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Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1914-1920
Published online before print August 21, 2003, doi: 10.1161/01.ATV.0000092328.66882.F5
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1914.)
© 2003 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Apolipoprotein A-I Deficiency Results in Markedly Increased Atherosclerosis in Mice Lacking the LDL Receptor

Ryan E. Moore; Masa-aki Kawashiri; Ken Kitajima; Anthony Secreto; John S. Millar; Domenico Pratico; Daniel J. Rader

From the University of Pennsylvania School of Medicine, Philadelphia, Pa.

Correspondence to Daniel J. Rader, University of Pennsylvania School of Medicine, 654 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104. E-mail rader{at}mail.med.upenn.edu


*    Abstract
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 *Abstract
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Objective— An inverse and independent association between plasma levels of apolipoprotein (apo) A-I and coronary heart disease in humans is well established. ApoA-I is the primary protein component of HDL and is thought to play an important role in mediating several of the atheroprotective effects of HDL. However, studies of the effects of apoA-I deficiency on the development of atherosclerosis in mice have not been definitive. We examined the effects of apoA-I deficiency on plasma lipids and atherosclerosis in LDL receptor-deficient mice fed a chow diet for up to 22 months.

Methods and Results— Both apoA-I-deficient (apoA-I-/-)/LDL receptor-deficient (LDLR-/-) and LDLR-/- mice had a similar moderate elevation of non-HDL cholesterol (non-HDL-C). Unlike previous studies of apoA-I deficiency in which the HDL-C levels were extremely low, the apoA-I-/-/LDLR-/- mice in this study had substantial levels of HDL-C that were similar to wild-type mice. Despite similar levels of non-HDL-C and substantial levels of HDL-C, apoA-I-/-/LDLR-/- mice develop significantly more atherosclerosis (up to a 5-fold increase) and oxidant stress (39% increase) than LDLR-/- mice.

Conclusions— These results demonstrate that despite normal levels of HDL-C, apoA-I deficiency is associated with a significant loss of protection from the formation of atherosclerosis in LDLR-/- mice fed a chow diet.


Key Words: arteriosclerosis • apolipoproteins • cholesterol • lipids • oxidant stress


*    Introduction
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 *Introduction
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Apolipoprotein A-I (apoA-I) is the primary protein component of HDL. ApoA-I plays a key role in lipid transport and metabolism and may play an important role in mediating several of the atheroprotective effects of HDL.1 Like plasma levels of HDL cholesterol (HDL-C),2 plasma levels of apoA-I are inversely associated with risk for coronary heart disease (CHD).3,4 Low levels of apoA-I have been identified as an independent risk factor for CHD in a human population lacking traditional risk factors such as total cholesterol >200 mg/dL or HDL-C <35 mg/dL.5 Several studies have shown apoA-I levels to be a similar or even stronger predictor of CHD than HDL-C.4–8

Studies in animals have consistently shown that increasing the level of apoA-I has antiatherogenic effects.1 Despite these data, the relationship between complete apoA-I deficiency and atherosclerosis remains uncertain. Because complete apoA-I deficiency in humans is rare, clinical studies have been limited to isolated cases and small kindreds. Results have been inconsistent; some studies have demonstrated markedly premature CHD in patients with apoA-I deficiency,9–15 whereas others have found CHD to be absent.16–18 Similar to humans, the effect of apoA-I deficiency on the development of atherosclerosis in animals has not been definitive. ApoA-I-/- mice have reduced levels of HDL-C,19 but when fed a chow diet, they do not spontaneously develop atherosclerosis.20 Furthermore, even when fed an atherogenic diet, apoA-I-/- mice develop only minimal atherosclerosis that is not increased relative to wild-type mice.20 Chow-fed apoA-I-/-/apoB transgenic mice also fail to develop significant atherosclerosis.21 Only when apoA-I-/-/apoB transgenic mice were fed a Western-type diet, which induced a marked increase in non-HDL-C ({approx}450 mg/dL) and an additional reduction in HDL-C ({approx}15 mg/dL), was apoA-I deficiency associated with a statistically significant 2-fold increase in atherosclerosis, but even then only in female mice.21 In one other study, apoA-I-/-/apoB-100 transgenic mice were fed a cholate-containing atherogenic diet and found to have a modest 39% increase in atherosclerosis relative to control mice.22

To test the hypothesis that apoA-I deficiency results in significantly increased atherosclerosis under a modest, physiological atherogenic stimulus, we generated mice that were deficient in both apoA-I and the LDL receptor (apoA-I-/-/LDLR-/-) and fed them a chow diet. Surprisingly, the level of HDL-C in apoA-I-/-/LDLR-/- mice was significantly greater than in apoA-I-/- mice and was similar to the level of HDL-C seen in wild-type mice. However, when compared with LDLR-/- mice, apoA-I-/-/LDLR-/- mice developed significantly greater oxidant stress and atherosclerosis. These studies indicate that apoA-I deficiency promotes atherosclerosis on the background of LDLR deficiency despite the presence of normal levels of HDL-C.


*    Methods
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 *Methods
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Animals
ApoA-I-/- and LDLR-/- mice on a C57BL/6 background were obtained from Jackson Laboratories (Bar Harbor, Maine). Mice were crossed to generate F1 apoA-I+/-/LDLR+/- mice. The F1 mice were crossed and genotyped for apoA-I and the LDLR using polymerase chain reaction assays (Jackson Laboratory). ApoA-I-/-/LDLR-/-, apoA-I+/+/LDLR-/-, and apoA-I-/-/LDLR+/+ mice were identified for use in the study. Both male and female mice fed a chow diet from the time of weaning through the time of euthanasia were used in the study. Mice were euthanized by intraperitoneal injection of ketamine/xylazine for collection of plasma and quantitation of atherosclerosis at 10, 16, and 22 months. Because significant sex differences were not observed for either plasma lipids or atherosclerosis, data from male and female mice were combined. Mice were handled according to the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee.

Plasma Lipid and Lipoprotein Analysis
Plasma cholesterol and triglyceride levels were measured enzymatically on a Cobas Fara II (Roche Diagnostic Systems Inc) using Sigma reagents as previously described.23 Pooled plasma 200 µL from wild-type, LDLR-/-, and apoA-I-/-/LDLR-/- mice or 400 µL pooled from apoA-I-/- mice was analyzed by FPLC gel filtration (Amersham Pharmacia Biotech) on 2 Superose 6 columns, as previously described.24 The cholesterol concentrations in the FPLC fractions were determined using an enzymatic assay (Wako Pure Chemical Industries Ltd). Data for apoA-I-/- mice is normalized to 200 µL of total pooled plasma, corresponding to the volume used in the other 3 mouse groups. The VLDL, LDL, and HDL FPLC peaks in 22-month-old mice were deconvoluted from the FPLC profile by fitting the sum of 3 Gaussian curves using SAAM II (SAAM Institute).

VLDL (d<1.006 mg/mL), LDL (d=1.006 to 1.063 mg/mL), and HDL (d=1.063 to 1.21) were isolated by sequential ultracentrifugation of 200 µL of pooled plasma samples from each mouse group.25 The lipoprotein fractions were analyzed for total cholesterol, esterified cholesterol, phospholipids, triglycerides, and protein using enzymatic assays (Wako Pure Chemical Industries Ltd and Pierce). Cholesterol values for each lipoprotein fraction were normalized to the total plasma cholesterol values.

SDS-PAGE was performed on VLDL, LDL, and HDL isolated from equal volumes of plasma by sequential ultracentrifugation on a 3% to 20% gradient gel under reducing conditions and stained for protein with GelCode Blue Stain Reagent (Pierce). The relative abundance of apolipoproteins in HDL was determined using densitometry of Coomassie-stained SDS-PAGE gels.

Western Blotting
Samples from 2 to 3 adjacent FPLC lipoprotein fractions were pooled and then subjected to SDS-PAGE under reducing conditions. Proteins were transferred to a nitrocellulose membrane, probed for murine apoA-II with a polyclonal rabbit anti-mouse apoA-II antibody (Biodesign International, Kennebunk, Maine), and detected with a peroxidase-conjugated goat anti-rabbit IgG antibody.

Atherosclerosis
The extent of atherosclerosis in the whole aorta was analyzed en face as described previously.26 Data are represented as the percent of the total aortic surface area that contained atherosclerotic lesion.

The extent of atherosclerosis in the aortic root was determined using methods previously described.26 The cross-sectional lesion area from serial sections of aortic roots that were stained with Oil Red O were measured by manual tracing of the lesion, which was identified by a combination of lipid staining and histological morphology. The acquisition of images and the analysis of lesion areas were both performed in a blinded fashion.

Isoprostane Analysis
For isoprostane measurement, total lipids from individual plasma samples were extracted with ice-cold Folch solution and chloroform/methanol (2:1, vol/vol), as described previously, and the organic phase was dried under nitrogen.27 The samples were hydrolyzed by the addition of aqueous KOH (15%), and total iPF2{alpha}-VI was measured as described previously.27

Statistical Analysis
The Mann-Whitney test was used for comparisons of plasma lipids, isoprostanes, and atherosclerosis between LDLR-/- and apoA-I-/-/LDLR-/- mice.


*    Results
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 *Results
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Plasma Lipids and Lipoproteins
At 10 months of age, both LDLR-/- and apoA-I-/-/LDLR-/- mice had elevated levels of non-HDL-C, which were not significantly different from each other when lipoproteins were separated by standard precipitation of apoB-containing lipoproteins23 (Table 1). The same trend was also observed in the non-HDL (VLDL+LDL) fractions of these groups when lipoproteins were separated by sequential density ultracentrifugation (Table 2). The elevated level of non-HDL-C in LDLR-/- and apoA-I-/-/LDLR-/- mice was also apparent when lipoproteins were separated by size using FPLC. Both LDLR-/- and apoA-I-/-/LDLR-/- mice had similarly elevated levels of LDL-C compared with wild-type or apoA-I-/- mice (please see Figure I, available online at http://atvb.ahajournals.org).


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  		TABLE 1. Plasma Lipid Values in 10-Month-Old Mice


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  		TABLE 2. Lipoprotein Separation by Ultracentrifugation in 10-Month-Old Mice

Ten-month-old apoA-I-/-/LDLR-/- mice had moderately lower levels of HDL-C compared with LDLR-/- mice as determined by precipitation of apoB containing lipoproteins (Table 1) and by sequential density ultracentrifugation (Table 2). Interestingly, although the level of HDL-C was somewhat decreased compared with LDLR-/- mice, apoA-I-/-/LDLR-/- mice had HDL-C levels that were much greater than apoA-I-/- mice and similar to wild-type mice (Tables 1 and 2 Up). FPLC demonstrated similar HDL-C profiles in wild-type and LDLR-/- mice, whereas apoA-I-/- and apoA-I-/-/LDLR-/- mice both had similar, reduced levels of HDL-C in the size range corresponding to HDL that contains apoA-I (please see online Figure I). It is noteworthy that the HDL peaks in apoA-I-/- and apoA-I-/-/LDLR-/- mice are shifted to the left, corresponding to larger-sized HDL particles.

There was a graded decrease in HDL-C levels of LDLR-/- mice with advancing age, whereas the opposite was observed in the HDL-C levels of apoA-I-/-/LDLR-/- mice. At 16 months of age, the level of HDL-C in LDLR-/- mice was 82 mg/dL, whereas in apoA-I-/-/LDLR-/- mice, it was 58 mg/dL (Table 3). By 22 months of age, the levels of HDL-C in LDLR-/- mice and apoA-I-/-/LDLR-/- mice were similar, 80±2.8 mg/dL for LDLR-/- versus 74±2.7 mg/dL for apoA-I-/-/LDLR-/- mice (Table 4). This same trend is also apparent in the FPLC profiles, where the peak corresponding to the population of larger HDL particles seen in apoA-I-/-/LDLR-/- mice increases in size as the mice grow older (please see online Figures I and II and Figure 1). To confirm that the peak seen in the FPLC tracing of apoA-I-/-/LDLR-/- mice was a population of large HDL, Western blotting was performed on the FPLC fractions in 22-month-old apoA-I-/-/LDLR-/- and LDLR-/- mice using an antibody against murine apoA-II (Figure 1). In LDLR-/- mice, HDL (as defined by presence of apoA-II) eluted within fractions 29 to 36 (the typical HDL peak), whereas in apoA-I-/-/LDLR-/- mice, HDL eluted within fractions 15 to 34, demonstrating the existence of larger HDL particles that overlap partially with the LDL size range. The overlapping large HDL and LDL peaks from the FPLC profiles of 22-month-old apoA-I-/-/LDLR-/- mice were deconvoluted by curve fitting (Figure 1). Integration of HDL peaks estimated that HDL-C levels in apoA-I-/-/LDLR-/- mice were 70% of those seen in LDLR-/- mice.


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  		TABLE 3. Plasma Lipid Values in 16-Month-Old Mice


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  		TABLE 4. Plasma Lipid Values in 22-Month-Old Mice



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  		Figure 1. FPLC cholesterol profiles in 22-month-old LDLR-/- and apoA-I-/-/LDLR-/- mice. Pooled plasma samples were separated by FPLC on 2 sequential Superose 6 columns, and cholesterol mass in each fraction was measured using an enzymatic assay kit. Peak labels in each graph represent the following: a, VLDL; b, LDL; c, apoA-I-/- HDL; and d, apoA-I+/+ HDL. The HDL peak from the apoA-I-/-/LDLR-/- FPLC profile was deconvoluted by fitting the sum of 3 Gaussian curves using SAAM II. Western blotting analysis was performed on 13 µL from adjacent FPLC fractions that were pooled and subjected to SDS-PAGE under reducing conditions. ApoA-II was detected using a primary antibody against murine ApoA-II. Lanes from the Western blot are aligned with the corresponding fractions in the FPLC cholesterol tracing that lie above. The first lane on the left contains control wild-type murine HDL that was isolated by sequential ultracentrifugation.

The non-HDL-C/HDL-C ratios were also surprisingly similar between apoA-I-/-/LDLR-/- and LDLR-/- mice. The non-HDL-C/HDL-C ratio ranged from 1.65 to 1.80 for LDLR-/- mice and from 2.76 to 3.47 for apoA-I-/-/LDLR-/- mice (Tables 1, 3, and 4UpUp). By 22 months of age, the non-HDL-C/HDL-C ratios were only 1.5 times greater in apoA-I-/-/LDLR-/- mice than LDLR-/- mice.

Lipoprotein Composition
The chemical composition of VLDL and LDL (data not shown) and HDL (Table 5) isolated by density ultracentrifugation did not differ greatly between LDLR-/- and apoA-I-/-/LDLR-/- mice. SDS-PAGE of VLDL and LDL revealed similar apolipoprotein composition in LDLR-/- and apoA-I-/-/LDLR-/- mice (data not shown). HDL apolipoprotein composition, on the other hand, differed between LDLR-/- and apoA-I-/-/LDLR-/- mice. The apolipoprotein composition of the HDL in LDLR-/- mice was similar to wild-type mice, whereas the HDL of apoA-I-/-/LDLR-/- had an increase in the percent composition of apo-II, apoA-IV, apoCs, and apoE, (Table 5) similar to what has been reported in apoA-I-/- mice20 (Table 5). Because equal volumes of plasma were subjected to FPLC, an increase in apoA-II can also be seen in the lipoprotein profiles of 22-month-old apoA-I-/-/LDLR-/- mice compared with LDLR-/- mice (Figure 1C).


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  		TABLE 5. HDL Composition in 10-Month-Old Mice

Aortic Atherosclerosis
Both LDLR-/- and apoA-I-/-/LDLR-/- mice had increased atherosclerosis with advancing age, but for each age that was investigated, apoA-I-/-/LDLR-/- mice had significantly more atherosclerosis than LDLR-/- mice (Figure 2). The magnitude of the difference between the 2 groups also increased with advancing age.



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  		Figure 2. Atherosclerosis in 10-, 16-, and 22-month-old mice. The percent of the total aortic surface area containing atherosclerotic lesion (percent lesion) was quantified by en face measurement of atherosclerotic lesions stained with Sudan IV. Average percent lesion in LDLR-/- mice (light bar) and apoA-I-/-/LDLR-/- mice (dark bar) at 10, 16, and 22 months of age is shown. At 10 months age, apoA-I-/-/LDLR-/- mice had a 2.6-fold increase in atherosclerosis compared with LDLR-/- mice (P=0.006). At 16 months age, apoA-I-/-/LDLR-/- mice had a 3.8-fold increase in atherosclerosis compared with LDLR-/- mice (P=0.006). At 22 month age, apoA-I-/-/LDLR-/- mice had a 5-fold increase in atherosclerosis compared with LDLR-/- mice (P<0.0001).

At 10 months of age, apoA-I-/- mice had nearly undetectable levels of atherosclerosis (data not shown). LDLR-/- mice had minimal atherosclerosis, with 1.56% of the aortic surface area containing lesion. ApoA-I-/-/LDLR-/- mice had 2.6 times more atherosclerosis than LDLR-/- mice, with 4.06% of the aortic surface area containing lesion (P=0.006) (Figure 2).

At 16 months of age, 2.47% of the aortic surface of LDLR-/- mice contained atherosclerotic lesion. Sixteen-month-old apoA-I-/-/LDLR-/- mice had 3.8 times more atherosclerosis as LDLR-/- mice, with 9.51% of the aortic surface area containing lesion (P=0.006) (Figure 2).

At 22 months of age, 3.61% of the aortic surface of LDLR-/- mice contained atherosclerotic lesion. Twenty-two-month-old apoA-I-/-/LDLR-/- mice, on the other hand, had a 5-fold increase in atherosclerosis compared with LDLR-/- mice, with 18.08% of the aortic surface area containing lesion (P<0.0001) (Figure 2).

Consistent with the en face results, analysis of the cross-sectional lesion area within the aortic root from a subset of 16-month-old mice demonstrated a 2.2-fold increase in atherosclerosis in apoA-I-/-/LDLR-/- mice compared with LDLR-/- mice (P=0.016). The lesions observed in LDLR-/- mice were early fatty streaks, composed primarily of lipid as determined by Oil Red O staining. The lesions in apoA-I-/-/LDLR-/- mice, on the other hand, were more advanced, containing both areas that were stained by Oil Red O as well as other areas that were mostly cellular with very little Oil Red O staining (Figure 3).



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  		Figure 3. Aortic root lesion morphology in 16-month-old mice. Oil Red O and hematoxylin staining of aortic root cross-sections from 16-month-old LDLR-/- and apoA-I-/-/LDLR-/- mice demonstrate increased atherosclerosis in apoA-I-/-/LDLR-/- mice. A, LDLR-/-, x40 magnification. B, LDLR-/-, x100 magnification. C, apoA-I-/-/LDLR-/-, x40 magnification. D, apoA-I-/-/LDLR-/-, x100 magnification.

Plasma Isoprostanes
Isoprostanes are formed by nonenzymatic free-radical-induced oxidation of arachidonic acid-containing phospholipids in cell membranes and LDL and are an established index of in vivo oxidant stress.28 To test the hypothesis that apoA-I-deficient mice experienced increased oxidant stress, plasma isoprostanes were measured in 22-month-old apoA-I-/-/LDLR-/- and LDLR-/- mice (Figure 4). ApoA-I-/-/LDLR-/- mice had a 39% increase in plasma isoprostanes compared with LDLR-/- mice (P<0.05), indicating that lack of apoA-I was associated with increased oxidant stress.



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  		Figure 4. Plasma isoprostanes in 22-month-old mice. Total plasma iPF2{alpha}-VI was measured in 22-month-old LDLR-/- and apoA-I-/-/LDLR-/- mice. ApoA-I-/-/LDLR-/- mice had a 39% increase in plasma iPF2{alpha}-VI compared with LDLR-/- mice (P<0.05), indicating that they experienced significantly increased oxidant stress.


*    Discussion
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 up arrowResults
 *Discussion
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In this report, we demonstrate that apoA-I deficiency resulted in a marked loss of protection against the formation of atherosclerosis in chow-fed LDLR-/- mice. LDLR-/- mice developed moderate hypercholesterolemia but were relatively resistant to the formation of atherosclerosis; in contrast, apoA-I-/-/LDLR-/- mice developed significant atherosclerosis despite similar non-HDL-C and well-preserved HDL-C levels. This study has some important differences from previous studies of atherosclerosis in apoA-I-deficient mice.20–22 We used LDLR-/- mice, fed them a chow diet, and carried out our experiment for 22 months to investigate the effects of apoA-I deficiency under a modest atherogenic stimulus. ApoA-I-/-/LDLR-/- mice had only moderately elevated levels of non-HDL-C ({approx}175 mg/mL), normal levels of HDL-C ({approx}60 mg/mL), and a non-HDL-C to HDL-C ratio of {approx}3. Other studies of atherosclerosis and apoA-I deficiency used younger apoA-I-/-/apoB transgenic mice that were fed an atherogenic diet, resulting in a marked increase in non-HDL-C ({approx}450 mg/dL) and extremely low levels of HDL-C ({approx}15 mg/dL). ApoA-I-/-/apoB transgenic mice that were fed an atherogenic diet therefore had a non-HDL-C to HDL-C ratio of {approx}30, which is an order of magnitude greater than the apoA-I-/-/LDLR-/- mice in this study. We believe that the moderate elevation of non-HDL-C in LDLR-/- mice fed chow is more similar to moderate hypercholesterolemia found within typical human populations and is therefore a more physiological condition under which to investigate the effects of apoA-I deficiency on atherosclerosis.

We were initially surprised by the relatively high level of HDL-C present in 10-month-old apoA-I-/-/LDLR-/- mice. The levels of HDL-C in previous reports of apoA-I deficiency in other mouse models have been lower. To confirm our results, we used multiple methods to measure HDL-C levels, including precipitation of apoB-containing lipoproteins (defining HDL as a lipoprotein lacking apoB), sequential density ultracentrifugation (defining HDL as a lipoprotein in the density range of 1.063 to 1.21 mg/mL), and FPLC (defining HDL based on size and the presence of apoA-II). Our results from the various methods were in general agreement with each other. When we examined 10-, 16-, and 22-month-old mice, we saw a trend of decreasing levels of HDL-C in LDLR-/- mice and increasing levels of HDL-C in apoA-I-/-/LDLR-/- mice with advancing age. By 22 months, the levels of HDL-C in the 2 groups were very similar. Taken together, our results demonstrate that (1) apoA-I-/-/LDLR-/- mice had significantly higher levels of HDL-C than apoA-I-/- mice (4- to 5-fold increase); (2) the levels of HDL-C in apoA-I-/-/LDLR-/- mice were equivalent to wild-type mice; and (3) by 22 months of age, apoA-I-/-/LDLR-/- mice had HDL-C levels that were similar to LDLR-/- mice (Tables 1, 3, and 4 UpUp). Unlike previous reports of apoA-I deficiency, the mice in our study lacked the LDLR. Our results indicate that in this metabolic milieu, the effect of apoA-I deficiency on HDL-C levels is less dramatic.

ApoA-I-/- mice have previously been reported to contain a significant population of HDL particles that are larger than HDL present in wild-type mice and therefore overlap with the LDL size range.20 The results in our study were consistent with this finding. In apoA-I-/-/LDLR-/- mice, apoA-II could be detected in FPLC fractions as early as fraction No. 15, which lies near the center of the LDL size range. In contrast, apoA-II was not detected in fractions earlier than fraction No. 29 in LDLR-/- mice (Figure 1). Western blotting of FPLC fractions for apoA-II also indicated that the level of apoA-II was considerably increased in the HDL of apoA-I-/-/LDLR-/- mice compared with LDLR-/- mice (Figure 1). In addition to larger size, HDLs previously described in apoA-I-/- mice were also reported to be enriched in apoA-II, apoA-IV, and apoE.20 HDL present in apoA-I-/-/LDLR-/- mice in our study was similar in composition to that reported in apoA-I-/- mice (Table 5). The LDLR has been described to be able to interact with large HDL particles (HDL1) enriched in apoE,29 the type of particles present in apoA-I-/- mice. One possible explanation for the increased levels of HDL-C seen in apoA-I-/-/LDLR-/- mice, compared with apoA-I-/- mice, is that there is no LDLR-mediated clearance of apoE-rich HDL in apoA-I-/-/LDLR-/- mice. This may have been responsible for the age-related accumulation of large HDLs in apoA-I-/-/LDLR-/- mice, seen both when HDL-C is measured by precipitation of apoB containing lipoproteins as well as in the increase in the peak corresponding to large HDL in the FPLC fractionation. Therefore, as opposed to other models of atherosclerosis associated with apoA-I deficiency in which levels of HDL-C were markedly reduced (12 to 23 mg/dL), HDL-C levels in apoA-I-/-/LDLR-/- mice were well maintained and closely approached the HDL-C levels in the LDLR-/- mice to which they were being compared.

Despite having relatively normal levels of HDL-C, apoA-I-/-/LDLR-/- mice developed up to 5 times more atherosclerosis than LDLR-/- mice. This suggests that the proatherogenic effects of apoA-I deficiency may be more than simply the lowering of the level of HDL-C and may be related to effects on the quality of HDL that is present in these mice. ApoA-I and apoA-I-rich HDL have been shown to exhibit several potentially antiatherogenic effects. Lipid-poor apoA-I is the major physiological acceptor of cellular cholesterol via ATP-binding cassette-A1 (ABCA1)30 and indeed has been shown to stabilize the ABCA1 protein.31 ApoA-I facilitates selective uptake of HDL cholesterol by the liver by activating lecithin-cholesterol acyltransferase32 and acting as a specific ligand for SR-BI.33 ApoA-I has also been shown to exhibit antiatherogenic properties,34 such as acting as an antioxidant,35,36 an antiinflammatory agent,37 and a stabilizer of prostacyclin.38 ApoA-I-/-/LDLR-/- mice have HDL that lacks apoA-I and is enriched in apoA-II. ApoA-II-rich HDL is not protected from oxidation39 and has been shown to be proatherogenic40 and proinflammatory.39 Consistent with this concept, apoA-I deficiency was associated with increased plasma isoprostanes in apoA-I-/-/LDLR-/- mice in our study. This study demonstrates that apoA-I deficiency results in significantly increased atherosclerosis in chow-fed LDLR-/- mice, even in the setting of well-maintained HDL-C levels, and suggests that apoA-I itself, independent of HDL-C, has properties that directly protect against atherosclerosis.


*    Acknowledgments
 
Supported by grants P01-HL59407, R01-HL55323, R01-HL57811, and P50-HL70128 from the National Heart, Lung, and Blood Institute of the NIH. R.E.M. was supported by F31-AI-10493 from the National Institute of Allergy and Infectious Diseases of the NIH. D.J.R. is an Established Investigator of the American Heart Association and a recipient of a Burroughs Wellcome Foundation Clinical Scientist Award in Translational Research. The authors would like to thank Anna Lillethun, Linda Morrell, Aisha Faruqi, Jennifer Dykhouse, and Christopher Long for excellent technical support. They would also like to thank Dawn Marchadier, Dr Uli Broedl, Dr Weijun Jin, Dr Jane Glick, and Dr Ellen Puré for valuable scientific input and discussion.

Received August 1, 2003; accepted August 13, 2003.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Rader DJ. High-density lipoproteins and atherosclerosis. Am J Cardiol. 2002; 90: 62–70.
  2. Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB. Incidence of coronary heart disease and lipoprotein cholesterol levels: the Framingham Study. JAMA. 1986; 256: 2835–2838.[Abstract]
  3. Rader DJ, Hoeg JM, Brewer HB Jr. Quantitation of plasma apolipoproteins in the primary and secondary prevention of coronary artery disease. Ann Intern Med. 1994; 120: 1012–1025.[Abstract/Free Full Text]
  4. Lamarche B, Moorjani S, Lupien PJ, Cantin B, Bernard PM, Dagenais GR, Despres JP. Apolipoprotein A-I and B levels and the risk of ischemic heart disease during a five-year follow-up of men in the Quebec cardiovascular study. Circulation. 1996; 94: 273–278.[Abstract/Free Full Text]
  5. Francis MC, Frohlich JJ. Coronary artery disease in patients at low risk: apolipoprotein AI as an independent risk factor. Atherosclerosis. 2001; 155: 165–170.[CrossRef][Medline] [Order article via Infotrieve]
  6. Maciejko JJ, Holmes DR, Kottke BA, Zinsmeister AR, Dinh DM, Mao SJ. Apolipoprotein A-I as a marker of angiographically assessed coronary-artery disease. N Engl J Med. 1983; 309: 385–389.[Abstract]
  7. Franzen J, Fex G. Low serum apolipoprotein A-I in acute myocardial infarction survivors with normal HDL cholesterol. Atherosclerosis. 1986; 59: 37–42.[CrossRef][Medline] [Order article via Infotrieve]
  8. Kwiterovich PO Jr, Coresh J, Smith HH, Bachorik PS, Derby CA, Pearson TA. Comparison of the plasma levels of apolipoproteins B and A-1, and other risk factors in men and women with premature coronary artery disease. Am J Cardiol. 1992; 69: 1015–1021.[CrossRef][Medline] [Order article via Infotrieve]
  9. Norum RA, Lakier JB, Goldstein S, Angel A, Goldberg RB, Block WD, Noffze DK, Dolphin PJ, Edelglass J, Bogorad DD, Alaupovic P. Familial deficiency of apolipoproteins A-I and C-III and precocious coronary-artery disease. N Engl J Med. 1982; 306: 1513–1519.[Abstract]
  10. Schaefer EJ, Heaton WH, Wetzel MG, Brewer HB Jr. Plasma apolipoprotein A-1 absence associated with a marked reduction of high density lipoproteins and premature coronary artery disease. Arteriosclerosis. 1982; 2: 16–26.[Abstract/Free Full Text]
  11. Karathanasis SK, Ferris E, Haddad IA. DNA inversion within the apolipoproteins AI/CIII/AIV-encoding gene cluster of certain patients with premature atherosclerosis. Proc Natl Acad Sci U S A. 1987; 84: 7198–7202.[Abstract/Free Full Text]
  12. Ordovas JM, Cassidy DK, Civeira F, Bisgaier CL, Schaefer EJ. Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11. J Biol Chem. 1989; 264: 16339–16342.[Abstract/Free Full Text]
  13. Matsunaga T, Hiasa Y, Yanagi H, Maeda T, Hattori N, Yamakawa K, Yamanouchi Y, Tanaka I, Obara T, Hamaguchi H. Apolipoprotein A-I deficiency due to a codon 84 nonsense mutation of the apolipoprotein A-I gene. Proc Natl Acad Sci U S A. 1991; 88: 2793–2797.[Abstract/Free Full Text]
  14. Ng DS, Leiter LA, Vezina C, Connelly PW, Hegele RA. Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia. J Clin Invest. 1994; 93: 223–229.
  15. Miccoli R, Bertolotto A, Navalesi R, Odoguardi L, Boni A, Wessling J, Funke H, Wiebusch H, Eckardstein A, Assmann G. Compound heterozygosity for a structural apolipoprotein A-I variant, apo A-I(L141R)Pisa, and an apolipoprotein A-I null allele in patients with absence of HDL cholesterol, corneal opacifications, and coronary heart disease. Circulation. 1996; 94: 1622–1628.[Abstract/Free Full Text]
  16. Funke H, von Eckardstein A, Pritchard PH, Karas M, Albers JJ, Assmann G. A frameshift mutation in the human apolipoprotein A-I gene causes high density lipoprotein deficiency, partial lecithin: cholesterol-acyltransferase deficiency, and corneal opacities. J Clin Invest. 1991; 87: 371–376.
  17. Romling R, von Eckardstein A, Funke H, Motti C, Fragiacomo GC, Noseda G, Assmann G. A nonsense mutation in the apolipoprotein A-I gene is associated with high-density lipoprotein deficiency and periorbital xanthelasmas. Arterioscler Thromb. 1994; 14: 1915–1922.[Abstract/Free Full Text]
  18. Takata K, Saku K, Ohta T, Takata M, Bai H, Jimi S, Liu R, Sato H, Kajiyama G, Arakawa K. A new case of apoA-I deficiency showing codon 8 nonsense mutation of the apoA-I gene without evidence of coronary heart disease. Arterioscler Thromb Vasc Biol. 1995; 15: 1866–1874.[Abstract/Free Full Text]
  19. Williamson R, Lee D, Hagaman J, Maeda N. Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I. Proc Natl Acad Sci U.S.A. 1992; 89: 7134–7138.[Abstract/Free Full Text]
  20. Li H, Reddick RL, Maeda N. Lack of apoA-I is not associated with increased susceptibility to atherosclerosis in mice. Arterioscler Thromb. 1993; 13: 1814–1821.[Abstract/Free Full Text]
  21. Voyiaziakis E, Goldberg IJ, Plump AS, Rubin EM, Breslow JL, Huang LS. ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice. J Lipid Res. 1998; 39: 313–321.[Abstract/Free Full Text]
  22. Hughes SD, Verstuyft J, Rubin EM. HDL deficiency in genetically engineered mice requires elevated LDL to accelerate atherogenesis. Arterioscler Thromb Vasc Biol. 1997; 17: 1725–1729.[Abstract/Free Full Text]
  23. Tsukamoto K, Hiester KG, Smith P, Usher DC, Glick JM, Rader DJ. Comparison of human apoA-I expression in mouse models of atherosclerosis after gene transfer using a second generation adenovirus. J Lipid Res. 1997; 38: 1869–1876.[Abstract]
  24. Tsukamoto K, Smith P, Glick JM, Rader DJ. Liver-directed gene transfer and prolonged expression of three major human ApoE isoforms in ApoE-deficient mice. J Clin Invest. 1997; 100: 107–114.[Medline] [Order article via Infotrieve]
  25. Brousseau T, Clavey V, Bard JM, Fruchart JC. Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles. Clin Chem. 1993; 39: 960–964.[Abstract/Free Full Text]
  26. Tangirala RK, Tsukamoto K, Chun SH, Usher D, Pure E, Rader DJ. Regression of atherosclerosis induced by liver-directed gene transfer of apolipoprotein A-I in mice. Circulation. 1999; 100: 1816–1822.[Abstract/Free Full Text]
  27. Pratico D, Tangirala RK, Rader DJ, Rokach J, FitzGerald GA. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat Med. 1998; 4: 1189–1192.[CrossRef][Medline] [Order article via Infotrieve]
  28. Pratico D, Lawson JA, Rokach J, FitzGerald GA. The isoprostanes in biology and medicine. Trends Endocrinol Metab. 2001; 12: 243–247.[CrossRef][Medline] [Order article via Infotrieve]
  29. Koo C, Innerarity TL, Mahley RW. Obligatory role of cholesterol and apolipoprotein E in the formation of large cholesterol-enriched and receptor-active high density lipoproteins. J Biol Chem. 1985; 260: 11934–11943.[Abstract/Free Full Text]
  30. Oram JF, Vaughan AM. ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Curr Opin Lipidol. 2000; 11: 253–260.[CrossRef][Medline] [Order article via Infotrieve]
  31. Arakawa R, Yokoyama S. Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from Thiol protease-mediated degradation. J Biol Chem. 2002; 277: 22426–22429.[Abstract/Free Full Text]
  32. Jonas A. Lecithin-cholesterol acyltransferase in the metabolism of high-density lipoproteins. Biochim Biophys Acta. 1991; 1084: 205–220.[Medline] [Order article via Infotrieve]
  33. Williams DL, Temel RE, Connelly MA. Roles of scavenger receptor BI and APO A-I in selective uptake of HDL cholesterol by adrenal cells. Endocr Res. 2000; 26: 639–651.[Medline] [Order article via Infotrieve]
  34. Shah PK, Kaul S, Nilsson J, Cercek B. Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: an idea whose time for testing is coming, part I. Circulation. 2001; 104: 2376–2383.[Free Full Text]
  35. Hayek T, Oiknine J, Dankner G, Brook JG, Aviram M. HDL apolipoprotein A-I attenuates oxidative modification of low density lipoprotein: studies in transgenic mice. Eur J Clin Chem Clin Biochem. 1995; 33: 721–725.[Medline] [Order article via Infotrieve]
  36. Navab M, Berliner JA, Subbanagounder G, Hama S, Lusis AJ, Castellani LW, Reddy S, Shih D, Shi W, Watson AD, Van Lenten BJ, Vora D, Fogelman AM. HDL and the inflammatory response induced by LDL-derived oxidized phospholipids. Arterioscler Thromb Vasc Biol. 2001; 21: 481–488.[Abstract/Free Full Text]
  37. Hyka N, Dayer JM, Modoux C, Kohno T, Edwards CK 3rd, Roux-Lombard P, Burger D. Apolipoprotein A-I inhibits the production of interleukin-1beta and tumor necrosis factor-alpha by blocking contact-mediated activation of monocytes by T lymphocytes. Blood. 2001; 97: 2381–2389.[Abstract/Free Full Text]
  38. Yui Y, Aoyama T, Morishita H, Takahashi M, Takatsu Y, Kawai C. Serum prostacyclin stabilizing factor is identical to apolipoprotein A-I (Apo A-I): a novel function of Apo A-I. J Clin Invest. 1988; 82: 803–807.
  39. Castellani LW, Navab M, Van Lenten BJ, Hedrick CC, Hama SY, Goto AM, Fogelman AM, Lusis AJ. Overexpression of apolipoprotein AII in transgenic mice converts high density lipoproteins to proinflammatory particles. J Clin Invest. 1997; 100: 464–474.[Medline] [Order article via Infotrieve]
  40. Warden CH, Hedrick CC, Qiao JH, Castellani LW, Lusis AJ. Atherosclerosis in transgenic mice overexpressing apolipoprotein A-II. Science. 1993; 261: 469–472.[Abstract/Free Full Text]



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