Yoshimune Shiratori

Retinoid is a collective term which indicates vitamin A (retinol) and its derivatives. Retinoid has a variety of functions such as growth promotion, maintenance of reproduction and dark adaptation. Retinoid also regulates cell differentiation and tissue morphogenesis. Sinceabnormality in the differentiation induces cellular atypia and that of the morphogenesis induces structural atypia, retinoid has important effects which inhibit carcinogenesis.

Macrophages in atherosclerotic lesions accumulate free cholesterol (FC) as well as cholesteryl ester and appear to have high rates of phospholipid (PL) synthesis and increased PL mass. Previous short term (i.e. ≤24 h) studies with cultured macrophages have shown that these cells respond to FC loading by up-regulating phosphatidylcholine biosynthesis. We propose that this response is adaptive by keeping the FC:PL ratio in the macrophages from reaching toxic levels. We further propose that one cause of macrophage necrosis, a prominent and important event in atherosclerosis, is an eventual decrease of this adaptive response. To explore these ideas, cultured macrophages were loaded with FC for up to 4 days and assayed for phosphatidylcholine biosynthesis, FC and PL mass, and cytotoxicity. For the first 24 h, cellular phosphatidylcholine biosynthesis and FC and PL mass increased 3-4-fold, and thus the FC:PL molar ratio was prevented from reaching very high levels; at this point, there were no overt signs of cytotoxicity. Over the next 24-48 h, however, phosphatidylcholine biosynthesis, and then phosphatidylcholine mass, began to decrease. Initially, the macrophages remained healthy and continued to accumulate FC, but eventually these macrophages, but not unloaded macrophages, became necrotic (swollen organelles and disrupted membranes). Lipoprotein dose studies indicated a close relationship between the onset of macrophage necrosis and the FC:PL ratio. To test further the causal nature of these relationships, cellular FC and PL mass were independently manipulated by using high density lipoprotein3 (HDL3) to decrease cellular FC and choline depletion to decrease cellular PC. As predicted by our hypotheses, HDL3 protected FC-loaded macrophages from necrosis, whereas choline depletion accelerated cytotoxic changes. These findings support the idea that the initial increase in phosphatidylcholine biosynthesis in FC-loaded macrophages is an adaptive response that prevents cholesterol-induced macrophage necrosis. We propose that an eventual failure of the PL response in foam cells may represent one cause of macrophage necrosis in advanced atherosclerotic lesions. Macrophages in atherosclerotic lesions accumulate free cholesterol (FC) as well as cholesteryl ester and appear to have high rates of phospholipid (PL) synthesis and increased PL mass. Previous short term (i.e. ≤24 h) studies with cultured macrophages have shown that these cells respond to FC loading by up-regulating phosphatidylcholine biosynthesis. We propose that this response is adaptive by keeping the FC:PL ratio in the macrophages from reaching toxic levels. We further propose that one cause of macrophage necrosis, a prominent and important event in atherosclerosis, is an eventual decrease of this adaptive response. To explore these ideas, cultured macrophages were loaded with FC for up to 4 days and assayed for phosphatidylcholine biosynthesis, FC and PL mass, and cytotoxicity. For the first 24 h, cellular phosphatidylcholine biosynthesis and FC and PL mass increased 3-4-fold, and thus the FC:PL molar ratio was prevented from reaching very high levels; at this point, there were no overt signs of cytotoxicity. Over the next 24-48 h, however, phosphatidylcholine biosynthesis, and then phosphatidylcholine mass, began to decrease. Initially, the macrophages remained healthy and continued to accumulate FC, but eventually these macrophages, but not unloaded macrophages, became necrotic (swollen organelles and disrupted membranes). Lipoprotein dose studies indicated a close relationship between the onset of macrophage necrosis and the FC:PL ratio. To test further the causal nature of these relationships, cellular FC and PL mass were independently manipulated by using high density lipoprotein3 (HDL3) to decrease cellular FC and choline depletion to decrease cellular PC. As predicted by our hypotheses, HDL3 protected FC-loaded macrophages from necrosis, whereas choline depletion accelerated cytotoxic changes. These findings support the idea that the initial increase in phosphatidylcholine biosynthesis in FC-loaded macrophages is an adaptive response that prevents cholesterol-induced macrophage necrosis. We propose that an eventual failure of the PL response in foam cells may represent one cause of macrophage necrosis in advanced atherosclerotic lesions. INTRODUCTIONCholesterol-loaded macrophages are prominent features of atherosclerotic lesions (1Schaffner T. Taylor K. Bartucci E.J. Fischer-Dzoga K. Beeson J.H. Glagov S. Wissler R.W. Am. J. Pathol. 1980; 100: 57-73Google Scholar, 2Gerrity R.G. Am. J. Pathol. 1981; 103: 181-190Google Scholar, 3Faggioto A. Ross R. Harker L. Arteriosclerosis. 1984; 4: 323-340Google Scholar), and there is increasing evidence that these cells play an important role both in early atherogenesis and in the clinical progression of advanced lesions (4Smith J.D. Trogan E. Ginsberg M. Grigaux C. Tian J. Miyata M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8264-8268Google Scholar, 5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar). Although cholesteryl ester accumulation in lesional macrophages (foam cells) is often emphasized, these cells also accumulate large amounts of FC, 1The abbreviations used are: FCfree cholesterolACATacyl-CoA:cholesterol O-acyltransferaseCTCTP:phosphocholine cytidylyltransferaseCon Aconcanavalin ADMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumHDL3high density lipoprotein3LDHlactate dehydrogenaseLDLlow density lipoproteinLPDSlipoprotein-deficient serumPBSphosphate-buffered salinePCphosphatidylcholinePLphospholipidlyso-PClysophosphatidylcholine. particularly in advanced atherosclerosis (7Katz S.S. Shipley G.G. Small D.M. J. Clin. Invest. 1976; 58: 200-211Google Scholar, 8Lundberg B. Atherosclerosis. 1985; 56: 93-110Google Scholar, 9Rapp J.H. Connor W.E. Lin D.S. Inahara T. Porter J.M. J. Lipid Res. 1983; 24: 1329-1335Google Scholar, 10Fowler S. Acta Med. Scand. Suppl. 1980; 642: 151-158Google Scholar). In this light, we have been interested in elucidating biological responses of macrophages to FC loading. One such response is the post-translational activation of the phosphatidylcholine (PC) biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CT), which leads to an increase in PC biosynthesis and in cellular PC mass (11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar, 12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar). This response is likely to be physiologically important, since increases in PC biosynthesis and mass have been noted to occur in lesional macrophages in vivo (13Buck R.C. Rossiter R.J. Arch. Pathol. 1951; 51: 224-230Google Scholar, 14Small D.M. Arteriosclerosis. 1988; 8: 103-129Google Scholar, 15Zilversmit D.B. Shore M.L. Ackerman R.F. Circulation. 1954; 9: 581-585Google Scholar, 16Day A.J. Wahlqvist M.L. Exp. Mol. Pathol. 1969; 11: 263-274Google Scholar, 17Wahlqvist M.L. Day A.J. Exp. Mol. Pathol. 1969; 11: 275-284Google Scholar).We have hypothesized that this PC response is initially adaptive (11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar, 12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar), since it would keep the cellular FC:PL ratio from getting too high and causing damage to cells (see Ref. 18Jackson R.L. Gotto Jr., A.M. Atheroscler. Rev. 1976; 1: 1-21Google Scholar). For example, membranes enriched with FC demonstrate inhibition of several membrane-bound enzymes (19Yeagle P.L. Biochim. Biophys. Acta. 1983; 727: 39-44Google Scholar, 20Ortega A. Mas-Oliva J. Biochem. Biophys. Res. Commun. 1986; 139: 868-874Google Scholar, 21Kashfi K. Dory L. Cook G.A. Biochem. Biophys. Res. Commun. 1991; 177: 1121-1126Google Scholar, 22Brasitus T.A. Dahiya R. Dudeja P.K. Bissonnette B.M. J. Biol. Chem. 1988; 263: 8592-8597Google Scholar), and cholesterol crystals may accumulate in such cells (14Small D.M. Arteriosclerosis. 1988; 8: 103-129Google Scholar). A corollary of our hypothesis is that an eventual blunting of this PC response would lead to cellular necrosis, and this scenario may be one cause of the necrosis of macrophages that is known to occur in advanced lesions (5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar, 23Ross R. Nature. 1993; 362: 801-809Google Scholar, 24Guyton J.R. Klemp K.F. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 4-11Google Scholar). Macrophage necrosis has been proposed to play an important role in plaque destabilization and thus clinical progression of lesions (5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar).The goal of the present study was to test these ideas using FC-loaded cultured macrophages. In our previous studies, the macrophages were FC-loaded for no longer than 24 h, at which point the PC biosynthetic response was still increasing, and the cells appeared healthy (11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar). In the present study, we have cultured these cells for longer periods, and we found that the PC biosynthetic response, but not the accumulation of FC, began to decrease after 24 h of culture. As predicted by our hypotheses, this event caused an increase in the cellular FC:PL ratio, and the cells subsequently showed signs of necrosis. Furthermore, removal of cellular FC prevented cytotoxicity, whereas premature blunting of the PL response accelerated cytotoxicity. These findings support the idea that the initial increase in PC biosynthesis in response to FC loading in macrophages is adaptive and raise the possibility that an eventual blunting of this response may lead to foam cell necrosis in advanced atherosclerotic lesions.DISCUSSIONAs summarized in Fig. 10, the findings in this report support the hypothesis that the initial rise in PC biosynthesis in FC-loaded macrophages is an adaptive response that keeps the FC:PL ratio from rising to toxic levels. This idea likely explains the lag in the onset of FC-mediated cytotoxicity observed in our study as well as that of Warner et al. (33Warner G.J. Stoudt G. Bamberger M. Johnson W.J. Rothblat G.H. J. Biol. Chem. 1995; 270: 5772-5778Google Scholar), which did not look at cellular PL metabolism. With prolonged FC loading, however, this adaptive response fails, the FC:PL ratio rises to cytotoxic levels, and macrophage necrosis ensues. Although we did not study the exact cause of macrophage death in these studies, high cellular FC levels are known to inhibit several critical membrane enzymes, including Na+/K+-ATPase activity (19Yeagle P.L. Biochim. Biophys. Acta. 1983; 727: 39-44Google Scholar), Ca2+/Mg2+-ATPase activity (20Ortega A. Mas-Oliva J. Biochem. Biophys. Res. Commun. 1986; 139: 868-874Google Scholar), carnitine palmitoyltransferase activity (21Kashfi K. Dory L. Cook G.A. Biochem. Biophys. Res. Commun. 1991; 177: 1121-1126Google Scholar), and alkaline phosphatase activity (22Brasitus T.A. Dahiya R. Dudeja P.K. Bissonnette B.M. J. Biol. Chem. 1988; 263: 8592-8597Google Scholar). Furthermore, as Small (14Small D.M. Arteriosclerosis. 1988; 8: 103-129Google Scholar) points out, excessive accumulation of cholesterol monohydrate crystals could lead to lysosomal rupture and cellular necrosis. In the case of membrane enzyme inhibition, the cause of FC-induced toxicity is probably related to perturbations of membrane fluidity, which could be compensated by increases in membrane phospholipid content (18Jackson R.L. Gotto Jr., A.M. Atheroscler. Rev. 1976; 1: 1-21Google Scholar). Cholesterol crystal formation would also be expected to be prevented by increases in cellular PL (14Small D.M. Arteriosclerosis. 1988; 8: 103-129Google Scholar). Interestingly, mouse peritoneal macrophages appear to require a higher FC:PL ratio than J774 macrophages to trigger cytotoxic changes (Fig. 7). Investigation into the mechanism of this relative resistance may shed additional light on how cells adapt to excess FC.The FC-loaded macrophages used in our studies are noted to have intracellular membrane whorls (see Ref. 11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar and Fig. 4, panel B), which probably represent the sites where most of the increased PL mass in these cells accumulate. Furthermore, filipin-labeling studies have shown that much of the FC that accumulates in cultured macrophages incubated with acetyl-LDL plus 58035 is localized in perinuclear lysosomes, presumably in lysosomal membranes (12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar). These findings suggest that our experimental model may reflect physiological events, since lesional macrophages in vivo have both intracellular membrane whorls (10Fowler S. Acta Med. Scand. Suppl. 1980; 642: 151-158Google Scholar) and accumulate FC in lysosomes (41Shio H. Haley N.J. Fowler S. Lab. Invest. 1979; 41: 160-167Google Scholar, 42Jerome W.G. Lewis J.C. Am. J. Pathol. 1985; 119: 210-222Google Scholar). These observations raise questions, however, about how the increased phospholipid might protect the cells from FC-mediated toxicity. First of all, where in the cell does the accumulation of FC cause problems? Many of the enzymes inhibited by excess FC are localized in the plasma membrane (see above), and Warner et al. (33Warner G.J. Stoudt G. Bamberger M. Johnson W.J. Rothblat G.H. J. Biol. Chem. 1995; 270: 5772-5778Google Scholar) have shown that FC export from the lysosomes is necessary for FC-mediated toxicity in macrophages. Thus, even though the bulk of FC appears to be in lysosomes, a critical amount of excess FC is probably in other cellular membranes, particularly the plasma membrane, and this localization is likely important for FC-mediated toxicity. Interestingly, we have shown that PC synthesis is still stimulated when lysosomal FC export is blocked (12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar), suggesting that lysosomal FC, while itself not initially toxic, may be the signal to “warn” the cell to protect other membranes from ensuing FC enrichment and damage. Eventually, massive lysosomal FC accumulation after very prolonged FC loading may result in cholesterol crystallization (43Tangirala R.K. Jerome W.G. Jones N.L. Small D.M. Johnson W.J. Glick J.M. Mahlberg F.H. Rothblat G.H. J. Lipid Res. 1994; 35: 93-104Google Scholar); consistent with this idea, cholesterol crystallization appears to be a relatively late effect of FC loading, since we have not observed it in our cells even after 3 days of incubation with acetyl-LDL plus 58035.How does the presence of intracellular membrane whorls pertain to the proposed ability of increased cellular PL to protect macrophages from FC-mediated toxicity? One possibility is that the membrane whorls serve as a “sink” for excess cellular cholesterol; for example, a critical amount of excess cholesterol from the sites of sensitive membrane enzymes (e.g. plasma membrane) might be transferred to the whorls, thus preventing inhibition of these enzymes (see above). Another idea is that the whorls represent a storage form of “excess” phospholipid in FC-loaded in this phospholipid would be transferred from the whorls to membranes in the cells that have a high FC:PL Ref. R.C. D.B. 1980; Scholar). such cholesterol phospholipid in found to play a role in the adaptive response of macrophages to FC loading, a in these might to FC-mediated important related to our studies but not are the of FC accumulation in vivo and the mechanism of blunting of the PC biosynthetic response with prolonged FC loading. the first cells several to the accumulation of excess These cellular cholesterol cholesterol of and of cholesterol biosynthesis, and cholesterol (e.g. synthesis in G.H. Mahlberg F.H. Johnson W.J. J. Lipid Res. 1992; Scholar, 1986; Scholar, 1992; Scholar). For macrophages large amounts of by other than the (e.g. the several of these are including of cholesterol biosynthesis, and of cholesterol into In cholesterol may be to of the cells to of it may be by the large amount of cholesterol in the the may be for example, in may of to I. H. M. Arterioscler. Thromb. Vasc. Biol. 1995; Scholar), itself may in advanced foam to PC biosynthesis, the initial in response to FC loading is to post-translational activation of the enzyme CTP:phosphocholine cytidylyltransferase (12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar); FC-mediated appears to a event the of and probably other cellular (12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar). cause of the eventual blunting of the PC biosynthesis response in our model and it is related to a in activity (e.g. changes in require further to our blunting of the PC biosynthesis response is an early event that subsequently a rise in the FC:PL ratio and FC-induced macrophage necrosis. We this idea on the that PC biosynthesis to at 24 h of FC loading (Fig. while the first signs of necrosis were 24 h is however, that the blunting of the PC biosynthesis response itself is a very early result of FC-induced which then an in the rise of the cellular FC:PL ratio. the of and the other membrane-bound PC biosynthetic enzymes to changes in membrane FC content in may shed light on this In it is also that the PC biosynthesis response to FC loading. For example, there is evidence that necrosis which is and by macrophages in response to a of P. Rev. 1992; Scholar, B. J. Lipid Res. 1994; 35: Scholar) and is known to be present in atherosclerotic lesions Rev. Biochem. 1983; Scholar, I. A.M. J. M. J. Lipid Res. 1981; Scholar), inhibit cellular PC biosynthesis in cells J. E. C. J. Clin. Invest. 1994; Scholar).We that the of this is that it at one for macrophage necrosis in atherosclerotic lesions. of macrophages in advanced atherosclerosis is to be an important event in progression (5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar, 23Ross R. Nature. 1993; 362: 801-809Google Scholar, 24Guyton J.R. Klemp K.F. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 4-11Google Scholar). For example, enzymes from these cells might to plaque rupture and eventual (5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar). mechanism of cellular necrosis in however, is not In to our other of free and and toxic example, Ref. J. Clin. Invest. 1995; Scholar). H. Exp. Mol. Pathol. Scholar) to test the of and depletion in lesional cell necrosis in the presence of by of lesions and lesions with Although both lesions a of and depletion and of necrosis was with the lesions. This the to that other than in to is necessary for necrosis and that the be related to in the lesions. This is consistent with our hypothesis as well as with other toxic In this we did the possibility that the of which be cytotoxic Biochim. Biophys. Acta. 1979; Scholar, K. P. R. J. Scholar), to the necrosis of macrophages. In studies, we found that levels are the onset of necrosis, but are in the after necrosis. Thus, we have no evidence that necrosis of FC-loaded macrophages, it may be an important of this event to cellular on PC. This may then the necrosis of cells to to be by this S. Proc. Natl. Acad. Sci. U. S. A. 1988; Scholar, N. J. Clin. Invest. 1992; Scholar, T. M. Ross R. Proc. Natl. Acad. Sci. U. S. A. 1994; that in a decrease in the PL content of lesional cells might necrosis and several the of on a which is known to decrease the phosphatidylcholine (PC) content of cells in R. J. Biol. Chem. 1985; Scholar) and in in vivo H. J. Biol. Chem. Scholar). Interestingly, when these were cholesterol and there was evidence of accelerated atherosclerosis in the and including increased necrosis Arch. Pathol. Scholar and Although the of the accelerated atherosclerosis in this is not known and may be it is to that at of the mechanism may be related to our hypothesis about the cytotoxicity of increased FC:PL goal of our is to test our in vivo by PL using mouse these to mouse of atherosclerosis, we to be to the important role of macrophage PL in macrophage necrosis and INTRODUCTIONCholesterol-loaded macrophages are prominent features of atherosclerotic lesions (1Schaffner T. Taylor K. Bartucci E.J. Fischer-Dzoga K. Beeson J.H. Glagov S. Wissler R.W. Am. J. Pathol. 1980; 100: 57-73Google Scholar, 2Gerrity R.G. Am. J. Pathol. 1981; 103: 181-190Google Scholar, 3Faggioto A. Ross R. Harker L. Arteriosclerosis. 1984; 4: 323-340Google Scholar), and there is increasing evidence that these cells play an important role both in early atherogenesis and in the clinical progression of advanced lesions (4Smith J.D. Trogan E. Ginsberg M. Grigaux C. Tian J. Miyata M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8264-8268Google Scholar, 5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar). Although cholesteryl ester accumulation in lesional macrophages (foam cells) is often emphasized, these cells also accumulate large amounts of FC, 1The abbreviations used are: FCfree cholesterolACATacyl-CoA:cholesterol O-acyltransferaseCTCTP:phosphocholine cytidylyltransferaseCon Aconcanavalin ADMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumHDL3high density lipoprotein3LDHlactate dehydrogenaseLDLlow density lipoproteinLPDSlipoprotein-deficient serumPBSphosphate-buffered salinePCphosphatidylcholinePLphospholipidlyso-PClysophosphatidylcholine. particularly in advanced atherosclerosis (7Katz S.S. Shipley G.G. Small D.M. J. Clin. Invest. 1976; 58: 200-211Google Scholar, 8Lundberg B. Atherosclerosis. 1985; 56: 93-110Google Scholar, 9Rapp J.H. Connor W.E. Lin D.S. Inahara T. Porter J.M. J. Lipid Res. 1983; 24: 1329-1335Google Scholar, 10Fowler S. Acta Med. Scand. Suppl. 1980; 642: 151-158Google Scholar). In this light, we have been interested in elucidating biological responses of macrophages to FC loading. One such response is the post-translational activation of the phosphatidylcholine (PC) biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CT), which leads to an increase in PC biosynthesis and in cellular PC mass (11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar, 12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar). This response is likely to be physiologically important, since increases in PC biosynthesis and mass have been noted to occur in lesional macrophages in vivo (13Buck R.C. Rossiter R.J. Arch. Pathol. 1951; 51: 224-230Google Scholar, 14Small D.M. Arteriosclerosis. 1988; 8: 103-129Google Scholar, 15Zilversmit D.B. Shore M.L. Ackerman R.F. Circulation. 1954; 9: 581-585Google Scholar, 16Day A.J. Wahlqvist M.L. Exp. Mol. Pathol. 1969; 11: 263-274Google Scholar, 17Wahlqvist M.L. Day A.J. Exp. Mol. Pathol. 1969; 11: 275-284Google Scholar).We have hypothesized that this PC response is initially adaptive (11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar, 12Shiratori Y. Houweling M. Zha X. Tabas I. J. Biol. Chem. 1995; 270: 29894-29903Google Scholar), since it would keep the cellular FC:PL ratio from getting too high and causing damage to cells (see Ref. 18Jackson R.L. Gotto Jr., A.M. Atheroscler. Rev. 1976; 1: 1-21Google Scholar). For example, membranes enriched with FC demonstrate inhibition of several membrane-bound enzymes (19Yeagle P.L. Biochim. Biophys. Acta. 1983; 727: 39-44Google Scholar, 20Ortega A. Mas-Oliva J. Biochem. Biophys. Res. Commun. 1986; 139: 868-874Google Scholar, 21Kashfi K. Dory L. Cook G.A. Biochem. Biophys. Res. Commun. 1991; 177: 1121-1126Google Scholar, 22Brasitus T.A. Dahiya R. Dudeja P.K. Bissonnette B.M. J. Biol. Chem. 1988; 263: 8592-8597Google Scholar), and cholesterol crystals may accumulate in such cells (14Small D.M. Arteriosclerosis. 1988; 8: 103-129Google Scholar). A corollary of our hypothesis is that an eventual blunting of this PC response would lead to cellular necrosis, and this scenario may be one cause of the necrosis of macrophages that is known to occur in advanced lesions (5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar, 23Ross R. Nature. 1993; 362: 801-809Google Scholar, 24Guyton J.R. Klemp K.F. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 4-11Google Scholar). Macrophage necrosis has been proposed to play an important role in plaque destabilization and thus clinical progression of lesions (5Libby P. Clinton S.K. Curr. Opin. Lipidol. 1993; 4: 355-363Google Scholar, 6Fuster V. Badimon L. Badimon J.J. Chesebro J.H. N. Engl. J. Med. 1992; 326: 242-250Google Scholar).The goal of the present study was to test these ideas using FC-loaded cultured macrophages. In our previous studies, the macrophages were FC-loaded for no longer than 24 h, at which point the PC biosynthetic response was still increasing, and the cells appeared healthy (11Shiratori Y. Okwu A.K. Tabas I. J. Biol. Chem. 1994; 269: 11337-11348Google Scholar). In the present study, we have cultured these cells for longer periods, and we found that the PC biosynthetic response, but not the accumulation of FC, began to decrease after 24 h of culture. As predicted by our hypotheses, this event caused an increase in the cellular FC:PL ratio, and the cells subsequently showed signs of necrosis. Furthermore, removal of cellular FC prevented cytotoxicity, whereas premature blunting of the PL response accelerated cytotoxicity. These findings support the idea that the initial increase in PC biosynthesis in response to FC loading in macrophages is adaptive and raise the possibility that an eventual blunting of this response may lead to foam cell necrosis in advanced atherosclerotic lesions.

PURPOSE: To determine the optimal b values required for diffusion-weighted (DW) imaging of the liver in the detection and characterization of benign and malignant hepatic lesions. MATERIALS AND METHODS: MR images obtained in 76 patients including 28 malignant hepatic lesions (21 hepatocellular carcinomas and 7 metastases) and 27 benign lesions (12 hemangiomas and 15 cysts) were reviewed. DW-echo planner images (EPIs; b values with 100, 200, 400, and 800 s/mm2) were reviewed solely first, and then with T2-weighted EPIs (b=0 s/mm2). RESULTS: Sensitivity for malignant lesions (74%) was highest on DW-EPIs with b value of 100 s/mm2 and T2-weighted EPIs combined (P<0.05), and sensitivity for benign lesions (87%) was highest on DW-EPIs with b value of 800 s/mm2 and T2-weighted EPIs (P<0.05). Specificities were comparably high for all sequences. The Az values for malignant lesions were 0.94, 0.90, 0.87, and 0.89, and those for benign lesions were 0.91, 0.89, 0.87, and 0.94 on DW-EPIs with b values of 100, 200, 400, and 800 and T2-weighted EPIs combined, respectively. Hepatic cysts were clearly distinguished with the cutoff ADC value of 2.5x10(-3) mm2/s using a b value of 400 s/mm2 or greater. CONCLUSION: DW-EPIs with middle b values were not required in the detection and characterization of benign and malignant hepatic lesions.

PURPOSE: To evaluate and compare total body weight (TBW), lean body weight (LBW), and estimated blood volume (BV) for the adjustment of the iodine dose required for contrast material-enhanced multidetector computed tomography (CT) of the aorta and liver. MATERIALS AND METHODS: Institutional review committee approval and written informed consent were obtained. One hundred twenty patients (54 men, 66 women; mean age, 64.1 years; range, 19-88 years) who underwent multidetector CT of the upper abdomen were randomized into three groups of 40 patients each: (a) TBW group (0.6 g of iodine per kilogram of TBW), (b) LBW group (0.821 g of iodine per kilogram of LBW), and (c) BV group (men, 8.6 g of iodine per liter of BV; women, 9.9 g of iodine per liter of BV). Change in CT number between unenhanced and contrast-enhanced images per gram of iodine and maximum hepatic enhancement (MHE) adjusted for iodine dose were examined for correlation with TBW, LBW, and BV by using linear regression analysis. RESULTS: In the portal venous phase, correlation coefficients for the correlation of change in CT number per gram of iodine with TBW for the aorta and liver were -0.71 and -0.79, respectively, in the TBW group; -0.80 and -0.86, respectively, in the LBW group; and -0.68 and -0.66, respectively, in the BV group. In the liver, they were marginally higher in the LBW group than in the BV group (P = .03). Adjusted MHE remained constant at 77.9 HU +/- 10.2 (standard deviation) in the LBW group with respect to TBW, but it increased in the TBW (r = 0.80, P < .001) and BV (r = 0.70, P < .001) groups as TBW increased. CONCLUSION: When LBW, rather than TBW or BV, is used, the iodine dose required to achieve consistent hepatic enhancement may be estimated more precisely and with reduced patient-to-patient variability.

Atheroma macrophages accumulate large amounts of free cholesterol (FC) as well as cholesteryl ester (CE). An important adaptive response to FC loading might be increased cellular phospholipid to accommodate the excess FC. To explore this idea, J774 macrophages were incubated for 48 h without lipid, with acetyl-low density lipoprotein to induce mostly CE loading, or with acetyl-low density lipoprotein plus an acyl-CoA:cholesterol O-acyltransferase inhibitor (58035) to induce marked FC loading. The total phospholipid content approximately doubled in FC-loaded versus control or CE-loaded macrophages, with phosphatidylcholine showing the largest increase (approximately 2.5-fold versus control). Electron micrographs revealed the presence of multiple intracellular membrane whorls in the FC-loaded macrophages but not in the control or CE-loaded macrophages. [3H]Choline incorporation into phosphatidylcholine was also greater in FC-loaded macrophages versus control or CE-loaded macrophages, whereas [3H]phosphatidylcholine degradation was similar in all of the macrophages. In these experiments and in others that used non-lipoprotein cholesterol, there was a very close correlation between cellular FC content and phosphatidylcholine biosynthesis. To determine the mechanism of increased phosphatidylcholine synthesis, FC-loaded and CE-loaded macrophages were pulsed with [3H]choline, then chased and assayed for labeled phosphatidylcholine biosynthetic precursors. The only major differences were a 2-fold greater disappearance of label from [3H]choline phosphate and a 5-fold greater appearance of label in CDP-[3H]choline in the FC-loaded macrophages. These data suggest a stimulation of CTP:phosphocholine cytidylyltransferase (CT), which was confirmed by microsomal CT assays. Further studies revealed that the increase in phosphatidylcholine biosynthesis in FC-loaded macrophages was: (a) reversible under conditions of high density lipoprotein3-mediated cellular cholesterol efflux; (b) not blocked by cycloheximide-induced protein synthesis inhibition; and (c) not associated with increased CT mRNA levels. Thus, FC loading of macrophages leads to an increase in phosphatidylcholine mass which is caused by increased phosphatidylcholine biosynthesis. The mechanism appears to be FC-mediated post-translational activation of CT. This adaptive response may be important for atheroma macrophage survival, and disruption of the response may lead to macrophage necrosis and lesion progression.