Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation.

Apolipoprotein (apo) E and the two B apolipoproteins, apoB48 and apoB100, are important proteins in human lipoprotein metabolism. Commonly occurring polymorphisms in the genes for apoE and apoB result in amino acid substitutions that produce readily detectable phenotypic differences in these proteins. We studied changes in apoE and apoB phenotypes before and after liver transplantation to gain new insights into apolipoprotein physiology. In all 29 patients that we studied, the postoperative serum apoE phenotype of the recipient, as assessed by isoelectric focusing, converted virtually completely to that of the donor, providing evidence that greater than 90% of the apoE in the plasma is synthesized by the liver. In contrast, the cerebrospinal fluid apoE phenotype did not change to the donor's phenotype after liver transplantation, indicating that most of the apoE in CSF cannot be derived from the plasma pool and therefore must be synthesized locally. The apoB100 phenotype (assessed with immunoassays using monoclonal antibody MB19, an antibody that detects a two-allele polymorphism in apoB) invariably converted to the phenotype of the donor. In four normolipidemic patients, we determined the MB19 phenotype of both the apoB100 and apoB48 in the "chylomicron fraction" isolated from plasma 3 h after a fat-rich meal. Interestingly, the apoB100 in the chylomicron fraction invariably had the phenotype of the donor, indicating that the vast majority of the large, triglyceride-rich apoB100-containing lipoproteins that appear in the plasma after a fat-rich meal are actually VLDL of hepatic origin. The MB19 phenotype of the apoB48 in the plasma chylomicron fraction did not change after liver transplantation, indicating that almost all of the apoB48 in plasma chylomicrons is derived from the intestine. These results were consistent with our immunocytochemical studies on intestinal biopsy specimens of organ donors; using apoB-specific monoclonal antibodies, we found evidence for apoB48, but not apoB100, in donor intestinal biopsy specimens.     

Evaluation of new apolipoprotein C-II and apolipoprotein C-III automatized immunoturbidimetric kits

BACKGROUND: Apolipoprotein C-II and apolipoprotein C-III play an important and complex role in plasma triglycerides metabolism, respectively, as inhibitor and activator of lipoprotein lipase. Thus, they appear to be suitable markers for clinical studies of triglyceride-rich lipoproteins and related cardiovascular risk. Our aim was to evaluate, for routine analysis, the accuracy to quantify these apolipoprotein in human sera. METHODS: Precision (intra- and inter-run), limit of detection and quantification, linearity, common interferents (lipids, haemoglobin, bilirubin) and reference intervals were determined according to guidelines of the French Society of Clinical Biology and ISO Norm 5725 specifications. RESULTS: Intra- and inter-run CVs were respectively less than 5.0% and 7.5%. Linearities extended from 10.8 mg/L to 112.9 mg/L for apolipoprotein C-II and from 31.8 mg/L to 375.5 mg/L for apolipoprotein C-III. Haemolysis (up to 227.6 micromol/L haemoglobin) and lipemia (up to 19.3 mmol/L triglycerides) do not interfere, contrary to bilirubin, which has a positive effect above 350 micromol/L. Comparison of methods shows good agreement between immunoturbidimetric and electro-immunodiffusion methods for measuring apolipoprotein C-III in 62 samples within a wide range (n = 62, r = 0.954, y = 3.81 x -14.4). CONCLUSION: This study shows the reliability of these kits for measuring apolipoprotein C-II and apolipoprotein C-III in human sera, and their suitability for routine analysis.     

Familial apolipoprotein A-I variants

Apolipoprotein (apo) A-I is the major apolipoprotein of HDL and is a single polypeptide chain with 243 amino acid residues. Familial apo A-I deficiency is a rare metabolic disorder of which 13 cases have been characterized at the molecular level. However, in subjects with apo A-I deficiency, coronary artery disease is not always present. To date, more than 40 genetically determined structural variants of apo A-I have been identified. Seven apo A-I mutations have been identified to cause hereditary amyloidosis. In our laboratory, we characterized ten apo A-I variants, including apo A-I(Vall56Glu) Oita. A minority of structural variants were associated with altered HDL cholesterol levels or increased risk for cardiovascular disease.     

Low diurnal variability of apolipoprotein A1, apolipoprotein B and apolipoprotein B/apolipoprotein A1 ratio during normal sleep and after an acute shift of sleep

ObjectiveThe aim of this study was to study the diurnal variation of the cardiovascular risk markers apolipoprotein A1 and B and apo B/apo A1 ratio.Design and methodsWe have studied the diurnal variation of apolipoprotein A1, apolipoprotein B and apo B/apo A1 ratio during night sleep and the day sleep conditions in seven healthy volunteers (age 22–32 yr). Samples were collected every hour to evaluate the effect of different sampling times on the test results.ResultsThe lowest diurnal coefficient of variation (CV) was observed for the apo B/apo A1 ratio, which usually was below 2% but also apolipoprotein A1, apolipoprotein B showed low CV. There were no significant differences between nightsleep and daysleep for any of the studied markers.ConclusionEven if there was a diurnal variation for these markers, the variation was very low. Thus, sampling does not have to be restricted to certain times of the day.     

Structural relationship of human apolipoprotein B48 to apolipoprotein B100.

Although the complete amino acid sequence of human apolipoprotein (apo) B100 is known (4536 amino acids), the structure of apo B48 has not been defined. The objective of our study was to define the structure of apo B48 and its relationship to apo B100. Antibodies were produced against 22 synthetic peptides corresponding to sequences in human apo B100. The levels of immunoreactivity of the antipeptides to apo B100 and apo B48 were used to define the structural relationship between these two species of apo B. Six antibodies from sequences in the amino-terminal half of apo B100, including antipeptide 2110-2129, bound to both apo B100 and apo B48. 15 other apo B-specific antipeptides from sequences carboxyl-terminal to residue 2152 bound to apo B100, but not to apo B48. Immunoblots of cyanogen bromide digests of apo B100 and apo B48 with antipeptides 2068-2091 and 2110-2129 detected a 16-KD fragment (residues 2016-2151) in the apo B100 digest and a fragment of identical size in the apo B48 digest. Because apo B48 appears to contain the apo B100 cyanogen bromide fragment 2016-2151 and because an antiserum specific for the peptide 2152-2168 does not bind to apo B48, we conclude that apo B48 represents the amino-terminal 47% of apo B100 and that the carboxyl terminus of apo B48 is in the vicinity of residue 2151 of apo B100.     

Gold-labeled nanoparticle-based immunoresonance scattering spectral assay for trace apolipoprotein AI and apolipoprotein B

BACKGROUND: Apolipoprotein AI (ApoAI) and ApoB are risk indicators of cardiovascular disease. We describe the use of immunoresonance scattering to measure the ApoAI and ApoB in serum. METHODS: We used a trisodium citrate method to prepare 9.0-nm gold nanoparticles labeled with goat anti-human ApoAI and ApoB antibodies. The immune reaction between gold-labeled antibodies and antigens took place in Na2HPO4-NaH2PO4 buffer solution (pH 6.4 for ApoAI and pH 6.0 for ApoB) in the presence of 75 g/L polyethylene glycol (PEG). We used a transmission electron microscope to observe the shape of the gold nanoparticles. Results were compared with those obtained by immunoturbidimetric methods. Twenty-five human serum samples were assayed by the immunoresonance scattering assay preset with the data indicated and by an immunoturbidimetric assay. RESULTS: The presence of PEG greatly enhanced the intensity of resonance-scattering peaks at 560 nm. The intensity (DeltaI) was proportional to concentration at 0.00833-0.3333 mg/L ApoAI and 0.00197-0.1972 mg/L ApoB. The detection limits were 2.04 and 0.96 microg/L for ApoAI and ApoB, respectively. The results for human serum samples were in agreement with those obtained with an immunoturbidimetric method. Linear regression analysis revealed a correlation coefficient, slope, and intercept of 0.915, 0.966, and 68.53 mg/L, respectively, for ApoAI and 0.919, 0.996, and 15.46 mg/L for ApoB. CONCLUSION: This method showed high sensitivity and good selectivity for quantitative determination of ApoAI and ApoB in human serum, with satisfactory results.     

载脂蛋白A5、载脂蛋白C3及载脂蛋白A5/C3比值与冠心病的关系

目的 研究载脂蛋白A5（apolipoprotein A5,ApoA5）、载脂蛋白C3（apolipoprotein C3,ApoC3）及载脂蛋白A5/C3（ApoA5/ApoC3）比值与血脂水平、冠心病的发生及冠状动脉病变支数的关系,通过调节载脂蛋白浓度水平预防冠心病的发生和发展。方法 统计冠心病患者与非冠心病患者年龄、性别、体重指数（body mass index, BMI）及吸烟情况,测定两组血清总胆固醇（total cholesterol, TC）、甘油三酯（triglyceride, TG）、高密度脂蛋白（high density lipoprotein, HDL)、低密度脂蛋白(low density lipoprotein, LDL)浓度水平,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA）测定血清载脂蛋白A5（ApoA5）、载脂蛋白C3（ApoC3）浓度水平,并计算ApoA5/ApoC3比值。按照TG水平进行危险因素分层,比较非冠心病组、冠心病组TG高值组与冠心病组TG正常值组ApoA5、ApoC3、ApoA5/ApoC3比值水平有无差异;研究血清ApoA5、ApoC3、ApoA5/ApoC3比值水平与冠心病病变支数及各项指标的相关性。结果 两组性别、TG水平差异有统计学意义（P<0.01）。冠心病组TG高值组和TG正常值组ApoA5/C3比值、ApoA5浓度水平均明显低于非冠心病组,TG高值组ApoC3浓度水平明显高于非冠心病组（P<0.01）。TG高值组ApoA5/C3比值、ApoA5浓度水平低于TG正常值组,TG高值组ApoC3浓度水平明显高于TG正常值组（P<0.01）。多因素二元Logistic回归分析表明,TG是ApoA5/C3比值、ApoA5浓度水平降低的独立危险因素［OR=0.074,95%CI（0.023,0.134）,Wald 19.650, SE 0.588, P<0.01; OR=0.039,95%CI（0.009,0.157）,Wald 20.641, SE 0.716, P<0.01］,TG是ApoC3浓度水平升高的独立保护因素(OR=9.084,95%CI（3.054,27.022）,Wald 15.736, SE 0.556, P<0.01);血清ApoA5/C3比值、ApoA5浓度水平与TG呈负相关,ApoC3浓度水平与TG呈正相关。结论 ①血清ApoA5/ApoC3比值、ApoA5浓度水平与冠心病的发生呈负相关;血清ApoC3浓度水平与冠心病的发生呈正相关;血清ApoA5和ApoC3分别是冠心病的保护因子和危险因素,ApoA5/ApoC3比值在一定程度上反映冠状动脉病变程度,可作为推测冠状动脉病变程度的依据。②TG水平是血清ApoA5/ApoC3比值、ApoA5浓度水平降低的独立危险因素,与血清ApoA5/ApoC3比值、ApoA5浓度水平呈负相关,TG水平是ApoC3浓度水平升高的保护因素,与ApoC3浓度水平呈正相关。     

Estimation of LDL-associated apolipoprotein B from measurements of triglycerides and total apolipoprotein B

BACKGROUND: VLDL and chylomicrons may interfere with measurements of apolipoprotein B (apo B) on LDL particles. Ultracentrifugation of samples enriched in chylomicrons and VLDL and subsequent measurement of apo B in the infranate fraction [density (d) = 1.006] removes this interference. This apo B fraction is called "LDL-apo B." METHODS: We retrospectively analyzed 64 895 measurements of triglycerides, total apo B, and LDL-apo B. Samples were ultracentrifuged, and 3 commercially available immunoassays that use different antibodies were used to measure LDL-apo B in the 1.006 infranate fraction. RESULTS: After adjusting for triglyceride concentration, we found total apo B and LDL-apo B measurements to be strongly correlated. We derived a simple linear equation for calculating LDL-apo B concentration (in milligrams per deciliter) from measurements of total apo B and triglycerides: LDL-apo B = apo B - 10 mg/dL - triglycerides/32. This equation accurately predicts LDL-apo B values within +/- 12% of the measured value in 75% of cases. CONCLUSIONS: Our equation provides a convenient means of estimating LDL-apo B from commonly available measurements of total apo B and triglycerides without the need for ultracentrifugation. LDL-apo B measurements were also independent of the different apo B antibodies in the 3 assays used in this study. An equation that predicts LDL-apo B particle number may be useful, regardless of the apo B assay used.     

Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice.

apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.     

Familial apolipoprotein E deficiency.

A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.     

The relationship between apolipoprotein E and serum oxidation-related variables is apolipoprotein E phenotype dependent

To examine the relationship between apolipoprotein E and serum oxidation status, we assayed apolipoprotein E level, apolipoprotein E phenotype, and levels of lipid peroxides and transition metal ions and their binding proteins in sera from apparently healthy individuals. The study group included 129 women aged 22–63 years and 53 men aged 22–56 years. Among subjects with apolipoprotein E 4/3 phenotype, lipid peroxide levels were higher compared with E 3/2 phenotype (786±182 nmol/l vs. 659±174 nmol/l,P=0.015), and ceruloplasmin levels were slightly higher compared with apolipoprotein E 3/3 phenotype (0.28±0.08 mg/l vs. 0.26±0.06 mg/l,P=0.035). In the study group as a whole, there were significant associations between serum apolipoprotein E level, and serum levels of ceruloplasmin (r=0.266,P<0.001) and ferritin (r=0.2,P<0.007). Among subjects with apolipoprotein E 4/3 phenotype, there was a significant association between serum apolipoprotein E and lipid peroxide levels (r=0.470,P<0.01), which was not apparent among subjects with E 3/3 or E 3/2 phenotypes. In multivariate analysis, apolipoprotein E phenotype was a small but significant independent contributor to variation in serum lipid peroxide levels. These data suggest that there may be heterogeneity among apolipoprotein E phenotypes in their relationships with serum lipid oxidation status.     

Human apolipoprotein B48 receptor

Unique candidate receptor membrane binding proteins, MBP 200 and 235, were identified in human monocyte-macrophages. The MBP binds and internizes chylomicrons, triglyceride (TG)-rich VLDL, and VLDL devoid of apo E, but not acetyl LDL, via a domain in apo B48. The MBP is named apo B48 receptor because the receptor-binding domain is within apo B48. The apo B48 receptor differs from the scavenger receptor family and LDL receptor family because it does not bind acetyl LDL and it binds VLDL devoid of apo E by an apo E-independent mechanism. Immunohisto-chemical studies indicate colocalization of anti-apo B48 receptor Ab and monocyte-macrophage specific Ab in human atherosclerotic lesion foam cells. This suggests that apo B48 receptor may contribute to foam cell formation and atherosclerosis in abnormal state with elevated TG-rich lipoproteins.     

Chylomicronemia due to apolipoprotein CIII overexpression in apolipoprotein E-null mice. Apolipoprotein CIII-induced hypertriglyceridemia is not mediated by effects on apolipoprotein E.

The mechanism of apolipoprotein (apo) CIII-induced hypertriglyceridemia remains uncertain. We crossed apoCIII transgenic and apoE gene knockout (apoE0) mice, and observed severe hypertriglyceridemia with plasma triglyceride levels of 4,521+/-6, 394 mg/dl vs. 423+/-106 mg/dl in apoE0 mice, P < 0.00001 for log(triglycerides [TG]). Cholesterols were 1,181+/-487 mg/dl vs. 658+/-151 mg/dl, P < 0.0001. Lipoprotein fractionation showed a marked increase in triglyceride-enriched chylomicrons+VLDL. This increase was limited to the lowest density (chylomicrons and Sf 100-400) subfractions. Intermediate density lipoproteins (IDL)+LDL increased moderately, and HDL decreased. There was no significant increase in triglyceride production in apoCIII transgenic/apoE0 mice. The clearance of VLDL triglycerides, however, was significantly decreased. Lipoprotein lipase in postheparin plasma was elevated, but activation studies suggested LPL inhibition by both apoCIII transgenic and apoCIII transgenic/apoE0 plasma. ApoCIII overexpression also produced a marked decrease in VLDL glycosaminoglycan binding which was independent of apoE. The predominant mechanism of apoCIII-induced hypertriglyceridemia appears to be decreased lipolysis at the cell surface. The altered lipoprotein profile that was produced also allowed us to address the question of the direct atherogenicity of chylomicrons and large VLDL. Quantitative arteriosclerosis studies showed identical results in both apoCIII transgenic/apoE0 and apoE0 mice, supporting the view that very large triglyceride-enriched particles are not directly atherogenic.     

In vivo metabolism of apolipoprotein S in humans. Comparison with apolipoprotein A-I metabolism

¹²⁵I-labelled apolipoprotein (Apo) S and ¹³¹I-labelled apolipoprotein A-I were injected i.v. into healthy volunteers. Blood samples and daily urine collections were drawn periodically for 15 days. Ninety-eight percent of ¹³¹I radioactivity and > 95% of ¹²⁵I radioactivity were found in HDL after Superose gel chromatography of plasma. About 10% of each radioactivity was recovered in the d 1.250 infranate after one ultracentrifugation. Affinity chromatography on monoclonal anti-Apo A-I Sepharose column separates two lipoprotein particles containing Apo S, one retained with Apo A-I (42.5%) and the other eluting without Apo A-I (57.5%).

Kinetic parameters were calculated according to exponential curve fitting. Mean transit time was about 7.0 days for both Apo A-I and Apo S. FCR of Apo S was 50% higher than FCR of Apo A-I. Synthetic rate of Apo S was about 150 times smaller than for Apo A-I.

As heterogeneity of HDL-S was suggested by both the results of affinity chromatography and the urinary data, a compartmental model was built which fitted adequately all data. Part of the model is common to HDL-A-I and HDL-S.     

Influence of apolipoprotein E polymorphism on apolipoprotein B-100 metabolism in normolipemic subjects.

This study examined apolipoprotein (apo) B metabolism in normolipemic subjects homozygous for the apo E2 (n = 4), apo E3 (n = 5), or apo E4 (n = 5) phenotype. Radioiodinated very low density lipoprotein (VLDL1) (ultracentrifuge flotation rate [Sf] 60-400) and VLDL2 (Sf 20-60) were injected into volunteers and the conversion of apo B was followed through intermediate density lipoprotein (IDL) to low density lipoprotein (LDL). Subjects homozygous for E3 converted approximately 50% of LVDL2 to LDL, the remainder being lost by direct catabolism. Those with the E2 phenotype produced less VLDL1, but converted more of it to VLDL2 (compared to E3 subjects). They displayed a characteristic dyslipidemia with the presence of slowly catabolized VLDL1 and VLDL2 remnants. LDL levels were low owing to increased direct catabolism of VLDL2 and IDL and a reduced efficiency of delipidation; only 25% of VLDL2 apo B was directed to LDL production. In contrast, E4 subjects converted more VLDL2 apo B to LDL than E3 subjects. About 70% of VLDL2 apo B was found in LDL; direct catabolism of VLDL and IDL was reduced as was the fractional catabolic rate of LDL (0.2 vs. 0.26 in E3 subjects). These changes in the VLDL----IDL----LDL metabolic cascade can in part be explained by alterations in hepatic LDL receptors with E2 subjects having higher and E4 subjects lower activities than those in E3 homozygotes.     

Comparison of four procedures for separating apolipoprotein A- and apolipoprotein B-containing lipoproteins in plasma

Because lipoproteins containing apolipoprotein A (ApoA-I + ApoA-II) or apolipoprotein B (ApoB) seem to exert opposite effects as risk factors for coronary heart disease, we decided to determine the separability of these two major plasma lipoproteins by procedures originally designed to separate high-density from low- and very-low-density lipoproteins. The presumably ApoB-free lipoproteins isolated from normal plasma by (a) ultracentrifugation at d = 1.063; precipitation with (b) heparin-Mn2+ or (c) phosphotungstate-Mg2+; or (d) immunoprecipitation with antibodies to ApoB were characterized by quantifying cholesterol and apolipoproteins A-I, A-II, B, C-II, C-III, D, E, F, and Lp(a). ApoA- and ApoB-containing lipoproteins were completely separated only by immunoprecipitation with antibodies to ApoB. The ApoB-containing lipoproteins isolated by other procedures always contained 4% to 20% of total plasma ApoA-I and differed substantially from one another with respect to the content of some of the minor apolipoproteins. Measuring apolipoproteins was more reliable than measuring cholesterol for monitoring this separation and for expressing the concentrations of ApoA- and ApoB-containing lipoproteins.     

Turbidimetric assay of apolipoprotein A-II

A turbidimetric assay for the quantification of apolipoprotein A-II in serum is described. Its adaptation for the Cobas Bio Analyser (Hoffmann La Roche) is reported. Regression analysis of apolipoprotein A-II values measured by turbidimetry revealed a good measure of agreement with the data obtained by radial immunodiffusion (RID) (r = 0.90, y = 0.97x + 0.021, n = 100) as well as with data obtained by immunonephelometry (r = 0.838, y = 0.925x + 0.029, n = 34). A variation coefficient of 3.2% (n = 18) was found in relation to the precision in the series and a variation coefficient of 3.5% (n = 36) in relation to day to day precision.     

Susceptibility to atherosclerosis in mice expressing exclusively apolipoprotein B48 or apolipoprotein B100.

All classes of lipoproteins considered to be atherogenic contain apo-B100 or apo-B48. However, there is a distinct paucity of data regarding whether lipoproteins containing apo-B48 or apo-B100 differ in their intrinsic ability to promote the development of atherosclerosis. To address this issue, we compared the extent of atherosclerosis in three groups of animals: apo-E-deficient mice (apo-B+/+apo-E-/-) and apo-E-deficient mice that synthesize exclusively either apo-B48 (apo-B48/48apo-E-/-) or apo-B100 (apo-B100/100apo-E-/-). Mice (n = 25 in each group) were fed a chow diet for 200 days, and plasma lipid levels were assessed throughout the study. Compared with the levels in apo-B+/+apo-E-/- mice, the total plasma cholesterol levels were higher in the apo-B48/48apo-E-/- mice and were lower in the apo-B100/100apo-E-/- mice. However, the ranges of cholesterol levels in the three groups overlapped. Compared with those in the apo-B+/+apo-E-/- mice, atherosclerotic lesions were more extensive in the apo-B48/48apo-E-/- mice and less extensive in the apo-B100/100apo-E-/- mice. Once again, however, there was overlap among the three groups. The extent of atherosclerosis in each group of mice correlated significantly with plasma cholesterol levels. In mice from different groups that had similar cholesterol levels, the extent of atherosclerosis was quite similar. Thus, susceptibility to atherosclerosis was dependent on total cholesterol levels. Whether mice synthesized apo-B48 or apo-B100 did not appear to have an independent effect on susceptibility to atherosclerosis.     

Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer

Elevation of HDL cholesterol, after adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. Previously, we observed transient human apo A-I expression after adenoviral gene transfer with a cytomegalovirus (CMV)-driven construct containing the human apo A-I cDNA. Therefore, the effects of promoters (CMV or 256 base pairs of the human apo A-I promoter), introns of the human apo A-I gene, and the liver-specific human apolipoprotein E (apo E) enhancer on adenovirus-mediated human apo A-I expression were evaluated in C57BL/6 mice. In the presence of the CMV promoter, human apo A-I introns prolonged expression above 20 mg/dl from 14 to 35 days. Addition of one, two, or four copies of the human apo E enhancer in these constructs resulted in a copy-dependent but transient increase in expression for 14 days. The apo A-I promoter induced 3.2-fold lower peak levels of human apo A-I than did the CMV promoter, but insertion of four apo E enhancers in the apo A-I promoter-driven construct resulted in human apo A-I levels above 20 mg/dl for 6 months. The decline between day 6 and day 35 of human apo A-I expression driven by the CMV promoter was due to (1) a 2.5-fold decline in transgene DNA levels that is not observed with apo A-I promoter-driven constructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decline in the human apo A-I mRNA/human apo A-I DNA copy number ratio between day 6 and day 35. Hepatotoxicity, as evidenced by up to 10-fold higher serum levels of transaminases on day 6 after gene transfer with CMV promoter-driven constructs than with apo A-I promoter-driven constructs, probably caused the accelerated decline of transgene DNA. In conclusion, gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA, and four apo E enhancers, all of human origin, is associated with low hepatotoxicity and with the absence of promoter shutoff resulting in human apo A-I expression above 20 mg/dl for up to 6 months.