Proc. Natl. Acad. Sci. USA Vol. 90, pp.

8314-8318, September 1993

Review Transgenic mouse models of lipoprotein metabolism and atherosclerosis

(apolpoprotein/diet/colester)

Jan L. Breslow
Laboratory of Biochemkal Genetcs and Metabolism, The Rockefeller UniversIty, 1230 York Avenue, New York, NY 10021-6399

Lipoprotein transport ABSTRACT genes have either been added to the germ line of mice by transgenic techniques or knocked out by homologous recombination in embryonic stem cells. The resultant over- or underexpression of these genes has resulted in new Insights about how these genes function In the body and their role In Hlpoprotein metabolism. Either singly or In combination, these genetic modifications can be used to engineer the mouse to make it a better model for human lipoprotein disorders and atherosclerosis.

mice, which have provided insights into Table 1. Lipoprotein transport proteins the control of lipoprotein transport gene Apolipoproteins expression as well as the effects of over- A-I, A-II, A-IV, B, C-I, C-Il, C-Ill, D, or underexpression of these genes on E, apo(a) lipoprotein metabolism. Finally, either Processing proteins singly or in combination these transgenes Lipoprotein lipase have been used to make the mouse a Hepatic lipase Lecithin cholesterol acyltransferase better model for human lipoprotein disCholesterol ester transfer protein (CETP) orders and atherosclerosis.

Expression of Lipoprotein Transport

Genes

Receptors LDL Receptor Chylomicron-remnant receptor Scavenger receptor

Lipoproteins are complex particles, composed of various lipids and proteins, that transport endogenous and dietary fats in the blood stream. There are five types of lipoprotein particles: from smallest to largest, HDL, LDL, IDL, VLDL (high, low, intermediate, and very low density lipoproteins), and chylomicrons. Abnormal lipoprotein levels are associated with several human diseases, most commonly atherosclerosis. Individuals with atherosclerotic coronary heart disease almost invariably have one or more of four lipoprotein abnormalities: increased LDL-C; decreased HDL-C, usually associated with increased levels of triglyceride-rich lipoproteins (VLDL); increased levels of IDL-C and chylomicron remnants; and high levels of an abnormal lipoprotein, called lipoprotein(a) [Lp(a)], which is a complex of LDL and a large glycoprotein called apolipoprotein(a) [apo(a)l. To better understand lipoprotein metabolism and its abnormalities, in the last decade genes have been isolated that code for proteins that directly interact with plasma lipids. There are approximately 17 such lipoprotein transport proteins, including apolipoproteins which coat lipoprotein particles, lipoproteinprocessing proteins, and lipoprotein receptors (Table 1). The genes coding for these proteins have all turned out to be single-copy in the human genome. They have been sequenced, mapped, and used as candidate genes to identify mutations underlying each of the lipoprotein phenotypes associated with susceptibility to coronary heart disease (1). The lipoprotein transport genes have also been used to make transgenic animals, principally

Cis-acting regions controlling transcriptional regulation of lipoprotein transport genes have been studied in transgenic mice. This approach has been particularly helpful in identifying the regions controlling tissue specific expression of two clusters of apolipoprotein genes. One cluster located on chromosome 11q23 consists ofthe A-I, C-IIl, and A-IV genes in that order. The C-Ill gene is transcribed in the opposite orientation from the other two genes (Fig. 1). The A-I and C-III genes are expressed primarily in liver and intestine, whereas the A-IV gene is expressed primarily only in intestine. Human genomic fragments introduced into transgenic mice have revealed that the liver control regions for A-I and C-III reside immediately 5' to each of the genes, whereas a region between the C-III and A-IV genes controls intestinal expression of all ofthe genes at this locus (Fig. 1) (2-4). The second cluster of apolipoprotein genes that has been studied in transgenic mice is located on chromosome 19q13 and consists of the E, C-I, and C-Il genes in that order. The genes are transcribed in the same orientation and there is a C-I pseudogene between the C-I and C-II genes. All three genes are expressed primarily in the liver, and the transgenic mouse experiments uncovered a 150-bp region between the C-I gene and the C-I pseudogene required for high-level liver expression of this entire locus (Fig. 1) (5-9). Thus, the transgenic mouse studies of both apolipoprotein gene clusters have revealed previously unsuspected but qualitatively different regulatory elements, an intestinal control element for the A-I, C-III, A-IV locus
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and a liver control element for the E, C-I, C-II locus. Transgenic mice have also been used to study nutritional regulation of CETP gene expression. Dietary cholesterol was shown to increase CETP transgene transcription when 3.2 kb of 5' and 2 kb of 3' natural flanking sequences were present (10). Thus, the transgenic experiments have defined a region containing a transcriptional element that is upregulated by dietary cholesterol, in contrast to the well-known sterol response element which downregulates the transcription of several genes in response to cholesterol.

Physiology of Lipoprotein Transport

Genes

Transgenic mouse lines have now been established that overexpress many of the lipoprotein transport genes, and new information about how these genes function has been produced in almost every case. In addition, recently, the technique of homologous recombination in embryonic stem cells has been used to knock out lipoprotein transport genes. This has made it possible to study the consequences of gene underexpression for lipoprotein metabolism and has provided other insights into lipoprotein transport gene function.
Abbreviations: HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; apo(a), apolipoprotein(a); Lp(a), lipoprotein(a); CETP, cholesterol ester transfer protein.

Review: Breslow
Chr 11
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Proc. Natl. Acad. Sci. USA 90 (1993)

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there was a single copy of the transgene and only 30-40% extra C-Ill in plasma, yet these mice had more than twice norA A L L mal triglyceride levels. The human C-Ill mice are the first animal model transgenic 1Kb of primary hypertriglyceridemia and, recently, the mechanism has been studied in detail (19). These animals accumulate E C-I Chr 19 C-IT C-L VLDL particles that are slightly larger than normal. The VLDL composition is L. appropriately triglyceride-rich, but there is altered apolipoprotein content with inFIG. 1. Tissue-specific regulation of apolipoprotein gene transcription. On chromosome creased C-III and diminished E. The (Chr) 11, liver control regions (L) lie immediately upstream of the genes encoding apolipopro- transgenic mice also have increased free teins A-I and C-III. Another region (I) controls the intestinal expression of all three genes in this acids. Metabolic studies indicate cluster. On chromosome 19, a region required for expression of the E, C-I, and C-II genes in the fatty the primary abnormality to be decreased liver (L) lies between the C-I gene and the C-I pseudogene (C-I'). VLDL fractional catabolic rate with a A-I Transgenic Mice. A-I is the major latter was not accompanied by a change small increase in VLDL triglyceride but HDL protein, comprising 70% of total in A-I mRNA levels. Thus over the wide not B production rate. In vitro the transHDL protein, and transgenic animals ex- range of A-I levels observed in the high- genic VLDL showed decreased LDL repressing the human A-I protein have fat-feeding and probucol experiments ceptor-mediated uptake by tissue culture been produced (2, 3, 11, 12). In trans- there were no changes observed in the cells but normal lipolysis by purified lilipase. Thus, the hypertriglycgenic mice and rats, A-I overexpression A-I fractional catabolic rates or in the A-I poprotein eridemia appears to be due to a prolonged was found to selectively increase HDL-C mRNA levels. Two interesting implicaVLDL residence time but not with the levels. This is compatible with studies in tions can be drawn from these studies: (i) accumulation of remnant particles. This humans, which show that HDL-C levels over the physiological range of A-I levels implies decreased in vivo lipolysis and correlate with A-I levels, and suggests there is no saturable A-I or HDL receptor tissue uptake, presumably secondary to that pharmacological interventions that and (ii) previously unrecognized, potent, altered surface apolipoprotein increase A-I production may raise posttranscriptional levels of regulation tion and/or elevated free fatty composiacid levHDL-C levels in humans and improve the exist which regulate A-I production in els. Human C-I transgenic mice have also lipoprotein profile. Human A-I expres- relevant clinical situations. been found to be mildly hypertriglycerision in the mouse also resulted in a deRecently, A-I gene knockout mice demic (7). Although this model has not crease in levels of mouse A-I, with some were created and, as expected, these been studied in detail, it raises the questransgenic lines having 80-90%oof plasma animals had reduced levels of HDL-C, tion of whether other apolipoprotein beA-I being of the human variety (11, 13). but the consequences of this for lipopro- sides C-III might also act in a similar Coincident with the expression of human tein metabolism have not been reported manner. A-I in the mouse, changes were also (16). The human C-III transgenic mouse exnoted in the physical properties of HDL. A-fl Transgenic Mice. A-II is the sec- periments prove that C-III overexpresMouse HDL normally consists of a single ond most abundant HDL protein, com- sion can cause hypertriglyceridemia and major size distribution of particles of prising 20%Wo of the total. Transgenic mice suggest that C-III gene expression may approximate mean diameter 10 nm. In expressing human A-II have been pro- regulate triglyceride levels in humans. human A-I transgenic mice there are two duced (17). Unlike the human A-I trans- Hypertriglyceridemia is common in humajor size distributions of particles, with genics, these animals do not have ele- mans, with one-third of middle-aged diameters approximately 10.3 and 8.8 vated HDL-C levels, nor do they have males affected, yet the known genetic nm. This corresponds to the two major diminished levels of mouse A-I or A-II. abnormalities, lipoprotein lipase and C-II size distributions of HDL particles in The lack of effect of excess A-II produc- deficiency, are quite rare, less than one in human plasma, HDL 2b and 3a, respec- tion on HDL-C levels is compatible both a million (1). Evidence for the involvetively. The transgenic mouse studies with clinical studies that fail to show a ment of the C-III gene in human hypershow that the structure of human versus correlation between plasma A-II and triglyceridemia has come from associamouse A-I is an important determinant of HDL-C levels and with the relatively tion studies. These have repeatedly HDL particle size distribution, and sug- normal HDL-C levels reported in an shown that C-III alleles with an Sst I gest an explanation for HDL subspecia- A-II-deficient patient. The human A-II restriction enzyme site in the 3' untranstion in humans. transgenic mice did show an alteration of lated region are more common in affected Human A-I transgenic mice have HDL particle size with the appearance of Caucasian hypertriglyceridemics than in served as a model system to examine the a population of 8.0-nm-diameter particles controls. More recently, five new sites of mechanisms whereby diet and drugs alter along with the normal-sized mouse HDL genetic variation have been identified in HDL-C and A-I levels. A high-fat diet particles. The protein of the smaller HDL the C-III gene promoter, and haplotyping increases HDL-C and A-I levels in hu- consisted almost entirely of human A-II, utilizing these and the Sst I site has mans, and this was mimicked in trans- whereas the larger particles consisted of revealed three classes of C-Ill alleles, genic mice (14). In these animals the main mouse A-I and human A-II. Thus A-II susceptible, neutral, and protective with metabolic effect was to increase HDL appears to affect the quality rather than regard to hypertriglyceridemia (20). If cholesterol ester and A-I transport rates the quantity of HDL. efforts to identify the causative mutawithout an increase in A-I mRNA levels. C-m Transgenic Mice. C-III is a pro- tions and prove that C-Ill expression In contrast, probucol decreases HDL-C tein in VLDL and HDL. Several trans- influences human triglyceride levels and A-I levels, and this effect was repro- genic mouse lines have been made with prove successful, the use of transgenic duced in transgenic mice (15). In probu- the C-III gene and triglyceride levels are animals to provide clues to the genes col-treated mice the HDL cholesterol es- proportional to C-III gene expression, as underlying complex human traits may ter fractional catabolic rate increased but measured by human C-III plasma con- become an important paradigm in human the A-I transport rate decreased. The centrations (18). In one transgenic line genetics.
A-IV

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Proc. Natl. Acad. Sci. USA 90 (1993)

E Transgenic Mice. E, a protein in VLDL and HDL, is a ligand for the LDL and chylomicron-remnant receptors and plays an important role in the clearance of lipoprotein particles from the plasma. The initial E transgenic mice were made with human E gene constructions containing limited amounts of natural flanking sequences (5, 6). These constructions lacked the liver control element discussed above, and as a result the animals produced had relatively low levels of E expression with no significant effect of transgene expression on lipoprotein levels. Transgenic mice have now been made with the rat E gene driven by the metallothionein promoter (21, 22). After zinc induction, these animals had a 4-fold increase in E levels accompanied by a significant decrease in VLDL and LDL cholesterol levels. Metabolic studies indicated a severalfold increase in the clearance rate of radiolabeled VLDL and LDL. In addition, these animals were resistant to diet-induced hypercholesterolemia. These studies indicate that E overexpression lowers fasting levels of atherogenic lipoproteins and decreases diet response. Mutant forms of E exist in the population which predispose to type III hyperlipoproteinemia. One of these, E3Leiden, which contains a tandem duplication of amino acids 120-126 of the normal E polypeptide of 299 residues, causes a dominantly inherited form of this disease. This E variant has been expressed in transgenic mice producing increased levels of cholesterol and triglyceride in the VLDL plus LDL fractions of plasma (23). Plasma lipid levels in these mice were extremely responsive to dietary cholesterol. The E3-Leiden mice appear to be a phenocopy of human type III hyperlipoproteinemia and will be a useful model in which to study the genetic and environmental factors which influence the expression of this disease. E knockout mice with a true null mutation have also been created (24-26). Homozygous deficient animals are viable and fertile. On a chow diet, which is very low in cholesterol (0.01%) and low in fat (4.5%), they have cholesterol levels of 400-500 mg/dl. Most of this is in the VLDL plus IDL lipoprotein fractions. When the homozygous E knockout mice are fed a Western-type diet, which has moderate amounts of cholesterol (0.15%) and fat (20%), they respond with cholesterol levels of approximately 1800 mg/dl, also mostly in the VLDL plus IDL lipoprotein fractions (26). On both diets triglyceride levels are minimally elevated, suggesting the presence of cholesterolenriched particles, probably similar to B-VLDL. Metabolic studies indicate a severe defect in lipoprotein clearance from plasma, as predicted from the known function of E as a ligand for

lipoprotein receptors. Heterozygous E knockout mice have diminished plasma E levels, normal fasting lipoprotein levels, and slightly delayed postprandial lipoprotein clearance. Thus, half-normal E expression in the mouse is nearly sufficient for normal lipoprotein metabolism. Apo(a) Transgenic Mice. Apo(a) is a large glycoprotein that is disulfidebonded to the B moiety of LDL to form Lp(a). Lp(a) is found in humans, Old World primates, and hedgehogs, but not in other species, and its function is unknown. Apo(a) resembles plasminogen, containing domains of plasminogen-like kringle IV, in multiple copies, and of plasminogen-like kringle V and protease, in single copies. Apo(a) alleles specify proteins that differ in size due to variation in the number of kringle IV-like domains. Mice do not express apo(a), and transgenic animals were made with a human apo(a) gene construction consisting of a cDNA containing 17 kringle IV-, kringle V-, and protease-coding regions driven by the transferrin promoter (27). Expression was achieved in all tissues analyzed, whereas the gene is normally expressed only in liver. Mean plasma levels equivalent to Lp(a) at 9 mg/dl were achieved, but, in contrast to humans, apo(a) was found in the lipoprotein-free fraction. Infusion of human LDL into these transgenic mice resulted in binding ofapo(a) to lipoproteins of the LDL density class. These experiments suggest that human apo(a) cannot bind to mouse B, perhaps due to the lack of conservation of a crucial cysteine. CETP Transgenic Mice. CETP exchanges cholesterol ester in HDL for triglycerides in VLDL and IDL. CETP activity is lacking in mouse plasma. A human CETP minigene driven by the mouse metallothionein I promoter was used to make a transgenic line with human-like levels of activity in plasma, which could be doubled by feeding zinc (28, 29). After zinc treatment, compared to control mice, these mice had lower levels of HDL-C and A-I, 35% and 24%, respectively, and a smaller HDL particle size (10-nm to 9.7-nm mean particle diameter). These effects of CETP were less than expected based on studies comparing normal and CETP-deficient humans. Subsequently, CETP was found to be more potent in mice expressing the human A-I transgene (29). In these experiments human CETP transgenic mice were crossed with human A-I transgenic mice. After zinc treatment, compared to the human A-I transgenic mice, the doubly transgenic mice had a large reduction in HDL-C and A-I levels, 66% and 42%, respectively, with an even smaller HDL particle size (10.4-, 8.8-, and 7.4-nm to 9.7-, 8.5-, and 7.3-nm mean particle diameter). In the doubly transgenic mice, 100% of the CETP was HDL-associated

versus 22% in the singly transgenic animals. Thus CETP overexpression can reduce HDL-C levels and particle size, and the effect is much more dramatic on the human A-I background. This implies a specific interaction of human CETP with human A-I or the particles it produces. The CETP-mediated exchange of triglycerides for HDL cholesterol ester is driven by the level of VLDL, which is quite low in the mouse. Therefore, the effect of CETP was also studied in hypertriglyceridemic human C-III transgenic mice also expressing human A-I (30). In these mice, human CETP gene expression reduced HDL-C and A-I to very low levels with a dramatic reduction in HDL particle size. This mimics the high-triglyceride low-HDL-C phenotype in humans, which is the most common lipoprotein disorder associated with susceptibility to coronary heart disease. The human A-I, C-III, CETP transgenic mice are the first animal model ofthis disorder. These animals provide insights into which genes may cause this abnormal phenotype in humans and present opportunities to study the mechanisms of the relationship between this lipoprotein abnormality and atherosclerosis susceptibility. Recently, a cynomolgus monkey CETP cDNA drive by the mouse metallothionein I promoter was used to make mice with very high levels of CETP (31). The monkey CETP transgenic mice showed a strong inverse correlation of CETP activity with HDL-C and A-I levels and HDL size, as previously shown, and also showed a positive correlation of CETP activity with B levels and the size of B-containing lipoproteins. The monkey CETP mice were also more dietresponsive than control animals. The experiments with human and monkey CETP transgenic mice confirm the proposed role of CETP in lipoprotein metabolism deduced from other systems and make it possible to test the effect of CETP on cholesterol homeostasis, particularly reverse cholesterol transport, and atherosclerosis. LDL Receptor Transgenic Mice. The LDL receptor mediates lipoprotein clearance from plasma through recognition of B and E on the surface of lipoprotein particles. LDL receptor transgenic mice were made with a human cDNA construction driven by the mouse metallothionein I promoter (32). After heavymetal induction, transgenic mice cleared injected radiolabeled LDL 8-10 times faster than control mice, and the plasma concentrations of LDL receptor ligands, B and E, declined by more than 90%. The increase in LDL clearance was primarily due to increased liver removal of LDL, mainly by parenchymal cells. This study shows that inappropriate overexpression

Review: Breslow of a receptor can lower plasma levels of its ligand. LDL receptor transgenic mice were also made with a human minigene construction driven by the transferrin promoter, resulting in animals with chronically elevated levels of LDL receptors (33). When the transgenic mice were challenged with a high-fat, highcholesterol diet they did not increase IDL or LDL levels and only slightly increased VLDL levels, whereas control mice significantly increased the levels of all three of these lipoprotein fractions. Thus, it appears that unregulated expression of LDL receptors can affect diet response. This suggests one possible mechanism for the variation in diet response in humans. Finally, LDL receptor transgenic mice have been used to study receptor sorting to the surfaces of epithelial cells (34). In these mice, transgenic LDL receptors localize to the basolateral surface of hepatocyes and intestinal epithelial cells but the apical surface of renal tubular epithelial cells. A signal presumably present in the coding sequence of the transgene apparently interacts in a tissuespecific manner to control sorting to cell membrane surfaces. LDL Receptor-Related Protein (LRP) Transgenic Mice. LRP is presumed to be the chylomicron-remnant receptor, but also functions in the cellular uptake of protease-inhibitor complexes. A knockout of the LRP gene has been accomplished, but homozygous deficiency is nonviable and heterozygous deficient mice are normal (35). Therefore, this model has not been useful in confirming the role of LRP in chylomicron-remnant metabolism in vivo.
Mouse Models of Atherosclerosis

Proc. Natl. Acad. Sci. USA 90 (1993) When certain strains of mice are fed the high-cholesterol diet for 4-5 months they develop foam-cell lesions in the region of the aortic sinus, whereas others do not. In this model, crosses between resistant and susceptible mouse strains have been used to identify atherosclerosis susceptibility loci (36). Transgenic techniques have now been used to further exploit this model. In these studies lipoprotein transport genes have been introduced into one of the susceptible strains, C57BL/6, to evaluate their effect on diet-induced atherosclerosis. In one study human A-I gene expression reduced aortic sinus foam-cell lesion area (37). These results suggest that A-I expression with its attendant increase in HDL-C levels can protect against atherosclerosis. In a second study human A-I and A-II transgenic mice were crossed, and there was less protection when both genes were expressed than with just human A-I gene expression (38). Since A-II gene expression does not increase HDL-C levels but rather increases the A-I-plus-A-IH and decreases the A-I-only HDL particles, this experiment suggests that not all HDL particles are equally antiatherogenic. Finally, in a variation on this type of study, the apo(a) transgene was introduced into outbred mice that do not get atherosclerosis (39). The apo(a) transgenic mice developed aortic sinus lesions. As previously noted, Lp(a) does not form in these mice. This implies that apo(a) is itself atherogenic. The homozygous E knockout mice have provided a new model of atherosclerosis (25, 26). These animals are outbred, representing a mixture of either C57BL/6 or BALB/c and 129-strain genetic backgrounds. When fed a chow diet, the E-deficient mice develop foamcell lesions in the aortic sinus by 10 weeks of age. Lesion area increases 3-fold after the mice consume a Western-type diet for 4 weeks, indicating that this atherosclerosis model is appropriately diet-responsive (26). Wild-type and heterozygous E knockout mice show no lesions under either diet condition. Closer examination of the lesions in these young E-deficient animals indicates, in addition to foam cells, smooth muscle or fibroblastic cells and collagen deposition. This suggests the potential for progression to more complicated human-like atherosclerotic lesions in this animal model. Thus the single genetic lesion causing E absence and severe hypercholesterolemia is sufficient to convert the mouse from a species that is highly resistant to one that is highly susceptible to atherosclerosis. These animals should be of great assistance in studies of diets, genes, and drugs influencing atherosclerosis and should greatly accelerate research in this field.

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The mouse is the best mammalian system for the study of genetic contributions to disease. This is because of the easy breeding, short generation time, and availability of inbred strains, many of which have interesting heritable phenotypes. Unfortunately, the mouse is highly resistant to atherosclerosis and that makes it difficult to use this animal model to identify the genes controlling this complex disease. In an attempt to overcome this problem and produce atherosclerotic lesions, mice have been fed an unphysiological diet consisting of 1.25% cholesterol, 15% fat, and 0.5% cholic acid. This diet-which contains 10-20 times the amount of cholesterol of a human diet and an unnatural constituent, cholic acid-is toxic when fed for a long time to mice. However, it does produce a sustained increase in the cholesterol level to 200-300 mg/dl, with the increase in cholesterol in the non-HDL lipoprotein fractions. This is in contrast to a chow diet, which in the mouse produces cholesterol levels of 60-80 mg/dl, mostly in HDL.

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