Steven H. Quarfordt

Targeted Replacement of the Mouse Apolipoprotein E Gene with the Common Human APOE3 Allele Enhances Diet-induced Hypercholesterolemia and Atherosclerosis

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: β-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.

Effect of apoproteins on hepatic uptake of triglyceride emulsions in the rat.

The addition of apoprotein E isolated from human very low density lipoproteins to both rat lymph chylomicrons and a triglyceride emulsion significantly increased the hepatic uptake of these particles in a nonrecycling isolated rat liver perfusion system. The cleared triglyceride was removed without apparent hydrolysis by the hepatocyte. When lymph chylomicrons were loaded with both Apo E and Apo C proteins by exposure to rat plasma, no increment in hepatic clearance was observed. Sequential evalutions of the influence of the C apoproteins on the hepatic clearance of both emulsions and chylomicrons revealed that the CIII (CIII-1) protein had a pronounced inhibitory effect on hepatic removal. The inhibition was observed for both Apo E-enriched chylomicrons and those containing little of this apoprotein.

Apo E structure determines VLDL clearance and atherosclerosis risk in mice

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 cholesterol in their diet. However, on all diets tested, the 4/4 mice had approximately twice the amount of cholesterol, apo E, and apo B-48 in their VLDL as did 3/3 mice. The 4/4 VLDL competed with human LDL for binding to the human LDL receptor slightly better than 3/3 VLDL, but the VLDL clearance rate in 4/4 mice was half that in 3/3 mice. On an atherogenic diet, there was a trend toward greater atherosclerotic plaque size in 4/4 mice compared with 3/3 mice. These data, together with our earlier observations in wild-type and human APOE*2-replacement mice, demonstrate a direct and highly significant correlation between VLDL clearance rate and mean atherosclerotic plaque size. Therefore, differences solely in apo E protein structure are sufficient to cause alterations in VLDL residence time and atherosclerosis risk in mice.

Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2.

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 (normolipidemic) mice that are expressing human apoE3 (3/3) made in an identical manner. The 2/2 mice are markedly defective in clearing beta-migrating VLDL particles, and spontaneously develop atherosclerotic plaques, even on a regular diet. An atherogenic diet, high in fat and cholesterol, exacerbates development of atherosclerosis and xanthomas in the 2/2 mice. Thus, comparisons between the 2/2 and 3/3 mice unequivocally demonstrate that a single amino acid difference (Arg158 Cys) in the apoE protein is sufficient to cause type III HLP and spontaneous atherosclerosis in mice.

Very low density lipoprotein triglyceride transport in type IV hyperlipoproteinemia and the effects of carbohydrate-rich diets

Transport of plasma-free fatty acids (FFA) and of fatty acids in triglycerides of plasma very low density lipoproteins (VLDL-TGFA) was studied in two normal subjects, five patients with type IV hyperlipoproteinemia, and two patients with type I hyperlipoproteinemia. After intravenous pulse-labeling with albumin-bound 1-palmitate-(14)C, specific radioactivity of plasma FFA and VLDL-TGFA were determined at intervals up to 24 hr. The results were analyzed using several different multicompartmental models each compatible with the experimental data. Fractional transport of VLDL-TGFA was distinctly lower (no overlap) in the type IV patients than in the control subjects, both on a usual balanced diet (40% of calories from carbohydrate) and on a high-carbohydrate diet (80% of calories). However, net or total transport of VLDL-TGFA in the type IV patients was not clearly distinguishable from that in the control subjects, there being considerable overlap on either diet. The results suggest that in this group of type IV patients the underlying defect leading to the increased pool size of VLDL-TGFA is not overproduction but a relative defect in mechanisms for removal of VLDL-TGFA. Since some of these type IV patients had only a moderate degree of hypertriglyceridemia at the time they were studied, and since it is not established that patients with the type IV phenotype constitute a biochemically homogeneous population, the present results should not be generalized. Four studies were done (in two control subjects and two type IV patients) in which the kinetic parameters in the same individual were determined on the balanced diet and on the high-carbohydrate diet. All subjects showed an increase in VLDL-TGFA pool size. Using two of the models for analysis, all showed an increase in net transport of VLDL-TGFA; using the third model, three of the four studies showed an increase in VLDL-TGFA transport. The results are compatible with the interpretation that the carbohydrate-induced increase in VLDL-TGFA, both in controls and type IV patients, is at least in part due to an increased rate of production of VLDL-TGFA. The magnitude of the increase was approximately the same in controls and patients. Thus, metabolic adjustment to a high-carbohydrate regimen in these type IV patients may not be basically different from that in normal controls; the higher levels of VLDL-TGFA reached may simply be another reflection of a defective removal mechanism. An alternative interpretation, compatible with the data, would involve both a carbohydrate-induced increase in fractional rate of release of VLDL-TGFA from liver to plasma and a decrease in fractional removal of VLDL-TGFA from plasma without increase in net production rate. The simpler hypothesis of a single primary effect on net VLDL-TGFA production from FFA seems more likely.

The Interaction of Heparin with an Apoprotein of Human Very Low Density Lipoprotein

An arginine-rich apoprotein obtained from human triglyceride-rich lipoprotein was isolated on a heparin affinity column when either the aqueousor urea-soluble apoproteins were applied to the column. Of all the aqueous- or urea-soluble apoproteins, only this arginine-rich protein exhibited a binding affinity to heparin. This protein was eluted from the column at sodium chloride concentrations above 0.35 M in the absence of urea and between 0.17-0.2 M when isolated in urea. The protein has been characterized by amino acid analysis, immunoelectrophoresis, dodecyl sulfate polyacrylamide electrophoresis, isoelectric focusing, and NH(2)-terminal analysis. It has the same amino acid composition, NH(2)-terminal, and molecular weight as previously described for human arginine-rich apoprotein. The triglyceride-rich lipoproteins of fasting normal humans were eluted as two fractions when applied to the heparin affinity column. A small amount was eluted in the unbound fraction and this species contained virtually no arginine-rich apoprotein. The bulk of the triglyceride-rich lipoproteins eluted in the bound fraction and contained appreciable amounts of arginine-rich apoprotein. The bound lipoproteins had more cholesterol and cholesterol ester and less triglyceride than the unbound. The isolated arginine-rich apoprotein was derivatized with phenylglyoxal with a resulting alteration of 75% of the arginine residues. This modified apoprotein did not bind to the heparin affinity column. Similar treatment of the whole triglyceride-rich lipoprotein produced a lipoprotein that was totally eluted in the unbound fraction.

Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E-deficient mice.

Dietary administration of probucol (0.5%, wt/wt) efficiently reduced total plasma cholesterol levels in apolipoprotein E-deficient mice (apoE-/-) by 40%, with decreases in high density lipoprotein (HDL) and apoAI by 70 and 50%, respectively. Paradoxically, however, aortic atherosclerotic plaques in the probucol-treated apoE-/- mice formed more rapidly than in the untreated apoE-/- mice, and the lesions were two to four times larger and more mature regardless of sex, age, and genetic background (P < 10(-)6). Histologically, lesions in probucol-treated mice contained increased fibrous materials and cells other than foam cells, and were commonly associated with focal inflammation and aneurysmal dilatation. Probucol treatment also accelerated lesion development in apoE+/- mice fed an atherogenic diet, indicating that the adverse effect is not dependent on the complete absence of apoE. Furthermore, mice lacking apoE and apoAI have plasma lipoprotein profiles very similar to the probucol-treated apoE-/- mice, but do not have accelerated plaque development. Thus, the enhanced atherosclerosis in the probucol-treated animals is unlikely to be caused by the reduction of HDL and apoAI levels. Our data indicate that a reduction in plasma cholesterol caused by probucol does not necessarily lead to an antiatherogenic effect.

The heterogeneity of rat high density lipoproteins

Abstract Both the Apo A-I and Apo E proteins are major components of the rat high density lipoproteins. High density lipoproteins containing predominantly Apo A-I but little Apo E were separated from high density lipoproteins containing mainly Apo E and virtually no Apo A-I by heparin affinity chromatography. High density lipoproteins with Apo A-I had the chemical composition, electrophoretic and ultracentrifugal properties previously noted for rat high density lipoproteins. The Apo E high density lipoproteins contained relatively more triglyceride and free cholesterol than Apo A-I high density lipoproteins but exhibited similar ultracentrifugal and identical electrophoretic properties. In the fasting rat, the Apo A-I fraction accounted for approximately 90% of the total high density lipoproteins and the Apo E for 10%.

On the lipoprotein abnormality in type III hyperlipoproteinemia

Abstract Two lipoprotein species were isolated by starch block electrophoresis from the very low density lipoproteins (VLDL) (Sf 20-400) of patients with type III hyperlipoproteinemia. One of these, α2-VLDL, had a content of lipid and protein and physical characteristics similar to VLDL from normal subjects or patients with other forms of hyperglyceridemia. The other species, β-VLDL, contained more cholesterol and less triglyceride in relation to the protein, than normal VLDL. Only the apoprotein of low density lipoprotein was immunochemically detectable in the β-VLDL; the proteins in the α2-VLDL reacted with antisera specific for low density lipoprotein and high density lipoprotein. The electrophoretic mobility of β-VLDL was similar to that of low density lipoprotein and significantly less than that of α2-VLDL. Isolated β-VLDL had a lesser mean flotation rate than α2-VLDL, but both α2- and β-VLDL were found throughout the Sf 20-400 flotation range. The relative quantities of α2- and β-VLDL could be varied by changing the diet or by heparin administration. Most of the VLDL from type III patients on a high carbohydrate diet was in the α2-VLDL form. During fasting, α2-VLDL fell and β-VLDL increased becoming the predominant species of VLDL. Heparin-induced acceleration of triglyceride clearance also increased β-VLDL and decreased α2-VLDL. These findings suggest a precursor-product relationship between the α2- and β-forms of VLDL.

Apoproteins in association with Intralipid incubations in rat and human plasma.

Intralipid was incubated with rat and human plasma and examined for changes in lipid and apoprotein composition. Upon incubation in rat plasma, Intralipid acquired an apoprotein complement similar to that found in chylomicrons following plasma incubation or in chylomicrons after alimentary lipemia. Since the apoproteins of lipoproteins probably govern their metabolism, these results suggest that Intralipid and chylomicrons undergo similar metabolic fates. This pattern is characterized by a predominance of Apo E (the arginine-rich apoprotein) and Apo C. Incubation of Intralipid with human plasma showed the uptake of Apo A-I and Apo A-IV as well. Density fractionation of the plasma into separate lipoprotein classes facilitated identification of high density lipoprotein as the major apoprotein donor to the Intralipid. When rat lipoprotein-free plasma (δ>1.21) was incubated with Intralipid, a different apoprotein pattern appeared in the particles of Sf>400 depending on whether the entire Intralipid preparation or only the Sf>400 fraction alone was incubated. The difference consisted of a virtual total absence of the arginine-rich protein on the Sf>400 particles in whole Intralipid incubations. Density fractionation of the Sf<400 particles of Intralipid and recombination of these fractions with the Sf>400 fraction before incubation revealed the major inhibitory fraction to be δ<1.006 (Sf 20–400).