterol: modi¢cations of low-density lipoprotein that increase its atherogenicity, New Engl. J. Med. 320 (1989) 915^924. [22] M. Krieger, J. Herz, Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP), Annu. Rev. Biochem. 63 (1994) 601^637. [23] I. Tabas, Nonoxidative modi¢cations of lipoproteins in atherogenesis, Annu. Rev. Nutr. 19 (1999) 123^139. [24] P.F.E.M. Nievelstein, A.M. Fogelman, G. Mottino, J.S. Frank, Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein, Arterioscler. Thromb. 11 (1991) 1795^1805. [25] H.F. Ho¡, R.E. Morton, Lipoproteins containing apo B extracted from human aortas: structure and function, Ann. NY Acad. Sci. 454 (1985) 183^194. [26] J.C. Khoo, E. Miller, P. McLoughlin, D. Steinberg, Enhanced macrophage uptake of low density lipoprotein after self-aggregation, Arteriosclerosis 8 (1988) 348^358. [27] A.G. Suits, A. Chait, M. Aviram, J.W. Heinecke, Phagocytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation, Proc. Natl. Acad. Sci. USA 86 (1989) 2713^2717. [28] X. Xu, I. Tabas, Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages, J. Biol. Chem. 266 (1991) 24849^24858. [29] X. Buton, Z. Mamdouh, R. Ghosh, H. Du, G. Kuriakose, N. Beatini, G.A. Grabowski, F.R. Max¢eld, I. Tabas, Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL). Prolonged cell-surface contact during which LDL-cholesteryl ester hydrolysis exceeds LDL-protein degradation, J. Biol. Chem. 274 (1999) 32112^32121.
BBAMCB 55719 28-11-00
172
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
[30] J.L. Goldstein, M.S. Brown, R.G.W. Anderson, D.W. Russell, W.J. Schneider, Receptor-mediated endocytosis: concepts emerging from the LDL receptor system, Annu. Rev. Cell Biol. 1 (1985) 1^39. [31] G.G. Klaus, Cytochalasin B; dissociation of pinocytosis and phagocytosis by peritoneal macrophages, Exp. Cell Res. 79 (1973) 73^78. [32] M. Koval, K. Preiter, C. Adles, P.D. Stahl, T.H. Steinberg, Size of IgG-opsonized particles determines macrophage response during internalization, Exp. Cell Res. 242 (1998) 265^ 273. [33] P.M. Henson, Interaction of cells with immune complexes: adherence, release of constituents, and tissue injury, J. Exp. Med. 134 (1971) Suppl. 114s+. [34] H. Maxeiner, J. Husemann, C.A. Thomas, J.D. Loike, J. El Khoury, S.C. Silverstein, Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized lowdensity lipoproteins and in secretion of H2 O2 , J. Exp. Med. 188 (1998) 2257^2265. [35] F. Liszt, K. Schnittker-Schulze, H.W. Stuhlsatz, H. Greiling, Composition of proteoglycan fragments from hyaline cartilage produced by granulocytes in a model of frustrated phagocytosis, Eur. J. Clin. Chem. Clin. Biochem. 29 (1991) 123^130. [36] Z. Werb, R. Takemura, P.E. Stenberg, D.F. Bainton, Directed exocytosis of secretory granules containing apolipoprotein E to the adherent surface and basal vacuoles of macrophages spreading on immobile immune complexes, Am. J. Pathol. 134 (1989) 661^670. [37] M.H. Eccles, A.M. Glauert, The response of human monocytes to interaction with immobilized immune complexes, J. Cell Sci. 71 (1984) 141^157. [38] C.G. Ragsdale, W.P. Arend, Neutral protease secretion by human monocytes. E¡ect of surface-bound immune complexes, J. Exp. Med. 149 (1979) 954^968. [39] R.B. Johnston Jr., J.E. Lehmeyer, L.A. Guthrie, Generation of superoxide anion and chemiluminescence by human monocytes during phagocytosis and on contact with surface-bound immunoglobulin G, J. Exp. Med. 143 (1976) 1551^1556. [40] Y. Lange, in: T.Y. Chang, D.A. Freeman (Eds.), Intracellular Cholesterol Tra¤cking, Kluwer, Boston, MA, 1998, pp. 15^27. [41] P.G. Pentchev, M.T. Vanier, K. Suzuki, M.C. Patterson, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1995, pp. 2625^2639. [42] P.G. Pentchev, E.J. Blanchette-Mackie, L. Liscum, Biological implications of the Niemann-Pick C mutation, Sub-Cell. Biochem. 28 (1997) 437^451. [43] T. Kobayashi, M.H. Beuchat, M. Lindsay, S. Frias, R.D. Palmiter, H. Sakuraba, R.G. Parton, J. Gruenberg, Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport, Nat. Cell Biol. 1 (1999) 113^118. [44] J.C. Cruz, S. Sugii, C. Yu, T.Y. Chang, Role of Niemann-
[45] [46]
[47]
[48]
[49]
[50]
[51]
[52] [53]
[54]
[55]
[56]
[57] [58]
[59]
Pick type C1 protein in intracellular tra¤cking of low density lipoprotein-derived cholesterol, J. Biol. Chem. 275 (2000) 4013^4021. Y. Lange, J. Ye, M. Rigney, T.L. Steck, J. Biol. Chem. (2000) in press. N. Bishop, P. Woodman, ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol tra¤cking, Mol. Biol. Cell 11 (2000) 227^239. G.H. Rothblat, F.H. Mahlberg, W.J. Johnson, M.C. Phillips, Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol e¥ux, J. Lipid Res. 33 (1992) 1091^1097. I. Bjo«rkhem, O. Andersson, U. Diczfalusy, B. Sevastik, R. Xiu, C. Duan, E. Lund, Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages, Proc. Natl. Acad. Sci. USA 91 (1994) 8592^8596. E. Lund, O. Andersson, J. Zhang, A. Babiker, G. Ahlborg, U. Diczfalusy, K. Einarsson, J. Sjovall, I. Bjorkhem, Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans, Arterioscler. Thromb. Vasc. Biol. 16 (1996) 208^212. M.S. Brown, J.L. Goldstein, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell 89 (1997) 331^340. T.Y. Chang, C.C.Y. Chang, D. Cheng, Acyl-coenzyme A:cholesterol acyltransferase, Annu. Rev. Biochem. 66 (1997) 613^638. J.L. Goldstein, M.S. Brown, Regulation of the mevalonate pathway, Nature 343 (1990) 425^430. J. Roitelman, R.D. Simoni, Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, J. Biol. Chem. 267 (1992) 25264^ 25273. M. Accad, S.J. Smith, D.L. Newland, D.A. Sanan, L.E. King Jr., M.F. Linton, S. Fazio, R.V. Farese Jr., Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1, J. Clin. Invest. 105 (2000) 711^719. K.W. Underwood, N.L. Jacobs, A. Howley, L. Liscum, Evidence for a cholesterol transport pathway from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane, J. Biol. Chem. 273 (1998) 4266^4274. E.B. Neufeld, A.M. Cooney, J. Pitha, E.A. Dawidowicz, N.K. Dwyer, P.G. Pentchev, E.J. Blanchette-Mackie, Intracellular tra¤cking of cholesterol monitored with a cyclodextrin, J. Biol. Chem. 271 (1996) 21604^21613. L. Liscum, N.J. Munn, Intracellular cholesterol transport, Biochim. Biophys. Acta 1438 (1999) 19^37. V.L. Meiner, S. Cases, H.M. Myers, E.R. Sande, S. Bellosta, M. Schambelan, R.E. Pitas, J. McGuire, J. Herz, R.V. Farese Jr., Disruption of the acyl CoA:cholesterol acyltransferase (ACAT) gene in mice: evidence suggesting multiple cholesterol esteri¢cation enzymes in mammals, Proc. Natl. Acad. Sci. USA 93 (1996) 14041^14046. D. Cheng, C.C.Y. Chang, X. Qu, T. Chang, Activation of
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72] [73]
[74]
acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system, J. Biol. Chem. 270 (1995) 685^695. C.C.Y. Chang, G.M. Doolittle, T.Y. Chang, Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. I. E¡ect of exogenous sterols, Biochemistry 25 (1986) 1693^1699. I. Tabas, G.C. Boykow, Protein synthesis inhibition in mouse peritoneal macrophages results in increased acyl coenzyme A:cholesterol acyl transferase activity and cholesteryl ester accumulation in the presence of native low density lipoprotein, J. Biol. Chem. 262 (1987) 12175^12181. X. Xu, I. Tabas, Lipoproteins activate acyl coenzyme A:cholesterol acyl transferase in macrophages only after cellular cholesterol pools are expanded to a critical threshold level, J. Biol. Chem. 266 (1991) 17040^17048. A.K. Okwu, X. Xu, Y. Shiratori, I. Tabas, Regulation of the threshold for lipoprotein-induced acyl-CoA:cholesterol Oacyltransferase stimulation in macrophages by cellular sphingomyelin content, J. Lipid Res. 35 (1994) 644^655. Y. Lange, J. Ye, M. Rigney, T.L. Steck, Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol, J. Lipid Res. 40 (1999) 2264^2270. A.L. Rozelle, L.M. Machesky, M. Yamamoto, M.H. Driessens, R.H. Insall, M.G. Roth, K. Luby-Phelps, G. Marriott, A. Hall, H.L. Yin, Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3, Curr. Biol. 10 (2000) 311^320. S. Green, D. Steinberg, O. Quehenberger, Cloning and expression in Xenopus oocytes of a mouse homologue of the human acyl coenzyme A:cholesterol acyltransferase and its potential role in metabolism of oxidized LDL, Biochem. Biophys. Res. Commun. 218 (1996) 924^929. N. Khelef, X. Buton, N. Beatini, H. Wang, V. Meiner, T.Y. Chang, R.V. Farese Jr., F.R. Max¢eld, I. Tabas, Immunolocalization of ACAT in macrophages, J. Biol. Chem. 273 (1998) 11218^11224. N. Khelef, T.T. Soe, O. Quehenberger, N. Beatini, I. Tabas, F.R. Max¢eld, Arterioscler. Thromb. Vasc. Biol. (2000) in press. M.S. Ladinsky, D.N. Mastronarde, J.R. McIntosh, K.E. Howell, L.A. Staehelin, Golgi structure in three dimensions: functional insights from the normal rat kidney cell, J. Cell Biol. 144 (1999) 1135^1149. S. Mayor, S. Sabharanjak, F.R. Max¢eld, Cholesterol-dependent retention of GPI-anchored proteins in endosomes, EMBO J. 17 (1998) 4626^4638. S. Mukherjee, T.T. Soe, F.R. Max¢eld, Endocytic sorting of lipid analogues di¡ering solely in the chemistry of their hydrophobic tails, J. Cell Biol. 144 (1999) 1271^1284. S. Mukherjee, F.R. Max¢eld, Tra¤c (2000) in press. M.S. Brown, Y.K. Ho, J.L. Goldstein, The cholesteryl ester cycle in macrophage foam cells: continual hydrolysis and reesteri¢cation of cytoplasmic cholesteryl esters, J. Biol. Chem. 255 (1980) 9344^9352. J.C. Khoo, K. Reue, D. Steinberg, M.C. Schotz, Expression
[75]
[76]
[77]
[78]
[79]
[80] [81]
[82]
[83]
[84] [85]
[86]
[87]
[88]
173
of hormone-sensitive lipase mRNA in macrophages, J. Lipid Res. 34 (1993) 1969^1974. J. Osuga, S. Ishibashi, T. Oka, H. Yagyu, R. Tozawa, A. Fujimoto, F. Shionoiri, N. Yahagi, F.B. Kraemer, O. Tsutsumi, N. Yamada, Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity, Proc. Natl. Acad. Sci. USA 97 (2000) 787^ 792. B. Lundberg, Chemical composition and physical state of lipid deposits in atherosclerosis, Atherosclerosis 56 (1985) 93^110. D.M. Small, M.G. Bond, D. Waugh, M. Prack, J.K. Sawyer, Physicochemical and histological changes in the arterial wall of nonhuman primates during progression and regression of atherosclerosis, J. Clin. Invest. 73 (1984) 1590^1605. J.H. Rapp, W.E. Connor, D.S. Lin, T. Inahara, J.M. Porter, Lipids of human atherosclerotic plaques and xanthomas: clues to the mechanism of plaque progression, J. Lipid Res. 24 (1983) 1329^1335. H. Shio, N.J. Haley, S. Fowler, Characterization of lipidladen aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester, Lab. Invest. 41 (1979) 160^167. P.L. Yeagle, Modulation of membrane function by cholesterol, Biochimie 73 (1991) 1303^1310. G.J. Warner, G. Stoudt, M. Bamberger, W.J. Johnson, G.H. Rothblat, Cell toxicity induced by inhibition of acyl coenzyme A:cholesterol acyltransferase and accumulation of unesteri¢ed cholesterol, J. Biol. Chem. 270 (1995) 5772^5778. I. Tabas, S. Marathe, G.A. Keesler, N. Beatini, Y. Shiratori, Evidence that the initial up-regulation of phosphatidylcholine biosynthesis in free cholesterol-loaded macrophages is an adaptive response that prevents cholesterol-induced cellular necrosis. Proposed role of an eventual failure of this response in foam cell necrosis in advanced atherosclerosis, J. Biol. Chem. 271 (1996) 22773^22781. D. Papahadjopoulos, Cholesterol and cell membrane function: a hypothesis concerning the etiology of atherosclerosis, J. Theor. Biol. 43 (1974) 329^337. D.M. Small, Progression and regression of atherosclerotic lesions, Arteriosclerosis 8 (1988) 103^129. R.K. Tangirala, W.G. Jerome, N.L. Jones, D.M. Small, W.J. Johnson, J.M. Glick, F.H. Mahlberg, G.H. Rothblat, Formation of cholesterol monohydrate crystals in macrophagederived foam cells, J. Lipid Res. 35 (1994) 93^104. R.Y. Ball, E.C. Stowers, J.H. Burton, N.R. Cary, J.N. Skepper, M.J. Mitchinson, Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma, Atherosclerosis 114 (1995) 45^54. G. Kellner-Weibel, P.G. Yancey, W.G. Jerome, T. Walser, R.P. Mason, M.C. Phillips, G.H. Rothblat, Crystallization of free cholesterol in model macrophage foam cells, Arterioscler. Thromb. Vasc. Biol. 19 (1999) 1891^1898. G. Kellner-Weibel, W.G. Jerome, D.M. Small, G.J. Warner, J.K. Stoltenborg, M.A. Kearney, M.H. Corjay, M.C. Phillips, G.H. Rothblat, E¡ects of intracellular free cholesterol
BBAMCB 55719 28-11-00
174
[89]
[90]
[91] [92]
[93] [94]
[95]
[96]
[97]
[98]
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174 accumulation on macrophage viability: a model for foam cell death, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 423^431. P.G. Pentchev, A.D. Boothe, H.S. Kruth, H. Weintroub, J. Stivers, R.O. Brady, A genetic storage disorder in BALB/C mice with a metabolic block in esteri¢cation of exogenous cholesterol, J. Biol. Chem. 259 (1984) 5784^5791. S.K. Loftus, J.A. Morris, E.D. Carstea, J.Z. Gu, C. Cummings, A. Brown, J. Ellison, K. Ohno, M.A. Rosenfeld, D.A. Tagle, P.G. Pentchev, W.J. Pavan, Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene, Science 277 (1997) 232^235. L. Liscum, J.J. Klansek, Niemann-Pick disease type C, Curr. Opin. Lipidol. 9 (1998) 131^135. H. Shio, N.J. Haley, S. Fowler, Characterization of lipidladen aortic cells from cholesterol-fed rabbits. II. Morphometric analysis of lipid-¢lled lysosomes and lipid droplets in aortic cell populations, Lab. Invest. 39 (1978) 390^397. R.W. St. Clair, Metabolism of the arterial wall and atherosclerosis, Atheroscler. Rev. 1 (1976) 61^117. Y. Shiratori, A.K. Okwu, I. Tabas, Free cholesterol loading of macrophages stimulates phosphatidylcholine biosynthesis and up-regulation of CTP:phosphocholine cytidylyltransferase, J. Biol. Chem. 269 (1994) 11337^11348. Y. Shiratori, M. Houweling, X. Zha, I. Tabas, Stimulation of CTP:phosphocholine cytidylyltransferase by free cholesterol loading of macrophages involves signaling through protein dephosphorylation, J. Biol. Chem. 270 (1995) 29894^ 29903. Y. Geng, P. Libby, Evidence for apoptosis in advanced human atheroma. Colocalization with interleukin-1-converting enzyme, Am. J. Pathol. 147 (1995) 251^266. M.J. Mitchinson, S.J. Hardwick, M.R. Bennett, Cell death in atherosclerotic plaques, Curr. Opin. Lipidol. 7 (1996) 324^ 329. D.K.M. Han, C.C. Haudenschild, M.K. Hong, B.T. Tinkle, M.B. Leon, G. Liau, Evidence for apoptosis in human atherogenesis and in a rat vascular injury model, Am. J. Pathol. 147 (1995) 267^277.
[99] M.M. Kockx, Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 1519^1522. [100] S. Bjorkerud, B. Bjorkerud, Apoptosis is abundant in human atherosclerotic lesions, especially in in£ammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability, Am. J. Pathol. 149 (1996) 367^380. [101] R. Kinscherf, M. Wagner, H. Kamencic, G.A. Bonaterra, D. Hou, R.A. Schiele, H. Deigner, J. Metz, Characterization of apoptotic macrophages in atheromatous tissue of humans and heritable hyperlipidemic rabbits, Atherosclerosis 144 (1999) 33^39. [102] V. Fuster, L. Badimon, J.J. Badimon, J.H. Chesebro, The pathogenesis of coronary artery disease and the acute coronary syndromes, New Engl. J. Med. 326 (1992) 242^250. [103] P.A. Berberian, W. Myers, M. Tytell, V. Challa, M.G. Bond, Immunohistochemical localization of heat shock protein-70 in normal-appearing and atherosclerotic specimens of human arteries, Am. J. Pathol. 136 (1990) 71^80. [104] A.G. Zaman, G. Helft, S.G. Worthley, J.J. Badimon, The role of plaque rupture and thrombosis in coronary artery disease, Atherosclerosis 149 (2000) 251^266. [105] J. Savill, Recognition and phagocytosis of cells undergoing apoptosis, Br. Med. Bull. 53 (1997) 491^508. [106] D. Zhang, W. Tang, P.M. Yao, C. Yang, B. Xie, S. Jackowski, I. Tabas, Macrophages de¢cient in CTP:phosphocholine cytidylyltransferase-K are viable under normal culture conditions but are highly susceptible to free cholesterol-induced death. Molecular genetic evidence that the induction of phosphatidylcholine biosynthesis in freecholesterol-loaded macrophages is an adaptive response. J. Biol. Chem. (2000) In press. [107] P.M. Yao, I. Tabas, Free cholesterol loading of macrophages induces apoptosis involving the Fas pathway, J. Biol. Chem. 275 (2000) 23807^23813.
BBAMCB 55719 28-11-00
Review
Cholesterol and phospholipid metabolism in macrophages Ira Tabas * Departments of Medicine and Anatomy and Cell Biology, Columbia University, 630 West 168th Street, New York, NY 10032, USA Received 20 April 2000; accepted 16 August 2000
Abstract Cholesterol-loaded macrophages are present at all stages of atherogenesis, and recent in vivo data indicate that these cells play important roles in both early lesion development and late lesion complications. To understand how these cells promote atherogenesis, it is critical that we understand how lesional macrophages interact with subendothelial lipoproteins, the consequences of this interaction, and the impact of subsequent intracellular metabolic events. In the arterial wall, macrophages likely interact with both soluble and matrix-retained lipoproteins, and a new challenge is to understand how certain consequences of these two processes might differ. Initially, the major intracellular metabolic route of the lipoproteinderived cholesterol is esterification to fatty acids, but macrophages in advanced atherosclerotic lesions progressively accumulate large amounts of unesterified, or free, cholesterol (FC). In cultured macrophages, excess FC accumulation stimulates phospholipid biosynthesis, which is an adaptive response to protect the macrophage from FC-induced cytotoxicity. This phospholipid response eventually decreases with continued FC loading, leading to a series of cellular death reactions involving both death receptor-induced signaling and mitochondrial dysfunction. Because macrophage death in advanced lesions is thought to promote plaque instability, these intracellular processes involving cholesterol, phospholipid, and death pathways may play a critical role in the acute clinical manifestations of advanced atherosclerotic lesions. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: Foam cell; Atherosclerosis ; Lipoprotein; Acyl-coenzyme A:cholesterol acyltransferase; Phosphatidylcholine; Cytidine 5P-triphosphate:phosphocholine cytidylyltransferase ; Apoptosis
1. Introduction A critical event in the developing atherosclerotic lesion is the entry of monocytes into the subendothelium of focal areas of the arterial wall [1]. It is likely Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; CE, cholesteryl ester; CT, CTP:phosphocholine cytidylyltransferase; ER, endoplasmic reticulum; FC, free cholesterol; HSL, hormone-sensitive lipase; PC, phosphatidylcholine; LDL, low-density lipoprotein; M-CSF, macrophage-colony stimulating factor; PL, phospholipid * Fax: +1-212-305-4834; E-mail: [email protected]
that this event is a biologic response to the focal subendothelial retention of lipoproteins [2,3]. After entry, the monocytes are exposed to endotheliumderived growth and di¡erentiation factors, such as macrophage-colony stimulating factor (M-CSF), leading to their di¡erentiation into macrophages [1]. Therefore, these focal areas of the arterial tree, like areas in the liver, lung, spleen, and in£ammatory lesions, become populated with di¡erentiated tissue macrophages. Arterial wall macrophages in developing atherosclerotic lesions, however, face a rather unique environment that distinguishes their fate from other tissue macrophages: in the subendothelial
1388-1981 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 0 ) 0 0 1 4 6 - 3
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
matrix, they encounter a virtual forest of matrixbound lipoprotein particles [2,3]. The macrophages react to this environment by internalizing and metabolizing these subendothelial lipoproteins, leading to the massive intracellular accumulation of lipoprotein-derived cholesterol [1,4]. Initially, the major storage form of cholesterol in these macrophages is cholesteryl fatty acid esters (CE), which are sequestered into membrane-bound cytoplasmic lipid droplets; these macrophages are often referred to as `foam cells' because of the `foamy' appearance of the cytoplasmic CE inclusions [1,4]. Other important metabolic events in these macrophages, particularly in advanced atherosclerotic lesions, is the accumulation of unesteri¢ed, or `free', cholesterol (FC) and increased rates of phospholipid biosynthesis [5,6]. Speci¢c consequences of atherosclerotic macrophages include both physical e¡ects, such as intimal thickening, and biological e¡ects, such as internalization of lipoproteins and secretion of biologically active molecules [1,7]. Examples of molecules secreted by macrophages include cytokines, growth factors, and pro-oxidants, which have been implicated in amplifying early atherogenic events, and proteases, which are thought to contribute to plaque instability, acute thrombosis, and acute clinical events [1,7]. Indeed, many studies, including recent in vivo investigations, have provided evidence that macrophage foam cells play roles both in early atherogenesis and in late lesional events [7^10]. In particular, mice with absent M-CSF and thus decreased tissue macrophages, including decreased arterial wall macrophages, have markedly decreased atherosclerotic lesion size [8]. Similarly, mice with genetic mutations in a chemokine for monocytes, monocyte chemotactic protein-1, or its monocyte receptor, CCR2, have less arterial wall macrophages and less atherosclerosis [9,10]. The role of lesional macrophages in these atherogenic events is critically in£uenced by processes related to intracellular cholesterol and phospholipid metabolism. For example, many of the critical physical properties of foam cell-rich lesions are due to the physicochemical properties of the intracellular CE droplets [11], and CE loading of macrophages may be an important stimulus for the secretion of matrix metalloproteinases in advanced lesions [12]. The accumulation of excess FC by macrophages leads to
165
important consequences related to cellular phospholipid metabolism and cell viability [5,6]. Thus, a clear understanding of these intracellular metabolic processes is essential to our elucidating how macrophages in£uence atherogenesis and to the development of novel anti-atherogenic therapeutic strategies directed at the lesional macrophage. This review will describe our current understanding of cholesterol and phospholipid metabolism in lesional macrophages and how these metabolic events might a¡ect speci¢c atherogenic processes. 2. The interaction of macrophages with atherogenic lipoproteins Cholesterol accumulation in lesional macrophages begins with the internalization of subendothelial lipoproteins. These lipoproteins enter the subendothelium from the plasma by either transcytosis through endothelial cells or possibly via `leakage' through transient gaps between endothelial cells [13^16]. Once in the subendothelial space, the lipoproteins can either egress back into the circulation or be retained on subendothelial matrix, principally by binding to proteoglycans and collagen [2,3]. As alluded to above, an increased tendency of lipoproteins to be focally retained by these subendothelial molecules, followed by biological responses to this retained material, appears to be a key event in the initiation of atherogenesis [2,3]. A major area of investigation in the cell biology of atherosclerosis is the exploration of how macrophages interact with lipoproteins. Most studies have explored this interaction by incubating soluble, monomeric lipoproteins with monolayers of macrophages plated on tissue culture plastic. This model, which emphasizes receptor-mediated endocytosis, may re£ect some aspects of lesional foam cell formation in early atherosclerotic lesions, where a portion of subendothelial lipoproteins are probably monomeric and in solution in the interstitial £uid [17]. In addition, the model can help us understand some post-uptake events, such as lipoprotein-CE hydrolysis in lysosomes and the threshold phenomenon of cholesterol esteri¢cation (see below). Based on this model, investigators have shown that native low-density lipoprotein (LDL) is a poor inducer of CE ac-
BBAMCB 55719 28-11-00
166
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
cumulation in cultured macrophages, which re£ects both the downregulation of the LDL receptor and peculiarities of intracellular metabolism of LDL-derived cholesterol [4,18]. It is possible, however, that the LDL receptor itself can contribute to foam cell formation, perhaps by mediating the uptake of atherogenic chylomicron remnant-like lipoproteins [4,19]. In this regard, Fazio and colleagues [20] have shown that LDL receptor knockout mice transplanted with LDL receptor-positive macrophages have larger lesion size than similar mice transplanted with LDL receptor-negative macrophages. The receptor-mediated endocytosis model has also revealed that macrophages can internalize large amounts of oxidatively modi¢ed LDL via a variety of receptors including type A and B scavenger receptors [18,21,22]. Interestingly, most forms of in vitro oxidized LDL are relatively weak inducers of macrophage CE accumulation [23], but studies with type A scavenger receptor knockout mice have suggested a role for this receptor in foam cell formation in vivo. It is possible, therefore, that this receptor and perhaps other oxidized LDL receptors mediate the uptake of other forms of atherogenic lipoproteins in the subendothelium (see below). Once lesions becomes established, the vast majority of lesional lipoproteins are avidly bound to matrix [17]. Moreover, both biochemical and morphological studies of human and animal lesions have shown that many of the matrix lipoproteins are in a fused or aggregated state [23^25]. This point is crucial, because cell culture studies have shown that aggregated and fused lipoproteins, unlike oxidized LDL, are among the most potent inducers of massive CE loading of macrophages [23,26^28]. Thus, the typical cell culture model described above may not accurately re£ect the lipoprotein engagement and uptake process ^ and unique consequences of this process ^ that probably occur in the majority of lesions. For example, using a new experimental system to better re£ect the interaction of macrophages with matrix-retained and aggregated LDL, we have shown that a unique event occurs during the initial engagement process. This event is characterized by prolonged contact between macrophage surface invaginations and the matrix-retained lipoproteins during which LDL-CE hydrolysis exceeds LDL-protein degradation [29]. This process, which does not require
the LDL receptor, is clearly di¡erent from events occurring during typical receptor-mediated endocytosis, where there is rapid uptake of ligand and parallel degradation of the CE and protein moieties of lipoproteins [30]. The receptor or receptors mediating this process and its possible physiologic signi¢cance remain to be determined. Similarities between the engagement of matrix-retained and aggregated lipoproteins and macrophage phagocytosis of immobilized ligand (often called `frustrated phagocytosis') may provide other examples of processes that distinguish macrophage-lipoprotein interactions in vivo from the uptake of monomeric, soluble lipoproteins. For example, speci¢c events related to reorganization of the actin cytoskeleton, mediated by speci¢c cell signaling pathways, can distinguish phagocytosis from endocytosis [31,32] and are thus likely involved when lesional macrophages engage subendothelial lipoproteins. Similarly, a feature that distinguishes phagocytosis, and particularly frustrated phagocytosis (above), from receptor-mediated endocytosis is a secretory response leading to the release of a number of bioactive molecules. The list includes potentially atherogenic molecules such as in£ammatory cytokines, reactive oxygen intermediates, lysosomal enzymes, neutral proteases, and even an apolipoprotein [33^ 39]. Thus, new models designed to study the interaction of macrophages with matrix-retained and aggregated lipoproteins are likely to reveal the role of speci¢c cell surface molecules, cell signaling reactions, and secretory events that are involved in macrophage foam cell formation in vivo. 3. Intracellular cholesterol metabolism in macrophages following the internalization of atherogenic lipoproteins The major pathway by which atherogenic lipoproteins deliver their cholesterol to cells is via endocytic or phagocytic delivery of the lipoproteins to degradative organelles (late endosomes, lysosomes, or phagolysosomes), followed by hydrolysis of the of the CE moiety to FC. This newly hydrolyzed FC, along with the original FC component of the lipoproteins, is then distributed to peripheral organelles. Most studies suggest that the initial transport of this
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
FC is to the plasma membrane [4,40], with subsequent transport to other organelles. The molecular basis of these transport processes is not fully known, but the protein that is defective in Niemann-Pick C disease, called npc1, clearly plays a role [41^44]. Two recent studies have suggested that FC is ¢rst transported to the plasma membrane and then to a type of endocytic vesicle in an npc1-independent process, followed by npc1-dependent tra¤cking either back to the plasma membrane or to other peripheral organelles [44,45]. Other molecules that may facilitate FC tra¤cking include lysophosphatidic acid [43], a protein called npc2 that has not yet been cloned but is defective in a rare type of Niemann-Pick C disease [41,42], and VPS4, which is a mammalian homologue of a yeast vacuolar sorting protein [46]. Several sites of post-plasma membrane FC transport are of particular importance in atherosclerotic macrophage biology. Recycling back to the plasma membrane may be critical in FC e¥ux from cells [47]. Transport to the mitochondria is necessary for conversion to 27-hydroxycholesterol by the mitochondrial enzyme sterol 27-hydroxylase [48]; Bjo«rkhem and colleagues [48,49] propose that this pathway is important for sterol e¥ux from macrophages. Transport to the endoplasmic reticulum (ER) is necessary for important negative feedback pathways [50] and for cholesterol esteri¢cation [51]. One feedback pathway occurs when FC interacts directly with the sterol sensing domain of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, a rate limiting enzyme in cholesterol biosynthesis, leading to the enzyme's degradation [52,53]. In another pathway, interaction of FC with a homologous sterol sensing domain in an ER protein called SCAP (sterol-response element binding protein (SREBP) cleavage activating protein) prevents a series of proteolytic cleavages that, in the absence of sterol, leads to activation of SREBP; proteolytically activated SREBP is a transcription factor for the LDL receptor gene as well as genes involved in endogenous cholesterol biosynthesis [50]. The cholesterol esteri¢cation reaction is particularly important in the atherosclerotic macrophage, because the CE resulting from this reaction is the major lipid that accumulates in lesional foam cells [4,51,54]. Via the plasma membrane pathway mentioned above, and perhaps also via another pathway
167
involving more direct transport of FC from the lysosomes to the ER [55,56], FC comes into contact with acyl-CoA:cholesterol acyltransferase (ACAT) [4,40, 51,57]; the form of ACAT in macrophages is called ACAT1, which distinguishes it from another form of ACAT in liver and intestine called ACAT2 [58]. Upon contacting ACAT, the activity of the enzyme is increased, most likely by a combination of substrate provision and allosteric activation [59]. An increase in the expression of ACAT in response to cholesterol is not likely to be a major factor in the increased activity in macrophages because there are only small changes in ACAT mRNA in response to elevated cholesterol [51], and cycloheximide actually stimulates overall ACAT activity [60,61]. This latter observation may indicate that a short-lived protein in cells acts to limit the cholesterol esteri¢cation pathway, but there has been no direct proof of this hypothesis. Tra¤cking of FC to ACAT is energy-dependent, requires an intact actin cytoskeleton, and probably involves vesicular transport [4,40]. Importantly, ACAT is stimulated when cellular FC levels reach a particular threshold level above the ambient cellular cholesterol concentration [62,63]. The Tabas and Lange laboratories have proposed that threshold phenomenon may involve the induction of the above-mentioned cholesterol transport pathway [4,64], and Lange and colleagues have recently shown that there is a dramatic rise in ER cholesterol content when threshold is reached [64]. The exact mechanism whereby a rise in cellular cholesterol triggers this putative vesicular transport pathway remains to be determined. Of potential interest in this regard, Rozelle et al. [65] have recently shown that decreasing cellular cholesterol content, perhaps by in£uencing the behavior of lipid-rich domains in the plasma membrane, can inhibit actin-propelled vesicular transport in cells. An important issue regarding the regulation and function of ACAT is its cellular location. Most investigators agree that the majority of the enzyme resides in the ER, perhaps concentrated in the rough ER, where it functions to prevent excess FC accumulation in the cell in general and ER in particular [51]. Two studies, however, have presented some data suggesting that a small portion of ACAT may be localized to the cell surface, where it may more directly
BBAMCB 55719 28-11-00
168
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
limit the FC content of the plasma membrane [66,67]. Curiously, cell surface ACAT in macrophages was found only when the cells were not attached to a substratum [67]. Finally, Tabas, Max¢eld, and colleagues [67,68] have demonstrated that another portion of ACAT in macrophages is localized to a paranuclear site in the vicinity of the transGolgi network and endocytic recycling compartment, which are both cholesterol-rich membranes. In a recent electron microscopic three-dimensional analysis of the Golgi apparatus [69], it was shown that elements of the ER enter into close apposition with trans elements of the Golgi. In fact, portions of the ER become morphologically indistinguishable from stacked Golgi cisternae, and this would provide an ideal site for transfer of cholesterol from a cholesterol-rich Golgi membrane to the ER. In terms of a potential function of ACAT in this paranuclear site, recent work has shown that cellular cholesterol content can in£uence endocytic tra¤cking [70^72], perhaps by in£uencing the FC content of the endocytic recycling compartment, trans-Golgi network, or both. Therefore, it is possible that paranuclear ACAT, by regulating the FC content in these organelles (i.e., converting FC into a neutral lipid), can directly in£uence endocytic tra¤cking. This section has dealt thus far with lipoproteincholesterol entering macrophages via endocytic or phagocytic pathways, where lipoprotein-protein degradation and CE hydrolysis occur in parallel. As mentioned in the previous section, however, macrophages interacting with matrix-retained and aggregated LDL demonstrate an initial period when LDL-CE uptake and hydrolysis exceeds LDL-protein degradation [29]. LDL-CE internalization and hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis [29]. The tra¤cking and metabolic fate of the cholesterol internalized by this process is not yet known. The LDL-CE is hydrolyzed by lysosomal acid lipase [29], suggesting initial tra¤cking to lysosomes. Thus, one would presume the cholesterol enters the same postlysosomal pathways as lipoprotein-cholesterol acquired by endocytosis or phagocytosis, but this remains to be experimentally proven. Finally, the CE from by the ACAT pathway can be rehydrolyzed to FC by a cytoplasmic neutral CE
hydrolase [73]. This `cholesteryl ester cycle' goes on continuously, but the ratio of CE hydrolysis to cholesterol esteri¢cation is increased during cholesterol e¥ux from cells, and the FC resulting from this hydrolysis is available for e¥ux [47,73]. The identity of this hydrolase enzyme is uncertain; previous evidence suggested that it was identical to hormone-sensitive lipase (HSL) [74], but a recent report showed that macrophages from HSL knockout mice are able to hydrolyze stored CE normally [75]. De¢nitive identi¢cation of neutral CE hydrolase in macrophages is important because its stimulation may be a way to accelerate reversal of foam cell formation in the presence of extracellular cholesterol acceptors like highdensity lipoprotein (HDL). 4. Free cholesterol accumulation and phospholipid biosynthesis in macrophages: possible relevance to foam cells in advanced atherosclerotic lesions As described above, a major metabolic pathway in lesional macrophages is the esteri¢cation of cholesterol. Macrophages in advanced lesions, however, have been shown to accumulate large amounts of FC [76^79], probably due to a combination of decreased FC e¥ux, decreased ACAT-mediated cholesterol esteri¢cation, and perhaps increased CE hydrolysis [5]. The potential relevance of this observation is that FC loading of cells in general and macrophages in particular is cytotoxic [80^82], and thus FC-induced toxicity may be an important cause of lesional macrophage death (below). The causes of cytotoxicity are almost certainly related to the inhibition of certain critical plasma membrane enzymes by a high FC:phospholipid (PL) ratio in the vicinity of these molecules [5,80,83]. At a normal FC:PL ratio, membranes contain areas of PL `packing defects', which provide `space' for integral membrane proteins to undergo conformational changes. In the presence of excess FC, however, these packing defects diminish, which restricts the conformational freedom of membrane proteins and inhibits their ability to function properly [80]. Other peripheral membrane proteins, particular those in the mitochondria, may also be adversely a¡ected by a high FC:PL ratio. In addition, another possible consequence of excess FC accumulation in macrophages is the precipitation of
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
intracellular cholesterol crystals [84^87], which may cause physical damage to cellular organelles. KellnerWeibel et al. [88] provided data to support the hypothesis that FC loading damages cells by a¡ecting proteins in peripheral organelles by showing that drugs that partially block FC transport out of lysosomes prevent FC-mediated toxicity in macrophages. Consistent with this ¢nding, we have recently shown that peritoneal macrophages from mice with the Niemann-Pick C mutation, which results in a similar block of FC transport (see above and [42,89^91]), are also protected from death by FC loading (D. Zhang, P.M. Yao and I. Tabas, manuscript in preparation). Another interesting observation related to the macrophages of advanced lesions is their accumulation of intracellular membrane whorls [92], and in situ labeling studies in experimental animals have demonstrated increased PL biosynthesis in lesional cells [93]. Based on these observations and those described above, we hypothesized that increased PL biosynthesis, by preventing the FC:PL ratio from reaching toxic levels, was an initial, transient adaptive response to intracellular FC accumulation. Indeed, we showed that cultured macrophages respond to FC loading by increasing their biosynthesis and mass of phosphatidylcholine (PC) [82,94,95]. The mechanism is activation of the rate limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT) [94]. FC activates CT by an incompletely characterized signaling pathway that requires dephosphorylation of CT itself and possibly other cellular proteins [95]. Macrophage sphingomyelin biosynthesis is also increased in FC-loaded macrophages [63], but the cellular pathways have not yet been de¢ned. If this response is adaptive, why does prolonged FC loading lead to macrophage death? Our studies have shown that shortly before the onset of toxicity in FC-loaded macrophages, CT activity begins to decrease [82]. Thus, the adaptive mechanism is not maintained with continuous FC loading. In fact, we can hasten the onset of toxicity by incubating the cells in choline-de¢cient medium, which inhibits PC biosynthesis at an early time point. To further test this point, we have used cell-speci¢c gene targeting to create mice whose major CT isoform, CTK, is absent in macrophages. Under normal growth conditions,
169
these macrophages are healthy due to a low level of PC biosynthesis e¡ected by the other CT isoform, CTL. With FC loading, however, these macrophages show markedly accelerated cell death [106]. These data indicate CT activity and a normal level of PC biosynthesis is necessary for macrophages to survive the initial period of FC loading. As alluded to above, the physiologic relevance of these studies may be related to an important event in advanced atherosclerotic lesions, namely, macrophage death (Fig. 1). Many studies have shown the presence of dead or dying macrophages in human and animal atherosclerotic lesions, particularly in advanced lesions [96^101]. On one hand, lesional macrophage death may limit the number of macrophages that accumulate in atheromata. On the other hand, macrophage death may contribute to lesion pathology by releasing plaque destabilizing enzymes (e.g., lysosomal proteases and matrix metalloproteinases) and pro-coagulant/thrombogenic molecules (e.g., tissue factor and phosphatidylserine). These events would be expected to contribute to plaque rupture and acute thrombosis, which precipitate most acute ischemic events [102]. In fact, plaque rupture often occurs in the vicinity of `necrotic' areas in lesions [102], and these areas have been shown to contain the debris of dead macrophages [86,103]. In this context, FC-induced toxicity may be an important cause of macrophage death in advanced atherosclerotic lesions [104]. Accad et al. [54] have recently studied hypercholesterolemic mice whose macrophage ACAT has been knocked out by gene targeting. The early atherosclerotic lesions in these mice are populated by CE-depleted macrophages, but the later lesions are mostly devoid of macrophages altogether. The authors speculated that there was progressive loss of lesional macrophages due to FCinduced toxicity [54]. Dying macrophages in lesions show morphologic and biochemical signs of both `necrosis' and `apoptosis' [97]. The potential physiologic implications of this distinction may relate to the fate of the dying cells: necrotic death is likely to result in the release of harmful cellular contents (above), whereas it is possible, though far from certain, that death by apoptosis may lead to `safe' disposal of the toxic cells via phagocytosis of apoptotic bodies [105]. In the case of FC-induced death, Rothblat and colleagues [88]
BBAMCB 55719 28-11-00
170
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
Fig. 1. Working hypothesis of how macrophage cholesterol and phospholipid metabolism may be related to macrophage death in atherosclerotic lesions. See text for details. MPs, macrophages.
showed morphologic features suggestive of apoptosis, and we recently extended these ¢ndings by showing the presence of biochemical signs of apoptosis, such as externalization of phosphatidylserine, DNA fragmentation, and caspase involvement [107]. Most remarkably, a portion of FC-induced death requires the Fas pathway, and FC loading induces the expression of cell surface Fas ligand [107]. Moreover, another pathway of apoptosis ^ mitochondrial dysfunction ^ is induced by FC loading [107]. Thus, two novel cellular e¡ects of FC loading in macrophages ^ induction of cell surface Fas ligand and mitochondrial dysfunction ^ appear to be responsible for at least a portion of the death that is induced by FC loading (Fig. 1). 5. Conclusion The prominence of cholesterol-loaded macrophages in all stages of atherosclerotic lesions has been appreciated for several decades, but only recently have in vivo models of genetically altered macrophage physiology taught us that these cells play critical roles in atherogenesis. This insight highlights the importance of investigating the biology of the cholesterol-loaded macrophage, with an emphasis on understanding cellular and molecular mechanisms that account for the atherogenicity of these cells. There can be no doubt that many important aspects related to the role of macrophages in atherosclerosis
are directly linked to the accumulation and metabolism of cholesterol by these cells. One particularly important example of this principle may be the cell biologic events that lead to excess free cholesterol accumulation, which in turn in£uence critical aspects of macrophage phospholipid metabolism and susceptibility to cell death. Circumstantial evidence linking macrophage death to necrotic core formation, plaque rupture, and acute thrombosis provides a potential link between the basic cellular processes of macrophage cholesterol and phospholipid metabolism and the most important clinical manifestation of atherosclerotic vascular disease. For the future, our increasing knowledge of lesional macrophage biology is likely to lead to novel therapeutic strategies that complement the lipid lowering strategies that are currently available. Moreover, if investigators' speculations about the role of macrophages in plaque rupture prove correct, work in this area may lead to novel therapies that speci¢cally aim to prevent the formation of rupture-prone lesions and acute vascular events. Acknowledgements The author expresses gratitude to colleagues in his laboratory who have worked on various aspects of the work reviewed herein, including Drs. Xiangxi Xu, Paul Skiba, Yoshimune Shiratori, Wei Tang, Xavier Buton, Pin Mei Yao, Dajun Zhang, and
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
Sana Sakr. The author's research has been supported in part by NIH Grants HL 57560, HL 54591, and HL 56984. References [1] R. Ross, Cell biology of atherosclerosis, Annu. Rev. Physiol. 57 (1995) 791^804. [2] K.J. Williams, I. Tabas, The response-to-retention hypothesis of early atherogenesis, Arterioscler. Thromb. Vasc. Biol. 15 (1995) 551^561. [3] K.J. Williams, I. Tabas, The response-to-retention hypothesis of atherogenesis, reinforced, Curr. Opin. Lipidol. 9 (1998) 471^474. [4] I. Tabas, in: D. Freeman, T.Y. Chang (Eds.), Intracellular Cholesterol Tra¤cking, Kluwer, Boston, MA, 1998, pp. 183^196. [5] I. Tabas, Free cholesterol-induced cytotoxicity. A possible contributing factor to macrophage foam cell necrosis in advanced atherosclerotic lesions, Trends Cardiovasc. Med. 7 (1997) 256^263. [6] I. Tabas, Phospholipid metabolism in cholesterol-loaded macrophages, Curr. Opin. Lipidol. 8 (1997) 263^267. [7] P. Libby, S.K. Clinton, The role of macrophages in atherogenesis, Curr. Opin. Lipidol. 4 (1993) 355^363. [8] J.D. Smith, E. Trogan, M. Ginsberg, C. Grigaux, J. Tian, M. Miyata, Decreased atherosclerosis in mice de¢cient in both macrophage colony-stimulating factor (op) and apolipoprotein E, Proc. Natl. Acad. Sci. USA 92 (1995) 8264^ 8268. [9] L. Boring, J. Gosling, M. Cleary, I.F. Charo, Decreased lesion formation in CCR23=3 mice reveals a role for chemokines in the initiation of atherosclerosis, Nature 394 (1998) 894^897. [10] L. Gu, Y. Okada, S.K. Clinton, C. Gerard, G.K. Sukhova, P. Libby, B.J. Rollins, Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-de¢cient mice, Mol. Cell 2 (1998) 275^281. [11] C.L. Lendon, M.J. Davies, G.V.R. Born, P.D. Richardson, Atherosclerotic plaque caps are locally weakened when macrophage density is increased, Atherosclerosis 87 (1991) 87^ 90. [12] Z.S. Galis, G.K. Sukhova, R. Kranzho«fer, S. Clark, P. Libby, Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading enzymes, Proc. Natl. Acad. Sci. USA 92 (1995) 402^406. [13] G.V. Born, New determinants of the uptake of atherogenic plasma proteins by arteries, Basic Res. Cardiol. 89 (1994) 103^106. [14] I. Snelting-Havinga, M. Mommaas, V.W. Van Hinsbergh, M.R. Daha, W.T. Daems, B.J. Vermeer, Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries, Eur. J. Cell Biol. 48 (1989) 27^36.
171
[15] E. Vasile, M. Simionescu, N. Simionescu, Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ, J. Cell Biol. 96 (1983) 1677^1689. [16] S. Weinbaum, S. Chien, Lipid transport aspects of atherogenesis, J. Biomech. Eng. 115 (1993) 602^610. [17] E.B. Smith, I.B. Massie, K.M. Alexander, The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin, Atherosclerosis 25 (1976) 71^84. [18] M.S. Brown, J.L. Goldstein, Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis, Annu. Rev. Biochem. 52 (1983) 223^261. [19] C. Koo, M.E. Wernette-Hammond, T.L. Innerarity, Uptake of canine-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor, J. Biol. Chem. 261 (1986) 11194^11201. [20] M.F. Linton, V.R. Babaev, L.A. Gleaves, S. Fazio, A direct role for the macrophage low density lipoprotein receptor in atherosclerotic lesion formation, J. Biol. Chem. 274 (1999) 19204^19210. [21] D. Steinberg, S. Parthasarathy, T.E. Carew, J.C. Khoo, J.L. Witztum, Beyond cholesterol: modi¢cations of low-density lipoprotein that increase its atherogenicity, New Engl. J. Med. 320 (1989) 915^924. [22] M. Krieger, J. Herz, Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP), Annu. Rev. Biochem. 63 (1994) 601^637. [23] I. Tabas, Nonoxidative modi¢cations of lipoproteins in atherogenesis, Annu. Rev. Nutr. 19 (1999) 123^139. [24] P.F.E.M. Nievelstein, A.M. Fogelman, G. Mottino, J.S. Frank, Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein, Arterioscler. Thromb. 11 (1991) 1795^1805. [25] H.F. Ho¡, R.E. Morton, Lipoproteins containing apo B extracted from human aortas: structure and function, Ann. NY Acad. Sci. 454 (1985) 183^194. [26] J.C. Khoo, E. Miller, P. McLoughlin, D. Steinberg, Enhanced macrophage uptake of low density lipoprotein after self-aggregation, Arteriosclerosis 8 (1988) 348^358. [27] A.G. Suits, A. Chait, M. Aviram, J.W. Heinecke, Phagocytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation, Proc. Natl. Acad. Sci. USA 86 (1989) 2713^2717. [28] X. Xu, I. Tabas, Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages, J. Biol. Chem. 266 (1991) 24849^24858. [29] X. Buton, Z. Mamdouh, R. Ghosh, H. Du, G. Kuriakose, N. Beatini, G.A. Grabowski, F.R. Max¢eld, I. Tabas, Unique cellular events occurring during the initial interaction of macrophages with matrix-retained or methylated aggregated low density lipoprotein (LDL). Prolonged cell-surface contact during which LDL-cholesteryl ester hydrolysis exceeds LDL-protein degradation, J. Biol. Chem. 274 (1999) 32112^32121.
BBAMCB 55719 28-11-00
172
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
[30] J.L. Goldstein, M.S. Brown, R.G.W. Anderson, D.W. Russell, W.J. Schneider, Receptor-mediated endocytosis: concepts emerging from the LDL receptor system, Annu. Rev. Cell Biol. 1 (1985) 1^39. [31] G.G. Klaus, Cytochalasin B; dissociation of pinocytosis and phagocytosis by peritoneal macrophages, Exp. Cell Res. 79 (1973) 73^78. [32] M. Koval, K. Preiter, C. Adles, P.D. Stahl, T.H. Steinberg, Size of IgG-opsonized particles determines macrophage response during internalization, Exp. Cell Res. 242 (1998) 265^ 273. [33] P.M. Henson, Interaction of cells with immune complexes: adherence, release of constituents, and tissue injury, J. Exp. Med. 134 (1971) Suppl. 114s+. [34] H. Maxeiner, J. Husemann, C.A. Thomas, J.D. Loike, J. El Khoury, S.C. Silverstein, Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized lowdensity lipoproteins and in secretion of H2 O2 , J. Exp. Med. 188 (1998) 2257^2265. [35] F. Liszt, K. Schnittker-Schulze, H.W. Stuhlsatz, H. Greiling, Composition of proteoglycan fragments from hyaline cartilage produced by granulocytes in a model of frustrated phagocytosis, Eur. J. Clin. Chem. Clin. Biochem. 29 (1991) 123^130. [36] Z. Werb, R. Takemura, P.E. Stenberg, D.F. Bainton, Directed exocytosis of secretory granules containing apolipoprotein E to the adherent surface and basal vacuoles of macrophages spreading on immobile immune complexes, Am. J. Pathol. 134 (1989) 661^670. [37] M.H. Eccles, A.M. Glauert, The response of human monocytes to interaction with immobilized immune complexes, J. Cell Sci. 71 (1984) 141^157. [38] C.G. Ragsdale, W.P. Arend, Neutral protease secretion by human monocytes. E¡ect of surface-bound immune complexes, J. Exp. Med. 149 (1979) 954^968. [39] R.B. Johnston Jr., J.E. Lehmeyer, L.A. Guthrie, Generation of superoxide anion and chemiluminescence by human monocytes during phagocytosis and on contact with surface-bound immunoglobulin G, J. Exp. Med. 143 (1976) 1551^1556. [40] Y. Lange, in: T.Y. Chang, D.A. Freeman (Eds.), Intracellular Cholesterol Tra¤cking, Kluwer, Boston, MA, 1998, pp. 15^27. [41] P.G. Pentchev, M.T. Vanier, K. Suzuki, M.C. Patterson, in: C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle (Eds.), Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1995, pp. 2625^2639. [42] P.G. Pentchev, E.J. Blanchette-Mackie, L. Liscum, Biological implications of the Niemann-Pick C mutation, Sub-Cell. Biochem. 28 (1997) 437^451. [43] T. Kobayashi, M.H. Beuchat, M. Lindsay, S. Frias, R.D. Palmiter, H. Sakuraba, R.G. Parton, J. Gruenberg, Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport, Nat. Cell Biol. 1 (1999) 113^118. [44] J.C. Cruz, S. Sugii, C. Yu, T.Y. Chang, Role of Niemann-
[45] [46]
[47]
[48]
[49]
[50]
[51]
[52] [53]
[54]
[55]
[56]
[57] [58]
[59]
Pick type C1 protein in intracellular tra¤cking of low density lipoprotein-derived cholesterol, J. Biol. Chem. 275 (2000) 4013^4021. Y. Lange, J. Ye, M. Rigney, T.L. Steck, J. Biol. Chem. (2000) in press. N. Bishop, P. Woodman, ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol tra¤cking, Mol. Biol. Cell 11 (2000) 227^239. G.H. Rothblat, F.H. Mahlberg, W.J. Johnson, M.C. Phillips, Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol e¥ux, J. Lipid Res. 33 (1992) 1091^1097. I. Bjo«rkhem, O. Andersson, U. Diczfalusy, B. Sevastik, R. Xiu, C. Duan, E. Lund, Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages, Proc. Natl. Acad. Sci. USA 91 (1994) 8592^8596. E. Lund, O. Andersson, J. Zhang, A. Babiker, G. Ahlborg, U. Diczfalusy, K. Einarsson, J. Sjovall, I. Bjorkhem, Importance of a novel oxidative mechanism for elimination of intracellular cholesterol in humans, Arterioscler. Thromb. Vasc. Biol. 16 (1996) 208^212. M.S. Brown, J.L. Goldstein, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell 89 (1997) 331^340. T.Y. Chang, C.C.Y. Chang, D. Cheng, Acyl-coenzyme A:cholesterol acyltransferase, Annu. Rev. Biochem. 66 (1997) 613^638. J.L. Goldstein, M.S. Brown, Regulation of the mevalonate pathway, Nature 343 (1990) 425^430. J. Roitelman, R.D. Simoni, Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, J. Biol. Chem. 267 (1992) 25264^ 25273. M. Accad, S.J. Smith, D.L. Newland, D.A. Sanan, L.E. King Jr., M.F. Linton, S. Fazio, R.V. Farese Jr., Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1, J. Clin. Invest. 105 (2000) 711^719. K.W. Underwood, N.L. Jacobs, A. Howley, L. Liscum, Evidence for a cholesterol transport pathway from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane, J. Biol. Chem. 273 (1998) 4266^4274. E.B. Neufeld, A.M. Cooney, J. Pitha, E.A. Dawidowicz, N.K. Dwyer, P.G. Pentchev, E.J. Blanchette-Mackie, Intracellular tra¤cking of cholesterol monitored with a cyclodextrin, J. Biol. Chem. 271 (1996) 21604^21613. L. Liscum, N.J. Munn, Intracellular cholesterol transport, Biochim. Biophys. Acta 1438 (1999) 19^37. V.L. Meiner, S. Cases, H.M. Myers, E.R. Sande, S. Bellosta, M. Schambelan, R.E. Pitas, J. McGuire, J. Herz, R.V. Farese Jr., Disruption of the acyl CoA:cholesterol acyltransferase (ACAT) gene in mice: evidence suggesting multiple cholesterol esteri¢cation enzymes in mammals, Proc. Natl. Acad. Sci. USA 93 (1996) 14041^14046. D. Cheng, C.C.Y. Chang, X. Qu, T. Chang, Activation of
BBAMCB 55719 28-11-00
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72] [73]
[74]
acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system, J. Biol. Chem. 270 (1995) 685^695. C.C.Y. Chang, G.M. Doolittle, T.Y. Chang, Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. I. E¡ect of exogenous sterols, Biochemistry 25 (1986) 1693^1699. I. Tabas, G.C. Boykow, Protein synthesis inhibition in mouse peritoneal macrophages results in increased acyl coenzyme A:cholesterol acyl transferase activity and cholesteryl ester accumulation in the presence of native low density lipoprotein, J. Biol. Chem. 262 (1987) 12175^12181. X. Xu, I. Tabas, Lipoproteins activate acyl coenzyme A:cholesterol acyl transferase in macrophages only after cellular cholesterol pools are expanded to a critical threshold level, J. Biol. Chem. 266 (1991) 17040^17048. A.K. Okwu, X. Xu, Y. Shiratori, I. Tabas, Regulation of the threshold for lipoprotein-induced acyl-CoA:cholesterol Oacyltransferase stimulation in macrophages by cellular sphingomyelin content, J. Lipid Res. 35 (1994) 644^655. Y. Lange, J. Ye, M. Rigney, T.L. Steck, Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol, J. Lipid Res. 40 (1999) 2264^2270. A.L. Rozelle, L.M. Machesky, M. Yamamoto, M.H. Driessens, R.H. Insall, M.G. Roth, K. Luby-Phelps, G. Marriott, A. Hall, H.L. Yin, Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3, Curr. Biol. 10 (2000) 311^320. S. Green, D. Steinberg, O. Quehenberger, Cloning and expression in Xenopus oocytes of a mouse homologue of the human acyl coenzyme A:cholesterol acyltransferase and its potential role in metabolism of oxidized LDL, Biochem. Biophys. Res. Commun. 218 (1996) 924^929. N. Khelef, X. Buton, N. Beatini, H. Wang, V. Meiner, T.Y. Chang, R.V. Farese Jr., F.R. Max¢eld, I. Tabas, Immunolocalization of ACAT in macrophages, J. Biol. Chem. 273 (1998) 11218^11224. N. Khelef, T.T. Soe, O. Quehenberger, N. Beatini, I. Tabas, F.R. Max¢eld, Arterioscler. Thromb. Vasc. Biol. (2000) in press. M.S. Ladinsky, D.N. Mastronarde, J.R. McIntosh, K.E. Howell, L.A. Staehelin, Golgi structure in three dimensions: functional insights from the normal rat kidney cell, J. Cell Biol. 144 (1999) 1135^1149. S. Mayor, S. Sabharanjak, F.R. Max¢eld, Cholesterol-dependent retention of GPI-anchored proteins in endosomes, EMBO J. 17 (1998) 4626^4638. S. Mukherjee, T.T. Soe, F.R. Max¢eld, Endocytic sorting of lipid analogues di¡ering solely in the chemistry of their hydrophobic tails, J. Cell Biol. 144 (1999) 1271^1284. S. Mukherjee, F.R. Max¢eld, Tra¤c (2000) in press. M.S. Brown, Y.K. Ho, J.L. Goldstein, The cholesteryl ester cycle in macrophage foam cells: continual hydrolysis and reesteri¢cation of cytoplasmic cholesteryl esters, J. Biol. Chem. 255 (1980) 9344^9352. J.C. Khoo, K. Reue, D. Steinberg, M.C. Schotz, Expression
[75]
[76]
[77]
[78]
[79]
[80] [81]
[82]
[83]
[84] [85]
[86]
[87]
[88]
173
of hormone-sensitive lipase mRNA in macrophages, J. Lipid Res. 34 (1993) 1969^1974. J. Osuga, S. Ishibashi, T. Oka, H. Yagyu, R. Tozawa, A. Fujimoto, F. Shionoiri, N. Yahagi, F.B. Kraemer, O. Tsutsumi, N. Yamada, Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity, Proc. Natl. Acad. Sci. USA 97 (2000) 787^ 792. B. Lundberg, Chemical composition and physical state of lipid deposits in atherosclerosis, Atherosclerosis 56 (1985) 93^110. D.M. Small, M.G. Bond, D. Waugh, M. Prack, J.K. Sawyer, Physicochemical and histological changes in the arterial wall of nonhuman primates during progression and regression of atherosclerosis, J. Clin. Invest. 73 (1984) 1590^1605. J.H. Rapp, W.E. Connor, D.S. Lin, T. Inahara, J.M. Porter, Lipids of human atherosclerotic plaques and xanthomas: clues to the mechanism of plaque progression, J. Lipid Res. 24 (1983) 1329^1335. H. Shio, N.J. Haley, S. Fowler, Characterization of lipidladen aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester, Lab. Invest. 41 (1979) 160^167. P.L. Yeagle, Modulation of membrane function by cholesterol, Biochimie 73 (1991) 1303^1310. G.J. Warner, G. Stoudt, M. Bamberger, W.J. Johnson, G.H. Rothblat, Cell toxicity induced by inhibition of acyl coenzyme A:cholesterol acyltransferase and accumulation of unesteri¢ed cholesterol, J. Biol. Chem. 270 (1995) 5772^5778. I. Tabas, S. Marathe, G.A. Keesler, N. Beatini, Y. Shiratori, Evidence that the initial up-regulation of phosphatidylcholine biosynthesis in free cholesterol-loaded macrophages is an adaptive response that prevents cholesterol-induced cellular necrosis. Proposed role of an eventual failure of this response in foam cell necrosis in advanced atherosclerosis, J. Biol. Chem. 271 (1996) 22773^22781. D. Papahadjopoulos, Cholesterol and cell membrane function: a hypothesis concerning the etiology of atherosclerosis, J. Theor. Biol. 43 (1974) 329^337. D.M. Small, Progression and regression of atherosclerotic lesions, Arteriosclerosis 8 (1988) 103^129. R.K. Tangirala, W.G. Jerome, N.L. Jones, D.M. Small, W.J. Johnson, J.M. Glick, F.H. Mahlberg, G.H. Rothblat, Formation of cholesterol monohydrate crystals in macrophagederived foam cells, J. Lipid Res. 35 (1994) 93^104. R.Y. Ball, E.C. Stowers, J.H. Burton, N.R. Cary, J.N. Skepper, M.J. Mitchinson, Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma, Atherosclerosis 114 (1995) 45^54. G. Kellner-Weibel, P.G. Yancey, W.G. Jerome, T. Walser, R.P. Mason, M.C. Phillips, G.H. Rothblat, Crystallization of free cholesterol in model macrophage foam cells, Arterioscler. Thromb. Vasc. Biol. 19 (1999) 1891^1898. G. Kellner-Weibel, W.G. Jerome, D.M. Small, G.J. Warner, J.K. Stoltenborg, M.A. Kearney, M.H. Corjay, M.C. Phillips, G.H. Rothblat, E¡ects of intracellular free cholesterol
BBAMCB 55719 28-11-00
174
[89]
[90]
[91] [92]
[93] [94]
[95]
[96]
[97]
[98]
I. Tabas / Biochimica et Biophysica Acta 1529 (2000) 164^174 accumulation on macrophage viability: a model for foam cell death, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 423^431. P.G. Pentchev, A.D. Boothe, H.S. Kruth, H. Weintroub, J. Stivers, R.O. Brady, A genetic storage disorder in BALB/C mice with a metabolic block in esteri¢cation of exogenous cholesterol, J. Biol. Chem. 259 (1984) 5784^5791. S.K. Loftus, J.A. Morris, E.D. Carstea, J.Z. Gu, C. Cummings, A. Brown, J. Ellison, K. Ohno, M.A. Rosenfeld, D.A. Tagle, P.G. Pentchev, W.J. Pavan, Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene, Science 277 (1997) 232^235. L. Liscum, J.J. Klansek, Niemann-Pick disease type C, Curr. Opin. Lipidol. 9 (1998) 131^135. H. Shio, N.J. Haley, S. Fowler, Characterization of lipidladen aortic cells from cholesterol-fed rabbits. II. Morphometric analysis of lipid-¢lled lysosomes and lipid droplets in aortic cell populations, Lab. Invest. 39 (1978) 390^397. R.W. St. Clair, Metabolism of the arterial wall and atherosclerosis, Atheroscler. Rev. 1 (1976) 61^117. Y. Shiratori, A.K. Okwu, I. Tabas, Free cholesterol loading of macrophages stimulates phosphatidylcholine biosynthesis and up-regulation of CTP:phosphocholine cytidylyltransferase, J. Biol. Chem. 269 (1994) 11337^11348. Y. Shiratori, M. Houweling, X. Zha, I. Tabas, Stimulation of CTP:phosphocholine cytidylyltransferase by free cholesterol loading of macrophages involves signaling through protein dephosphorylation, J. Biol. Chem. 270 (1995) 29894^ 29903. Y. Geng, P. Libby, Evidence for apoptosis in advanced human atheroma. Colocalization with interleukin-1-converting enzyme, Am. J. Pathol. 147 (1995) 251^266. M.J. Mitchinson, S.J. Hardwick, M.R. Bennett, Cell death in atherosclerotic plaques, Curr. Opin. Lipidol. 7 (1996) 324^ 329. D.K.M. Han, C.C. Haudenschild, M.K. Hong, B.T. Tinkle, M.B. Leon, G. Liau, Evidence for apoptosis in human atherogenesis and in a rat vascular injury model, Am. J. Pathol. 147 (1995) 267^277.
[99] M.M. Kockx, Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects, Arterioscler. Thromb. Vasc. Biol. 18 (1998) 1519^1522. [100] S. Bjorkerud, B. Bjorkerud, Apoptosis is abundant in human atherosclerotic lesions, especially in in£ammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability, Am. J. Pathol. 149 (1996) 367^380. [101] R. Kinscherf, M. Wagner, H. Kamencic, G.A. Bonaterra, D. Hou, R.A. Schiele, H. Deigner, J. Metz, Characterization of apoptotic macrophages in atheromatous tissue of humans and heritable hyperlipidemic rabbits, Atherosclerosis 144 (1999) 33^39. [102] V. Fuster, L. Badimon, J.J. Badimon, J.H. Chesebro, The pathogenesis of coronary artery disease and the acute coronary syndromes, New Engl. J. Med. 326 (1992) 242^250. [103] P.A. Berberian, W. Myers, M. Tytell, V. Challa, M.G. Bond, Immunohistochemical localization of heat shock protein-70 in normal-appearing and atherosclerotic specimens of human arteries, Am. J. Pathol. 136 (1990) 71^80. [104] A.G. Zaman, G. Helft, S.G. Worthley, J.J. Badimon, The role of plaque rupture and thrombosis in coronary artery disease, Atherosclerosis 149 (2000) 251^266. [105] J. Savill, Recognition and phagocytosis of cells undergoing apoptosis, Br. Med. Bull. 53 (1997) 491^508. [106] D. Zhang, W. Tang, P.M. Yao, C. Yang, B. Xie, S. Jackowski, I. Tabas, Macrophages de¢cient in CTP:phosphocholine cytidylyltransferase-K are viable under normal culture conditions but are highly susceptible to free cholesterol-induced death. Molecular genetic evidence that the induction of phosphatidylcholine biosynthesis in freecholesterol-loaded macrophages is an adaptive response. J. Biol. Chem. (2000) In press. [107] P.M. Yao, I. Tabas, Free cholesterol loading of macrophages induces apoptosis involving the Fas pathway, J. Biol. Chem. 275 (2000) 23807^23813.
BBAMCB 55719 28-11-00