Apolipoprotein (apo) E, a constituent of several lipoproteins, is a ligand for the low density lipoprotein receptor, and this interaction is important for maintaining cholesterol and triglyceride homeostasis. We have used a gene replacement strategy to generate mice that express the human apoE3 isoform in place of the mouse protein. The levels of apoE mRNA in various tissues are virtually the same in the human apoE3 homozygous (3/3) mice and their littermates having the wild type mouse allele (؉/؉). Total cholesterol and triglyceride levels in fasted plasma from the 3/3 mice were not different from those in the ؉/؉ mice, when maintained on a normal (low fat) chow diet. We found, however, notable differences in the distribution of plasma lipoproteins and apolipoprotein E between the two groups: ␤-migrating lipoproteins and plasma apoB100 levels are decreased in the 3/3 mice, and the apoE distribution is shifted from high density lipoproteins to larger lipoprotein particles. In addition, the fractional catabolic rate of exogenously administered remnant particles without apoE was 6-fold slower in the 3/3 mice compared with the ؉/؉ mice. When the 3/3 and ؉/؉ animals were fed a high fat/high cholesterol diet, the 3/3 animals responded with a dramatic increase (5-fold) in total cholesterol compared with the ؉/؉ mice (1.5-fold), and after 12 weeks on this same diet the 3/3 animals developed significantly (at least 13-fold) larger atherosclerotic plaques in the aortic sinus area than the ؉/؉ animals. Thus the structural differences between human APOE3 and mouse ApoE proteins are sufficient to cause an increased susceptibility to dietary-induced hypercholesterolemia and atherosclerosis in the 3/3 mice.

Apo E is a 34-kDa plasma protein important for the metabolism of plasma lipoproteins (1). Like other apolipoproteins, apo E contains multiple 22-amino acid repeats that form amphipathic helices, enabling it to associate with the surface of plasma lipoproteins. Apo E also contains a stretch of basic residues (136-150) that is important for high-affinity binding to the LDL receptor and subsequent endocytosis of the associated lipoprotein particle (2). In addition, apo E mediates lipoprotein interactions with LDL receptor-related protein (LRP) (3), the VLDL receptor (4), other lipoprotein receptors (5), endothelial heparin sulfate (6), and plasma lipases (7,8). The phenotype of severe hyperlipidemia and spontaneous development of atherosclerosis in mice lacking apo E clearly demonstrates the central role of apo E in mammalian lipid metabolism (9, 10).In humans, the APOE gene is polymorphic and has 3 alleles: APOE*2, APOE*3, and APOE*4. These alleles have frequencies of 7%, 77%, and 15%, respectively, in the general population (11). The APOE*3 allele codes for cysteine at position 112 and for arginine at 158. The APOE*2 allele codes cysteines at both positions, whereas the APOE*4 allele codes for arginines at both positions. Various population-based studies have suggested that the different APOE alleles have distinct influences on lipid metabolism in humans. Possession of at least 1 copy of the APOE*2 allele has been associated with higher plasma apo E (12) and lower plasma cholesterol, LDL cholesterol, and apo B levels (11) when compared with APOE*3 homozygotes. The APOE*2 allele is also associated with lower risk of coronary artery disease (13), except in 5-10% of APOE*2 homozygotes who develop type III hyperlipoproteinemia and premature atherosclerosis (14). On the other hand, the presence of at least 1 APOE*4 allele is associated with lower plasma apo E (12) and increased plasma cholesterol, LDL cholesterol, and apo B levels (11), and a greater risk of coronary artery disease (13), when compared with APOE*3 homozygotes. Davignon et al. (11) estimate that the apo E polymorphism accounts for 2.8% of the variation of risk for atherosclerosis, which is a large contribution for a single locus in this complex, polygenic disease.The availability of a well-defined model system should benefit studies of the role of the human apo E polymorphism in lipid metabolism and atherosclerosis. To develop such a model, we have used gene targeting to replace the murine Apoe gene with the 3 human APOE alleles. These mice retain the murine Apoe regulatory sequences and solely produce human apo E proteins with different We have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform. Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and choles...

To study isoform-specific effects of apolipoprotein E (apoE) in vivo, we generated mice with a human APOE*2 allele in place of the mouse Apoe gene via targeted gene replacement in embryonic stem cells. Mice expressing human apoE2 (2/2) have virtually all the characteristics of type III hyperlipoproteinemia. Their plasma cholesterol and triglyceride levels are both twice to three times those in (

The effect of hepatic lipase (HL) deficiency on the susceptibility to atherosclerosis was tested using mice with combined deficiencies in HL and apoE. Mice lacking both HL and apoE (hhee) have a plasma total cholesterol of 917 ؎ 252 mg/dl (n ‫؍‬ 24), which is 184% that of mice lacking only apoE (HHee; 497 ؎ 161 mg/dl, n ‫؍‬ 20, p < 0.001). The increase in cholesterol was mainly in ␤-migrating very low density lipoproteins, although high density lipoprotein cholesterol (HDLc) was also increased (53 ؎ 37 versus 20 ؎ 13 mg/dl, p < 0.01). Despite the increase in plasma cholesterol, we found that HL deficiency significantly decreased aortic plaque sizes in female mice fed normal chow (31 ؋ 10 3 ؎ 22 ؋ 10 3 m 2 in hhee versus 115 ؋ 10 3 ؎ 69 ؋ 10 3 m 2 in HHee, p < 0.001). Reduction of plaque sizes was also observed in female heterozygous apoE-deficient mice fed an atherogenic diet (2 ؋ 10 3 ؎ 2.5 ؋ 10 3 m 2 in hhEe versus 56 ؋ 10 3 ؎ 49 ؋ 10 3 m 2 in HHEe, p < 0.01). Changes in aortic lesion size were not apparent in the small number of male mice studied. In HHee females, both HDLc and the capacity of high density lipoprotein (HDL) particles to promote cholesterol efflux from cultured cells were 26% of the wild type. The absence of HL in hhee females partially restored HDLc levels to 57% and cholesterol efflux to 55% of the wild type. Circulating pre-␤ 1 -migrating HDL were present in all mutants, suggesting that there are alternative pathways in the formation of these pre-␤-HDL not involving apoE, HL, or cholesteryl ester transfer protein. The improved capacity to promote cholesterol efflux, together with increased HDL, may explain why these animals can overcome the increase in atherogenic lipoproteins.In circulation, nascent lipoproteins are remodeled by removal of core lipids and transfer of surface proteins and lipids prior to their removal through receptor-mediated mechanisms (1). Hepatic lipase (HL) 1 is a 60-kDa lipolytic enzyme involved in the processing of chylomicrons, intermediate density lipoproteins (IDL), and high density lipoproteins (HDL) (2). HL efficiently hydrolyzes both triglycerides (TG) and phospholipids, while lipoprotein lipase is mainly responsible for hydrolysis of plasma TG. The preferred enzymatic substrates for HL are intermediate-size particles, such as IDL, and HDL 2 . Changes observed in lipoprotein profiles in humans with congenital HL deficiencies are the presence of ␤-migrating very low density lipoproteins (␤-VLDL) and larger HDL (3). The ␤-VLDL that accumulate in HL deficiency are more TG-rich than ␤-VLDL in typical type III hyperlipoproteinemia subjects (4). We previously reported that HL-deficient mice have a mild dyslipidemia with increased cholesterol and phospholipid, and the plasma contains large HDL floating in the 1.02-1.04 g/ml density range (5). Consistent with these observations, overexpression of HL decreases HDL cholesterol and HDL particle size in mice (6) and decreases HDL cholesterol and IDL in rabbits (7).Both HL and cholesteryl ester transfer protein (CETP) parti...

Objective-Peroxisome proliferator-activated receptor (PPAR) ␣ and ␥ are nuclear receptors that may modulate atherogenesis, not only by correcting metabolic disorders predisposing to atherosclerosis but also by directly acting at the level of the vascular wall. The accumulation of lipid-laden macrophages in the arterial wall is an early pivotal event participating in the initiation and promotion of atherosclerotic lesion formation. Because PPAR␣ and ␥ modulate macrophage gene expression and cellular function, it has been suggested that their ligands may modulate atherosclerosis development via direct effects on macrophages. In this report, we investigated the effect of a PPAR␣ ligand (fenofibrate) and 2 PPAR␥ ligands (rosiglitazone and pioglitazone) on atherogenesis in a dyslipidemic nondiabetic murine model that develops essentially macrophage-laden lesions. Methods and Results-Mice were fed a Western diet supplemented or not with fenofibrate (100 mpk), rosiglitazone (10 mpk), or pioglitazone (40 mpk) for 10 weeks. Atherosclerotic lesions together with metabolic parameters were measured after treatment. Fenofibrate treatment significantly improved lipoprotein metabolism toward a less atherogenic phenotype but did not affect insulin sensitivity. Contrarily, rosiglitazone and pioglitazone improved glucose homeostasis, whereas they did not improve lipoprotein metabolism. Fenofibrate treatment significantly decreased the accumulation of lipids and macrophages in the aortic sinus. However, surprisingly, neither rosiglitazone nor pioglitazone had an effect on lesion lipid accumulation or macrophage content. Key Words: atherosclerosis Ⅲ foam cells Ⅲ peroxisome proliferator-activated receptors ␣ and ␥ Ⅲ ligands Ⅲ murine model P eroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors regulating the expression of genes that control lipid and glucose homeostasis, thus modulating the major metabolic disorders predisposing to atherosclerosis. 1 Moreover, PPARs exert additional antiinflammatory and lipid-modulating effects in the arterial wall, therefore being interesting molecular targets for the treatment of atherosclerosis. 2 PPARs are the targets of 2 classes of drugs currently used in clinical practice: fibrates (fenofibrate, clofibrate, ciprofibrate, and gemfibrozil) are PPAR␣ agonists, Conclusion-These results indicate that in a dyslipidemic See page 1763whereas thiazolidinediones (TZDs) (rosiglitazone and pioglitazone) are potent PPAR␥ activators. 3 Although the beneficial effects of fibrate treatment on coronary events and atherogenesis in humans are well-documented through epidemiological and clinical intervention studies, 4 -6 results of outcome trials with PPAR␥ agonists in humans are still awaited to answer whether treatment with TZDs translates into a therapeutic benefit in atherosclerotic cardiovascular disease. The majority of clinical studies performed to date assessed the effects of TZDs in diabetic patients and most suggested vascular protective effects of PPAR␥ ligands as ...