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(Circulation Research. 2004;95:998.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Plasma Cholesteryl Esters Provided by Lecithin:Cholesterol Acyltransferase and Acyl-Coenzyme A:Cholesterol Acyltransferase 2 Have Opposite Atherosclerotic Potential

Richard G. Lee, Kathryn L. Kelley, Janet K. Sawyer, Robert V. Farese, Jr, John S. Parks, Lawrence L. Rudel

From the Arteriosclerosis Research Program (R.G.L., K.L.K., J.K.S., J.S.P., L.L.R.), Departments of Pathology and Biochemistry, Wake Forest University School of Medicine Winston-Salem, NC; Gladstone Institute of Cardiovascular Disease (R.V.F.), San Francisco, Calif.

Correspondence to Lawrence L. Rudel, PhD, Department of Pathology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157. E-mail lrudel{at}wfubmc.edu

	Abstract

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Evidence suggests that ACAT2 is a proatherogenic enzyme that contributes cholesteryl esters (CEs) to apoB-containing lipoproteins, whereas LCAT is an antiatherogenic enzyme that facilitates reverse cholesterol transport by esterifying free cholesterol on HDL particles. We hypothesized that deletion of LCAT and ACAT2 would lead to absence of plasma CEs and reduced atherosclerosis. To test this hypothesis, ACAT2^–/– LCAT^–/– LDLr^–/–, ACAT2^–/– LDLr^–/–, and LCAT^–/– LDLr^–/– mice were fed a 0.15% cholesterol diet for 20 weeks. In comparison to LDLr^–/– mice, the total plasma cholesterol (TPC) of ACAT2^–/– LCAT^–/– LDLr^–/– mice was 67% lower because of the complete absence of plasma CEs, leading to 94% less CE accumulation in the aorta. In the LCAT^–/– LDLr^–/– mice, TPC and atherosclerosis were significantly higher because of increased accumulations of ACAT2-derived CE. In ACAT2^–/– LDLr^–/– mice, again compared with LDLr^–/– mice, TPC was 19% lower, whereas atherosclerosis was 88% lower. Therefore, the absence of ACAT2 led to a significant reduction in TPC although benefits in reduction of atherosclerosis were much more pronounced. Overall, the data suggest that ACAT2-derived CE is the predominant atherogenic lipid in blood, and that an important goal for prevention of atherosclerosis is to limit ACAT2-derived CE accumulation in lipoproteins.

Key Words: LCAT • ACAT2 • atherosclerosis • cholesterol • cholesteryl esters • lipoproteins

	Introduction

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Arterial degeneration known as atherosclerosis underlies coronary heart disease, the number one cause of premature death in the United States.¹ Since the early studies of atherosclerosis, accumulation of plasma lipoprotein cholesteryl esters (CEs) in the intima of arteries has been thought to be central to the development of the disease.² This accumulation occurs when the arterial influx of CEs within the core of apoB lipoproteins originating from the liver exceeds the efflux of free cholesterol (FC) on HDL particles. Efflux of cholesterol onto HDL is driven by a diffusion gradient of FC, itself maintained by the esterification of cholesterol within the HDL particle. Two enzymes are thought to be responsible for the synthesis of plasma CE, ie, acyl-CoA:cholesterol acyltransferase 2 (ACAT2)^3–5 and lecithin:cholesterol acyltransferase (LCAT).⁶

ACAT is an integral membrane protein localized to the rough ER that catalyzes a reaction in which the fatty acid of an acyl-CoA molecule, typically oleoyl-CoA, is esterified to cholesterol, generating CE.⁷ The initial evidence for existence of an ACAT enzyme specific to the liver and small intestine came from characterization of ACAT1 KO mice in which esterification activity was absent in the adrenal, but was not significantly reduced in the liver and small intestine.⁸ This evidence led to the discovery of a second ACAT isoform, designated ACAT2, specific to the liver and small intestine of monkeys, mice, and humans.^9–11 When immunofluorescent analysis was performed on nonhuman primate liver and jejunum using antibodies specific for ACAT1 and ACAT2, ACAT2 was the only isoform found in hepatocytes and enterocytes, the cells that secrete and assemble apoB lipoproteins.¹² A similar finding has recently been made for human liver.¹³ In addition, several types of evidence suggest that ACAT2, and not ACAT1, may be proatherogenic because of its ability to synthesize CEs for incorporation into apoB-containing lipoproteins that are secreted into the plasma.^14,15 The recent observation of reduced atherosclerosis in the apoE^–/– ACAT2^–/– double knockout mouse supports this suggestion.¹⁶

The other enzyme important in synthesis of plasma lipoprotein CE is LCAT, a glycoprotein that is secreted by the liver into the blood. The most potent activator of LCAT is apoA-I, and most, but not all, of the LCAT mass is associated with HDL.¹⁷ By esterifying HDL free cholesterol, LCAT is thought to promote reverse cholesterol transport by maintaining a FC gradient between HDL and peripheral tissues¹⁸ and by generating spherical HDL that can accumulate more CE mass in the core of the particle. Deletion of LCAT would be expected to enhance atherosclerosis by interfering with this process, and this has been observed in LCAT^–/– mice.¹⁹ However the antiatherogenic properties of LCAT have been called into question because of independent studies showing that deletion of LCAT had opposite effects on the development of atherosclerosis.^19,20 Whereas the focus of most investigations of LCAT has been on its role in HDL metabolism, there is growing interest in the participation of LCAT in the potentially proatherogenic process of apoB particle CE synthesis because of findings that it may also synthesize many of the long chain (>18 carbons) CE species in apoB containing lipoproteins.^21,22

An objective of the present study was to establish whether LCAT and ACAT2 are the only two genes responsible for synthesis of plasma CE. Additionally, we hypothesized that the two enzymes may have opposing roles, ie, synthesis of CE by LCAT is antiatherogenic because of its role in reverse cholesterol transport and synthesis of polyunsaturated CE, whereas ACAT2 promotes atherosclerosis by promoting accumulation of saturated and monounsaturated CE in apoB-containing lipoprotein particles. In this scenario, gene deletion of LCAT alone should have a proatherogenic effect, whereas gene deletion of ACAT2 should have an antiatherogenic effect. Deletion of both enzymes together should prevent atherosclerosis through a major reduction in plasma CE levels. To test these hypotheses, we developed LDLr^–/– mice, a genetically engineered mouse model that facilitates atherosclerosis evaluation, with deletions of LCAT, ACAT2, or both enzymes together.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and Diets
Single LDLR^–/–, double ACAT2^–/– LDLr^–/– and LCAT^–/– LDLr^–/–, and triple ACAT2^–/– LCAT^–/– LDLr^–/– KO male mice were created as described in the expanded Materials and Methods (see online data supplement available at http://circres.ahajournals.org). A diet with trans monounsaturated fatty acid-enriched fat (10% of energy as fat) supplemented with cholesterol (0.18% w/w) was then fed daily for 20 weeks (online Table 1). Lipids and lipoproteins were analyzed as described in the online data supplement.

Cholesterol Absorption Analysis by Dual Fecal Radioisotope Method
After 8 weeks on diet, cholesterol absorption was performed using the dual fecal isotope method as described previously.²³ The protocol is described in detail in the expanded Materials and Methods section (see online data supplement).

Analysis of Liver Lipid Composition
At the time of analysis, 100 mg of liver was thawed and minced, and lipids were extracted in 2:1 chloroform: methanol at room temperature overnight. The protein was quantitatively separated from the lipid extract which was then dried down under N₂ and redissolved in a measured volume of CHCl₃:MeOH, 2:1 v/v. Dilute H₂SO₄ was added, vortexed, and centrifuged to split the phases. The aqueous upper phase was aspirated and discarded and an aliquot of the bottom phase was removed and dried down. 1% Triton X-100 in CHCl₃ was then added, and the solvent was evaporated. Deionized H₂O was then added to each tube and vortexed until the solution was clear. Lipids were then quantified using enzymatic assays described for plasma lipid analysis (see online data supplement).

Quantification of Atherosclerosis
Atherosclerosis was evaluated by both morphometric²⁴ and biochemical²⁵ methods as described previously. A detailed description is provided in the expanded Materials and Methods section.

Statistical Analyses
Data were evaluated using 1-way ANOVA for genotype with post hoc analyses by Fisher protected least significant difference test. Statistical significance was considered at P<0.05. The outcomes for post hoc analyses are as indicated.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our first objective was to develop an atherosclerosis-susceptible mouse that lacked the LCAT and ACAT2 genes, which was accomplished by breeding mice with the desired traits. At 6 to 7 weeks of age, males of each genotype including LDLr^–/–, ACAT2^–/– LDLr^–/–, LCAT^–/– LDLr^–/–, and ACAT2^–/– LCAT^–/– LDLr^–/–, began eating an atherogenic diet that was continued for 20 weeks. Body weights were monitored periodically throughout the study. It was found that the ACAT2^–/– LDLr^–/– mice gained significantly more weight in comparison to each of the other groups, which were not significantly different with respect to weight gain (online Figure 1).

Genotype-specific differences in total plasma cholesterol levels were already apparent two weeks into the diet period (Figure 1), with LCAT^–/– LDLr^–/– and LDLr^–/– mice having significantly higher TPC levels than ACAT2^–/– LDLr^–/– and ACAT2^–/– LCAT^–/– LDLr^–/– mice (averages of 541 and 620 mg/dL versus 252 and 164 mg/dL, respectively). As time on diet progressed to week 20, the TPC of LCAT^–/– LDLr^–/– and ACAT2^–/– LDLr^–/– mice steadily increased, whereas the TPC of the LDLr^–/– mice and ACAT2^–/– LCAT^–/– LDLr^–/– mice exhibited only modest increases, leading to significant differences in TPC among all genotypes by end of the study (Table 1). The plasma FC concentrations of LCAT^–/– LDLr^–/– mice were 2-fold higher when compared with the other three genotypes. The apparently higher FC than TPC concentrations in ACAT2^–/– LCAT^–/– LDLr^–/– KO mice represents experimental error. In ACAT2^–/– LDLr^–/– mice, plasma CEs were present at lower concentrations than control LDLr^–/– mice. Deletion of the LCAT gene resulted in significantly higher ACAT2-derived plasma CE concentrations when compared with other groups, whereas deletion of both ACAT2 and LCAT led to a complete absence of CEs in the plasma. Triglyceride levels for the ACAT2^–/– LDLr^–/– and LCAT^–/– LDLr^–/– mice were significantly higher than LDLr^–/– controls and ACAT2^–/– LCAT^–/– LDLr^–/– mice.

View larger version (14K): [in this window] [in a new window]   Figure 1. Total plasma cholesterol concentrations of KO mice during diet feeding. LDLr–/– (), ACAT2–/– LDLr–/– (), LCAT–/– LDLr–/– (), and ACAT2–/– LCAT–/– LDLr–/– () were fed a 0.18% diet for 20 weeks with time points taken at 2, 8, 12, and 16 weeks and TPC of each time point was determined as described in Material and Methods. The number of animals analyzed in each genotype is as indicated for Table 1. Each point represents the average with error bars representing SEM.

View this table: [in this window] [in a new window]   Table 1. Plasma Lipid Measurements of KO Mice

The distribution of cholesterol in the lipoprotein fractions was determined by size separation of plasma on a HPLC column with cholesterol measurement via an online cholesterol assay (Figure 2). The percent of cholesterol found in the VLDL-, LDL-, and HDL-sized peaks was calculated (see Materials and Methods) and multiplied by the total cholesterol concentrations to determine VLDL cholesterol (VLDL-C), LDL cholesterol (LDL-C), and HDL cholesterol (HDL-C) concentrations (Table 2). When compared with LDLr^–/– controls, LCAT^–/– LDLr^–/– mice had 300% more VLDL-C, 50% less LDL-C, and no HDL-C, whereas ACAT2^–/– LDLr^–/– mice had 75% less VLDL-C, similar amounts of LDL-C, and 50% more HDL-C. In addition, the LDL retention time of the ACAT2^–/– LDLr^–/– mice (33.4±0.2 minutes.) was significantly longer on the HPLC column than the LDL of LDLr^–/– mice (30.8±0.3), indicating that the LDL particles were of a smaller size. ACAT2^–/– LCAT^–/– LDLr^–/– mice had similar levels of VLDL-C and very low LDL-C and HDL-C compared with LDLr^–/– mice.

View larger version (15K): [in this window] [in a new window]   Figure 2. Separation of plasma lipoprotein fractions by HPLC and analysis by online cholesterol assay. 30 µL of plasma from the terminal blood sample of (A) LDLr–/–, (B) ACAT2–/– LDLr–/– (aarr), (C) LCAT–/– LDLr–/– (llrr), and (D) ACAT2–/– LCAT–/– LDLr–/– (aallrr) mice was applied to a HPLC Superose 6 column and cholesterol content in the eluate was determined by online cholesterol assay. Each panel represents a representative sample from each genotype. Regions included in each lipoprotein class are indicated as VLDL, LDL, and HDL.

View this table: [in this window] [in a new window]   Table 2. Distribution of Cholesterol in VLDL, LDL, and HDL Size Fractions

To determine whether the differences in the TPC concentrations could be at least partially explained by differences in dietary cholesterol absorption, the percent absorption was quantified in subsets of the animals via the dual fecal radioisotope method (online Figure 2). Compared with the percent cholesterol absorption of the LDLr^–/– mice (65±4%), ACAT2^–/– LDLr^–/– (49±2%), LCAT^–/– LDLr^–/– (51±3%), and ACAT2^–/– LCAT^–/– LDLr^–/– (41±2%) mice all absorbed significantly less dietary cholesterol. However, these differences among groups generally were not proportional to the average TPC concentrations shown in Table 1.

The fact that ACAT2 prefers saturated and monounsaturated fatty acids and LCAT prefers polyunsaturated fatty acids for esterification to cholesterol provided the opportunity to determine which enzyme was synthesizing the CE in the apoB-containing lipoproteins of the mice. ApoB lipoproteins were isolated by ultracentrifugation and the CE and PL of the particles were subjected to FAME analysis to determine the fatty acid (FA) species composition (Figure 3). The ratio of the percentages of saturated+monounsaturated FA to polyunsaturated FA in the apoB lipoprotein CE was significantly higher in LCAT^–/– LDLr^–/– compared with LDLr^–/– mice, indicating the increased contribution of ACAT2-derived CE. This was supported by the fact that both the percent of palmitate (17.1±0.6% versus 7.0±0.4%) and oleate (50.3±0.8% versus 31.2±0.8%) in CEFA of apoB lipoproteins were higher in the LCAT^–/– LDLr^–/– mice. Conversely, the ratio was significantly lower in ACAT2^–/– LDLr^–/– when compared with LDLr^–/– mice, indicating an increased contribution of LCAT-derived CE. This was evident by the enrichment in polyunsaturated fatty acids such as linoleate (30.2±0.7% versus 22.5±0.7) and arachidonate (28.8±1.4% versus 10.8±0.6%) in apoB lipoprotein CE of ACAT2^–/– LDLr^–/– versus LDLr^–/– mice, respectively. There were no significant differences in the ratio of saturated+monounsaturated to polyunsaturated fatty acids of the PL among the three genotypes, indicating that there were no substantial differences in the fatty acid substrate pools for LCAT among the groups of mice.

View larger version (36K): [in this window] [in a new window]   Figure 3. Cholesteryl ester and phospholipid fatty acid methyl ester (FAME) analysis of lipoproteins with d<1.063. Plasma from LDLr–/– (rr, n=5), ACAT2–/– LDLr–/– (aarr, n=5), and LCAT–/– LDLr–/– (llrr, n=4) was adjusted to a solution density of 1.063 g/mL with KBr and ultracentrifugation was performed. After centrifugation, tops were isolated, lipids were extracted and separated by TLC. CE and PL bands were scraped and FAME were created via transesterification and analyzed by GLC. A, CE fatty acid ratio expressed as the percent of fatty acids that are saturated and monounsaturated divided by the percent that are polyunsaturated. B, Phospholipid fatty acid ratio expressed as percent of fatty acids that are saturated and monounsaturated divided by the percent that are polyunsaturated. Different letters denote statistically significant differences (P<0.05) between values, whereas values with the same letter are not significantly different (P>0.05).

The liver lipid compositions of the mice were also measured (Table 3). Hepatic FC concentrations of the LCAT^–/– LDLr^–/– mice were significantly higher than FC concentrations in the other groups of mice where FC concentrations were comparable. Hepatic CE concentrations of the ACAT2^–/– LDLr^–/– and ACAT2^–/– LCAT^–/– LDLr^–/– mice were lower by 84% and 98%, respectively, when compared with LDLr^–/– controls and CE concentrations were significantly higher in the LCAT^–/– LDLr^–/– mice. Regression analysis demonstrated that a positive correlation existed between hepatic CE concentrations and week 20 TPC (r=0.81) (online Figure 3). A significant difference in liver TG concentration was the 200% to 300% higher value in the ACAT2^–/– LDLr^–/– mice. A significantly lower hepatic TG concentration was found in the triple KO mice. There were only small differences in hepatic PL concentrations among the genotypes with the LCAT deficient strains having slightly higher values. Real-time PCR analysis of hepatic ACAT2 and LCAT mRNA expression showed that absence of hepatic LCAT mRNA did not have an effect on the expression of hepatic ACAT2 mRNA, and vice versa (online Figure 4).

View this table: [in this window] [in a new window]   Table 3. Hepatic Lipid Compositions of KO Mice

In an effort to explain the higher TG levels in livers of ACAT2^–/– LDLr^–/– mice, we looked at hepatic sterol regulatory binding protein-1c (SREBP-1c) mRNA levels by real-time PCR. SREBP-1c is a sterol-sensitive transcription factor important in the synthesis of FA and TG.²⁶ We found no significant differences in SREBP-1c mRNA levels among the four genotypes (data not shown).

To determine whether the rather large differences in lipid levels led to an overall change in the size of the liver, the liver weight as a percentage of total body weight was calculated (data not shown). Significant differences were observed including an increase in the percent liver weight in LCAT^–/– LDLr^–/– mice when compared with LDLr^–/– control mice (6.16±0.24% versus 5.56±0.12%, respectively) and a decrease in ACAT2^–/– LCAT^–/– LDLr^–/– mice compared with controls (4.59±0.11%). No differences in percent liver weight were found in ACAT2^–/– LDLr^–/– mice (5.64±0.11%) when compared with controls.

Two methods were used to quantify the amount of atherosclerosis that developed in the animals. Initially, the percent of aortic surface covered with atherosclerotic plaque was determined (Figure 4A). The aortae of LCAT^–/– LDLr^–/– mice had significantly (3-fold) more of the surface area covered with lesion when compared with those of LDLr^–/– control mice (15.3±1.0% versus 4.9±0.8%), whereas the aortae of both the ACAT2^–/– LDLr^–/– mice (1.1±0.8%) and ACAT2^–/– LCAT^–/– LDLr^–/– mice (0±0%) had significantly less surface area with lesion. The greatest extent of lesion involvement typically occurred within the proximal third of the aorta (including the arch).

View larger version (36K): [in this window] [in a new window]   Figure 4. Measurements of atherosclerosis end points in mice. A, Morphometric analysis of aortas isolated from LDLr–/– (rr, n=10 and n=17 for A and B, respectively), ACAT2–/– LDLr–/– (aarr, n=9 and n=12 for A and B, respectively), LCAT–/– LDLr–/– (llrr n=9 for both panels), and ACAT2–/– LCAT–/– LDLr–/– (aallrr, n=9 for both panels) mice. Values are expressed as percent surface area of aorta covered in lesion. B, GLC analysis of CE extracted from aortas expressed as µg of CE/mg of aortic protein. Different letters denote statistically significant differences (P<0.01) between values, whereas values with the same letter are not significantly different (P>0.01).

Lipids were then extracted from the whole aorta and the aortic CE accumulation was quantified by GLC (Figure 4B). Results were similar to the morphometric analysis, with aortae of LCAT^–/– LDLr^–/– mice having over 200% more CE accumulation (67.5±11.2 versus 23.8±1.9 µg CE/mg aortic protein) than LDLr^–/– mice, whereas ACAT2^–/– LDLr^–/– and ACAT2^–/– LCAT^–/– LDLr^–/– mice had significantly less aortic CE accumulation (2.9±0.8 and 1.4±0.8 µg CE/mg protein, respectively). Across all animals, there was a significant correlation between percent surface area involvement and µg CE/mg protein of r=0.79, a result similar to that found in an earlier study using the same experimental protocols.²⁷ Regression analysis showed that the positive correlation between TPC and aortic CE accumulation (r=0.75) and the positive correlation between hepatic CE concentration and aortic CE concentration (r=0.78) were of a similar magnitude, whereas the correlation between plasma CE and aortic CE concentrations was also statistically significant (r=0.65) (Figure 5).

View larger version (17K): [in this window] [in a new window]   Figure 5. Regression analysis of (A) total plasma cholesterol and aortic cholesteryl ester content, (B) plasma cholesteryl ester and aortic cholesteryl ester content, and (C) hepatic CE content and aortic CE content. Shown are least squares best fit regression lines representing data from all LDLr–/– (), ACAT2–/– LDLr–/– (), LCAT–/– LDLr–/– (), and ACAT2–/– LCAT–/– LDLr–/– () mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study allowed us to determine the importance of LCAT and ACAT2 in both the synthesis of plasma CE and the development of atherosclerosis. We found that deletion of both LCAT and ACAT2 genes led to the complete absence of plasma CE, indicating that there are no compensatory mechanisms, such as ACAT1, to synthesize plasma CE. A striking observation was that in ACAT2^–/– LDLr^–/– mice, a 25% lower plasma CE concentration was associated with an 88% decrease in aortic CE accumulation. This observation suggests that the antiatherosclerotic effects of the loss of ACAT2 extend beyond decreases in plasma cholesterol concentration. Earlier data have suggested that the cholesteryl oleate and cholesteryl palmitate synthesized by ACAT2 are particularly atherogenic^25,28,29 presumably because of the limited ability of macrophages to mobilize cholesterol from these CE.³⁰ When even more of these CE accumulated in plasma lipoproteins, as in the LCAT^–/– LDLr^–/– mice (Figure 3A), the extent of atherosclerosis was even higher (Figure 4). Higher plasma cholesterol concentrations as well as modified CE composition characterized the LCAT^–/– mice and likely contributed to the higher extent of atherosclerosis, as would the decreased ability to transport cholesterol from the periphery to the liver via the hypothesized reverse cholesterol transport pathway.¹⁸

The effects of the gene deletion of ACAT2 or LCAT had contrasting effects on plasma lipoproteins. Deletion of ACAT2 lowered TPC because of a decrease in plasma CEs, and the plasma lipoprotein profile showed a lower VLDL cholesterol and a higher HDL cholesterol (Table 2) as well as a smaller-sized LDL particle. All of these changes are consistent with the decreased atherogenicity observed in these animals. It is interesting to note that in the mouse strains that synthesize plasma CEs (all genotypes but the ACAT2^–/– LCAT^–/– LDLr^–/–), the extent of atherosclerosis generally followed the concentrations of VLDL-C. Because VLDL contain primarily ACAT2-derived CEs, this may be another indication that ACAT2-derived CEs promote atherosclerosis. It also was noteworthy that LDL-C levels in the ACAT2^–/– LDLr^–/– mice were not significantly different than in LDLr^–/– mice even though the LDL particles were smaller in ACAT2^–/– LDLr^–/– mice. Analysis of apoB lipoprotein CEs in ACAT2-deficient mice showed enrichment in polyunsaturated fatty acids. These findings suggest that LCAT is able to compensate for the loss of ACAT2-derived CEs in the LDL fraction although it was only able to partially compensate in the VLDL fraction. The resulting LDL particles are smaller with less cholesteryl ester per particle presumably resulting from the lower CEs in VLDL. It has been hypothesized that the compensatory increase in LCAT activity in ACAT2^–/– mice may also contribute to the increase in HDL cholesterol.¹⁵

Another factor contributing to the reduced atherosclerosis extent in ACAT2-deficient mice may be the replacement of cholesteryl ester with triglyceride in the core of apoB-containing lipoproteins. However, hypertriglyceridemia has also been considered as a risk factor for atherosclerosis, leading to speculation that enrichment of apoB-containing lipoprotein particles with triglyceride could be proatherogenic. In ACAT2 gene deletion experiments in the apoE^–/– mice, triglyceride levels were not elevated, making it difficult to ascertain the effects of elevated plasma triglycerides and triglyceride-rich lipoproteins.¹⁶ However in the LDLr^–/– background of these studies, deletion of ACAT2 led to mild hypertriglyceridemia when compared with controls, yet atherosclerosis decreased dramatically in these mice. These data provide a clear indication that, when compared with plasma CEs, triglyceride is less effective in promoting atherosclerosis development. The elevation of plasma apoB-containing lipoprotein triglyceride in the ACAT2^–/– LDLr^–/– mice may be associated with the increase in body mass, and weight gain may be a possible negative side effect of ACAT2 inhibition. The mechanism resulting in the increase in triglyceride accumulation in the ACAT2^–/– mice is unclear at the present time and needs further study. Further, it is unclear why deletion of LCAT together with the deletion of ACAT led to lower plasma TG levels.

Loss of LCAT led to an increase in TPC because of increases in both FC and CEs (Table 1). Furthermore, the atherogenicity of the lipoprotein profile was greater apparently because of the increased VLDL cholesterol and the complete absence of HDL. The relative TPC concentrations observed in the LCAT^–/– LDLr^–/– mice differ from the results of Furbee et al,¹⁹ who saw no difference in TPC when compared with LDLr^–/– controls, and Lambert et al²⁰ who saw decreases in TPC when compared with controls. The use of trans fatty acid enriched fat in the diet of this study may partly explain the contradictory results. In the LCAT^–/– LDLr^–/– mice, our atherosclerosis results agree with those generated by Furbee et al¹⁹ in that the absence of LCAT led to increased atherosclerosis in comparison to LDLr^–/– control mice. It has been argued that loss of LCAT would be antiatherosclerotic because of the decreases in LDL-C concentrations.⁶ We speculate that the proatherogenic effects of the loss of LCAT, through limiting the ability to maintain the FC gradient between peripheral tissue and HDL necessary for reverse cholesterol transport, may outweigh the antiatherogenic effects of the decrease in LDL. Further, in LCAT deficiency in our study, VLDL cholesterol concentrations were greatly increased with ACAT2-derived cholesteryl esters even though LDL cholesterol concentrations were decreased (Table 2). This shift, together with the cholesteryl ester composition changes and HDL depletion, were sufficient to counterbalance any apparently anti-atherogenic effects of reduced LDL cholesterol concentrations.

Analysis of cholesterol absorption resulted in all three KO animals having significantly decreased cholesterol absorption (20%) when compared with LDLr^–/– controls when they were fed the 0.18% cholesterol, trans fat diet (Figure 2). In ACAT2^–/– LDLr^–/– mice fed a chow diet (0.02% cholesterol), cholesterol absorption was not significantly different from that in wild-type mice but when ACAT2^–/– mice were fed an 1.25% cholesterol diet, cholesterol absorption was 85% less than in wild-type mice, leading the authors to speculate that a compensatory mechanism occurs with increased amounts of cholesterol in the absence of ACAT2.¹⁵ A possible compensatory mechanism is related to results generated by Iqbal et al³¹ in CaCo-2 cells. An apoB-independent secretion pathway apparently involving ABCA1, suggests that HDL may be a cholesterol acceptor in the absorption pathway. In the absence of ACAT2, it is possible that cholesterol that would normally be secreted in chylomicrons is transported to the site within the enterocyte to be effluxed to apoA-I via ABCA1. This pool of cholesterol may be expanded when cholesterol-enriched diets are fed. This also could provide a possible explanation for the decrease of cholesterol absorption in the LCAT^–/– LDLr^–/– mice as the absence of HDL in these mice may inhibit the apoB-independent secretion pathway thereby inhibiting cholesterol absorption. Further investigation is necessary to determine whether the cell culture results will carry over to in vivo models.

Deletion of ACAT2 led to depletion of CE in the liver, whereas deletion of LCAT led to CE enrichment in the liver when compared with controls. Hepatic CE enrichment taken together with the increased VLDL-C in the plasma of the LCAT^–/– LDLr^–/– mice suggests that ACAT2 expression is upregulated in the liver of the LCAT^–/– LDLr^–/– mice. Recently, it has been shown that ACAT2 is upregulated, primarily through posttranscriptional mechanisms, by the presence of increased levels of dietary cholesterol.³² It is not clear to us at this time why the absence of LCAT resulted in increased cholesterol concentrations in the liver of these mice. The positive correlation between hepatic CE content and TPC, as well as between hepatic CE and aortic CE is another indication that hepatic ACAT2 activity contributes importantly in the development of atherosclerosis.

In conclusion, these studies in mouse models suggest that there are only two enzymes that possess the ability to synthesize CE, ACAT2 and LCAT. Loss of the ability to synthesize plasma cholesteryl esters did not cause obvious detrimental effects but did eliminate the appearance of atherosclerotic lesions in the aorta. Our findings support the long-standing hypothesis that accumulation of CE from blood plasma in the intima of arteries is central to the development of atherosclerosis. Our data further support the importance of ACAT2 in the development of atherosclerosis. This enzyme facilitates cholesterol absorption in the intestine and incorporation of atherogenic CEs into apoB-containing plasma lipoproteins, which then appear to accumulate in the aortic intima promoting atherosclerotic lesion development. The fact that the decrease in TPC was disproportionately less than the decrease in atherosclerosis in ACAT2 KO mice suggests that other factors, such as increased HDL-C cholesterol, increased polyunsaturated CE, and smaller LDL particles with fewer CE per particle, contribute important antiatherosclerotic effects in the presence of ACAT2 deficiency.

	Acknowledgments

 
This work was made possible with the support of the National Institutes of Health, including National Heart, Lung, and Blood Institute grants HL-49373, HL-054176, and HL-24736. R.G.L. was supported by National Institutes of Health training grants HL-07668 and HL-07115–28. This work represents a part of that completed by R.G.L. for a PhD degree at Wake Forest University.

	Footnotes

 
Original received July 16, 2004; revision received September 20, 2004; accepted September 30, 2004.

	References

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