Macrophage Lipoprotein Lipase Promotes Foam Cell Formation and Atherosclerosis in Low Density Lipoprotein Receptor-deficient Mice*

The role of macrophage lipoprotein lipase (LPL) expression in atherosclerotic lesion formation was examined in low density lipoprotein receptor (LDLR 2 / 2 ) mice using dietary conditions designed to induce either fatty streak lesions or complex atherosclerotic lesions. First, LDLR 2 / 2 mice chimeric for macrophage LPL expression were created by transplantation of lethally irradiated female LDLR 2 / 2 mice with LPL 2 / 2 ( n 5 12) or LPL 1 / 1 ( n 5 14) fetal liver cells as a source of hematopoietic cells. To induce fatty streak lesions, these mice were fed a Western diet for 8 weeks, resulting in severe hypercholesterolemia. There were no differences in plasma post-heparin LPL activity, serum lipid levels, or lipoprotein distribution between these two groups. The mean lesion area in the proximal aorta in LPL 2 / 2 3 LDLR 2 / 2 mice was significantly reduced by 33% compared with LPL 1 / 1 3 LDLR 2 / 2 mice, and a similar reduction (38%) in lesion area was found by en face analysis of the aortae. To induce complex atherosclerotic lesions, female LDLR 2 / 2 mice were lethally irradiated, transplanted with LPL 2 / 2 ( n 5 14), LPL 1 / 2 ( n 5 Analysis— examine the relationship between serum and the extent of atherosclerosis, linear regression analysis and correlation coefficient using Scientific serum cholesterol levels average serum level while on analysis of correlation between serum cholesterol in

Lipoprotein lipase (LPL) 1 is the rate-limiting enzyme for hydrolysis of lipoprotein triglycerides (1). The majority of LPL synthesis occurs in adipose and muscle tissues, and LPL is then transported to the luminal surface of the vascular endothelium where it is bound to heparan sulfate proteoglycans (2). LPL is also synthesized by macrophages and macrophage-derived foam cells in atherosclerotic lesions (3,4). LPL has been proposed to play a dual role in atherogenesis. The efficient lipolysis of triglyceride-rich lipoproteins, the promotion of rapid clearance of post-prandial lipoproteins, and the generation of material for HDL formation are viewed as antiatherogenic effects of LPL (5). In contrast, local LPL activity in the artery wall has been proposed to promote atherosclerosis (6). Recently, LPL has been proposed to influence atherogenesis by mechanisms that are independent from its catalytic actions on the plasma lipoproteins. An increasing amount of evidence indicates that LPL also functions as a ligand, associating with lipoproteins and promoting their binding to the LDLR-related protein (7,8), LDLR (9,10), VLDL receptor (11), and extracellular proteoglycans (12,13). LPL may promote atherogenesis by increasing the binding and retention of LDL cholesterol by proteoglycans of the subendothelial matrix (14 -16).
Macrophage-derived foam cells are present in all stages of atherosclerosis and are believed to play an important role in both the initiation and progression of atherosclerotic lesions (17). In addition, lesions rich in macrophage-derived foam cells may be more prone to plaque rupture (18). A number of in vitro experiments have suggested that macrophage LPL expression may promote foam cell formation (19 -21). Recently, we have reported that C57BL/6 mice reconstituted with LPL Ϫ/Ϫ macrophages develop significantly less atherosclerosis than control mice wild type for macrophage LPL expression (22). These studies demonstrated for the first time that macrophage LPL expression promotes foam cell formation and atherosclerotic lesion formation in vivo (22). However, C57BL/6 mice clearly have limitations as a model for human atherosclerosis. In response to an atherogenic diet, C57BL/6 mice develop mild hypercholesterolemia with ␤-VLDL accumulation and modest fatty streak lesions located exclusively in the proximal aorta (23). In contrast, LDL receptor-deficient (LDLR Ϫ/Ϫ ) mice have enhanced susceptibility to diet-induced atherosclerosis (24,25). On a high fat diet, LDLR Ϫ/Ϫ mice develop severe hypercholesterolemia with VLDL, IDL, and LDL accumulation and extensive atherosclerotic lesions throughout the aorta (24,25). Fur-thermore, the extent of atherosclerosis in LDLR Ϫ/Ϫ mice can be modulated by varying the duration of the high fat diet, providing the opportunity to study the impact of macrophage LPL expression at different stages of atherosclerotic lesion formation. The effect of LPL-induced lipid changes on atherogenesis has been investigated previously by overexpressing human LPL in LDLR Ϫ/Ϫ mice. LDL receptor-deficient mice overexpressing human LPL were protected from hypercholesterolemia (26) and on a high fat diet, they had reduced (18-fold compared with the control) atherosclerotic lesions (27). The results are consistent with a beneficial effect of human LPL overexpression on the plasma lipoprotein profile. The goal of our current study is to examine the role of macrophage LPL in atherogenesis in LDLR Ϫ/Ϫ mice using dietary conditions designed to induce either fatty streak lesions or complex atherosclerotic lesions.
In the current study, the contribution of macrophage LPL expression to atherosclerotic lesion formation was examined in LDLR Ϫ/Ϫ mice. LDLR Ϫ/Ϫ mice chimeric for macrophage expression of LPL were generated by transplantation of lethally irradiated LDLR Ϫ/Ϫ mice with LPL Ϫ/Ϫ , LPL ϩ/Ϫ , and LPL ϩ/ϩ fetal liver cells (FLC) as a source of hematopoietic cells. The mice were fed the Western diet for 8 or 19 weeks to induce fatty streak lesions or complex atherosclerotic lesions, respectively. Our results demonstrate that macrophage LPL expression promotes atherogenesis during the stage of fatty streak formation. However, the impact of macrophage LPL on the extent of lesion area in the proximal aorta is lost in the setting of complex atherosclerotic lesions.

EXPERIMENTAL PROCEDURES
Animal Procedures-A colony of mice with LPL gene inactivation by homologous recombination (28) is established in our facility. Mice heterozygous for inactivation of the LPL gene were at the 7th or higher backcross into the C57BL/6 background. Recipient LDLR Ϫ/Ϫ mice were originally purchased from Jackson Laboratories (Bar Harbor, ME) and were at the 10th backcross into the C57BL/6 background. All mice were maintained in microisolator cages on a rodent chow diet containing 4.5% fat (PMI no. 5010, St. Louis, MO) and autoclaved acidified (pH 2.8) water. The Western type diet contains 21% milk fat and 0.15% cholesterol (Teklad, Madison, WI). Animal care and experimental procedures were carried out according to the regulations and under the approval of Vanderbilt University's Animal Care Committee.
FLC Collection-FLC were obtained and identified as described previously (22). Briefly, female and male LPL ϩ/Ϫ mice were mated, the pregnant mice were sacrificed, and embryos were isolated on day 14 of gestation. A single cell suspension of FLC in RPMI medium (Life Technologies, Inc.) containing 2% fetal calf serum (fetal calf serum) was prepared. FLC were counted in a hemocytometer and cryopreserved in RPMI containing 10% Me 2 SO and 25% fetal calf serum. To identify the LPL genotype and the sex of the fetuses, a PCR reaction with primer sets specific for mouse LPL gene, Neo insert, and Zfy gene of the Y chromosome was performed as described (22). A Rapidcycler (Idaho Technology, Idaho Falls, OH) was used with the following parameters: 40 s at 94°C for the first cycle, and then 30 cycles of 15 s at 94°C, 35 s at 55°C, and 59 s at 72°C.
FLC Transplantation-Shortly before and for 2 weeks following transplantation, all recipient mice were given 100 mg/liter neomycin and 10 mg/liter polymyxin B sulfate (both from Sigma) in acidified water. FLCs were thawed rapidly, washed in RPMI 1640 containing 2% fetal bovine serum, and counted. Recipient LDLR Ϫ/Ϫ mice were lethally irradiated (9 Gy) from a cesium gamma source, and 4 h later, 5 ϫ 10 6 cells in 300 l of RPMI 1640 medium were injected into the tail vein.
Reverse Transcriptase PCR-To verify genotype changes in LDLR Ϫ/Ϫ mice transplanted with FLC, total RNA was extracted from bone marrow of the mice using an Atlas Pure Total RNA Isolation kit (CLON-TECH). RNA was reverse-transcribed by the Moloney murine leukemia virus reverse transcriptase in the presence of RNase inhibitor and random hexamers (all from Promega, Madison, WI) at 37°C for 60 min. Primers specific for mouse LPL (GTG GCC GAG AGC GAG AAC AT and GCT TTC ACT CGC ATC CTC TC) and ␤-actin (TCA GAA GGA CTC CTA TGT GG and TCT CTT TGA TGT CAC GCA CG) were used to amplify products of 197 and 500 base pairs, respectively. The LPL primers were designed to span an intron of the gene to distinguish genomic DNA contamination resulting in an 1138-base pair PCR product. Amplification was performed in the Rapidcycler using the following parameters: 40 s at 94°C for the first cycle, and then 30 cycles of 15 s at 94°C, 45 s at 55°C, and 1 min 20 s at 72°C.
Serum Cholesterol and Triglyceride Analysis-Mice were fasted for 4 h, and blood samples were collected by retro-orbital venous plexus puncture under metofane anesthesia. Serum was separated by centrifugation, and 1 mM phenylmethylsulfonyl fluoride was added (Sigma). The serum total cholesterol and triglycerides were determined using Sigma kit no. 352 and 339 adapted for microtiter plate assay. HDL cholesterol concentration was measured on an automated ACE analyzer using the Direct HDL Test (no. 10981) from Schiapparelli Biosystems, Inc. (Fairfield, NJ).
Lipoprotein Separation-Serum from mice was subjected to fast performance liquid chromatography analysis using a Superose 6 column from Amersham Pharmacia Biotech on a Waters high pressure liquid chromatography system, model 600 (Milford, MA). A 100-l aliquot of serum was injected onto the column and separated with a buffer containing 0.15 M NaCl, 0.01 M Na 2 HPO 4 , 0.1 mM EDTA, pH 7.5, at a flow rate of 0.5 ml/min. Forty 0.5-ml fractions were collected and tubes 11-40 were analyzed for cholesterol.
LPL Activity Assay-After a 4-h fast, mice were injected with 200 units heparin (Sigma), 30 min later blood was drawn from retro-orbital plexus, and the plasma was separated and frozen. LPL enzyme activity was determined as the salt inhibitable ability of triplicate samples to hydrolyze a radiolabeled triolein emulsion as noted (28).
Quantitation of Arterial Lesions-After 8 or 19 weeks on the Western diet, mice were sacrificed and flushed with saline by injection through the left ventricle. The aorta was dissected from the proximal aorta to the iliac bifurcation, and the aortae were pinned out in an en face preparation as described previously (29). The heart with the proximal aorta was embedded in OCT and snap-frozen in liquid N 2 . Cryosections of 10-micron thickness were cut from the region of the proximal aorta starting from the end of the aortic sinus and for 300 m distally, according to the method of Paigen et al. (30), adapted for computer analysis (31).
Immunocytochemistry-To detect LPL protein and macrophages in the arterial lesions, 5-micron serial cryosections of the proximal aorta were fixed in cold acetone and incubated overnight at 4°C with either a chicken antibody to recombinant human LPL (a gift of Dr. Lawrence Chan, Baylor College, Houston, TX) reacting with mouse LPL or with monoclonal rat antibody to mouse macrophages, MOMA-2 (Accurate Chemical & Scientific Corp., Westbury, NY) (32) as described previously (22).
In Situ Hybridization-A 141-base HindIII-PstI fragment of the mouse LPL cDNA (a gift of Dr. R. Zechner, University of Graz, Austria) (23) was subcloned into the pBlueScript SK (Promega Corp). Antisense and sense riboprobes for LPL were prepared using 35 S-uridine (RNA Transcription kit, Stratagene, La Jolla, CA). The sections were fixed with 4% paraformaldehyde-phosphate-buffered saline and processed as described previously (22).
Statistical Analysis-To examine the relationship between serum cholesterol and the extent of atherosclerosis, linear regression analysis was performed and the correlation coefficient was calculated using the SigmaStat 2.0 program (Jandel Scientific Inc.). Mean serum cholesterol levels representing an average serum cholesterol level for individual mice while on the Western diet were used for analysis of the correlation between serum cholesterol levels and the extent of atherosclerosis in the proximal aorta. The statistical significance of differences in mean aortic lesion areas between the groups were determined using the Student's t test.
A change in macrophage genotype of transplanted mice was verified by reverse transcriptase PCR using total RNA extracted from bone marrow at sacrifice. LPL mRNA expression was detected in bone marrow from cells in LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ but not in LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice (Fig. 1).
Eight weeks after transplantation, the mean total serum cholesterol and triglyceride levels did not differ between the groups on a chow diet containing 4.5% fat in either experiment (Tables I and II). When the mice were fed the Western diet, levels of serum cholesterol or triglyceride did not differ between the groups in either experiment (Tables I and II), except for an elevation in serum triglycerides in the LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice after 12 weeks of Western diet that did not persist (Table II).
Analysis of aortic sections of the proximal aorta after 8 weeks of the Western diet revealed prominent lesions consisting mainly of foam cells of macrophage origin. Immunocytochemistry studies of the distribution of LPL in the aortic lesions demonstrated that the macrophage-derived foam cells expressed LPL in the lesion area of LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice but not in LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice (data not shown). In contrast, the proximal aorta of mice after 19 weeks of the Western diet contained complicated lesions with relatively few macrophages, which were located predominantly on the luminal side of the lesion (Fig. 3A). Immunocytochemistry revealed that lesion cells staining with the anti-mouse macrophage antibody, MOMA-2 (Fig. 3A) also reacted with a chicken anti-human antibody to LPL (Fig. 3C) in LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ and LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ mice but not in LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice (Fig. 3,  B and D). These studies demonstrated that the arterial macrophages in the atherosclerotic lesions were donor-derived.
Examination of the advanced lesions for LPL mRNA using in situ hybridization demonstrated that macrophages, but not endothelial and smooth muscle cells, expressed LPL in lesions of LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice (Fig. 4A). No specific LPL message was detected with the sense control (Fig. 4B). In the lesion area of LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice mRNA LPL expression was detected only in myocardial cells (data not shown).
After 19 weeks of the Western diet, visual inspection of the en face aortae revealed a dramatic difference in the extent of atherosclerotic lesions between the groups, with less atherosclerosis in the LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice (Fig. 6). Surprisingly, quantitative analysis of the extent of atherosclerosis in the proximal aorta revealed no differences in the extent of lesion area between LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ , LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ , and LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice with mean lesion areas (m 2 Ϯ S.E.) of 421,303 Ϯ 25,701, 461,243 Ϯ 47,554, and 372,897 Ϯ 46,663, respectively (Fig. 7A). The ratio of macrophage area/Oil Red O-staining area was 0.16 -0.22, indicating that the lesions were complex and contained few macrophages. In contrast, quantitative analysis of the of the aortic lesion area by the en face approach revealed a dose-dependent effect of LPL expression by macrophages with a stepwise decrease in the mean aortic lesion area of LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ , LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ , and LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice (5.9 Ϯ 0.8%, 3.5 Ϯ 0.5% and 1.8 Ϯ 0.2%, respectively; Fig. 7B). The extent of atherosclerosis is significantly reduced in LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ mice and LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice compared with LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice by the Student's t test (p ϭ 0.014 and p Ͻ 0.0001, respectively). Furthermore, the extent of atherosclerosis in the LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice is also significantly reduced compared with LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ mice (p ϭ 0.004). There was no correlation between the extent of lesion area in the proximal aorta and the aorta en face (n ϭ 26; r ϭ 0.14; p Ͻ 0.50; Fig. 7C). In both the short and long term experiments, there was no correlation between the mean lesion area in the proximal aorta and serum total cholesterol levels (n ϭ 26; r ϭ 0.36; p ϭ 0.11, and n ϭ 41; r ϭ 0.07; p ϭ 0.69, respectively) or HDL cholesterol levels (r ϭ 0.24; p ϭ 0.38 and r ϭ 0.14; p ϭ 0.48, respectively). DISCUSSION In the present study, the role of macrophage LPL expression was examined in LDLR Ϫ/Ϫ mice transplanted with LPL Ϫ/Ϫ or LPL ϩ/ϩ FLC under dietary conditions designed to induce atherosclerotic lesions of varying severity, from macrophage-derived foam cells to more complex atherosclerotic lesions. In the first experiment, LDLR Ϫ/Ϫ mice reconstituted with either LPL Ϫ/Ϫ or LPL ϩ/ϩ macrophages were fed the Western diet for 8 weeks to induce foam cell lesion formation. The mice developed severe hypercholesterolemia because of accumulations of VLDL-IDL-LDL cholesterol (Fig. 2), but levels of serum cholesterol and triglycerides did not differ between the two groups (Table I). Thus, macrophage LPL does not significantly influence plasma lipoprotein metabolism even under conditions of severe hypercholesterolemia in LDLR Ϫ/Ϫ mice. The mean aortic lesion area in the LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice was significantly reduced compared with the LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice using either the en face analysis of aortic lesion area (38% reduction; Fig. 5B) or the Paigen approach to the analysis of lesions in cross-sections of the proximal aorta (33% reduction; Fig. 5A). These results extend our previous findings in C57BL/6 mice (22) by demonstrating that even in conditions of extreme hypercholesterolemia because of the absence of the LDLR, macrophage LPL expression promotes foam cell formation and atherosclerotic lesion development in vivo.
In the second experiment, LDLR Ϫ/Ϫ mice reconstituted with either LPL ϩ/ϩ , LPL ϩ/Ϫ , or LPL Ϫ/Ϫ macrophages were fed the Western diet for 19 weeks to induce complex atherosclerotic lesions. After 19 weeks of the Western diet, a dose-dependent reduction in atherosclerotic lesion area was seen in en face analyses of the aortae of LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ (69%) and LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ (41.5%) mice compared with LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice. Again, these results strongly support a proatherogenic role for macrophage LPL expression in vivo. Surprisingly, the extent of the lesion area in the proximal aorta did not differ between LDLR Ϫ/Ϫ mice reconstituted with LPL ϩ/ϩ , LPL ϩ/Ϫ , or LPL Ϫ/Ϫ macrophages fed the Western diet for 19 weeks. This may be explained by the fact that mice develop atherosclerotic lesions first in the proximal aorta and the lesions progress distally, resulting in more advanced lesions in the proximal than the distal aorta. After 19 weeks on the Western diet, the lesions were extremely complicated in the proximal aorta (Fig.  3). The fact that no correlation was found between the extent of lesion area in the proximal aorta and the en face approach in these mice after 19 weeks on the Western diet (Fig. 7C) is consistent with the proposition that atherosclerotic lesions in the proximal aorta were more advanced and thus out of step with lesions in the rest of the aorta. In support of this hypothesis, we have recently observed that, after 15 weeks on the Western diet, LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ mice already have lesions in the proximal aorta that are complex, whereas lesions in the abdominal aorta are fatty streaks containing only macrophagederived foam cells (data not shown). The ratio of macrophage area/Oil Red O-staining area in the proximal aorta of these mice was 4 times less compared with the ratio in mice fed the Western diet for 8 weeks. In contrast, after 8 weeks on the Western diet the lesions in the proximal aorta consisted almost entirely of macrophage-derived foam cells, and there was a significant correlation in the extent of lesion area measured by the two independent techniques (Fig. 5C). Tangirala et al. (33) have previously reported a significant correlation between the extent of lesions in the entire aorta and in the proximal aorta of LDLR Ϫ/Ϫ mice on a high fat diet. Our results indicate that the correlation between the extent of lesion area as determined by en face analysis and the Paigen approach is lost when lesions in the proximal aorta become complicated.
LPL is the rate-limiting enzyme for the hydrolysis of lipoprotein triglycerides. The majority of LPL is synthesized by the muscle and adipose tissues, and LPL is transported to the vascular endothelium, where it hydrolyzes lipoprotein triglycerides. We have previously reported that levels of serum lipids, lipoproteins, and post-heparin plasma LPL activity do not differ between C57BL/6 mice reconstituted with LPL Ϫ/Ϫ or LPL ϩ/ϩ macrophages (22). These studies demonstrated that macrophage LPL expression does not significantly contribute  to the metabolism of plasma lipoproteins or the pool of LPL attached to the vascular endothelium in vivo. However, it is possible that a contribution of macrophage LPL to lipoprotein metabolism could be detected under conditions where LPL is limiting, such as in LPL ϩ/Ϫ mice or under conditions of severe dyslipidemia. In the current studies, LDLR Ϫ/Ϫ mice transplanted with LPL Ϫ/Ϫ FLC did not differ from the mice reconstituted with LPL ϩ/ϩ or LPL ϩ/Ϫ FLC in plasma LPL activity, plasma lipid levels, or lipoprotein distribution when fed a high fat high cholesterol diet (Tables I and II). These results indicate that macrophage expression of LPL does not contribute signif-icantly to the lipolysis of plasma lipoproteins, even in the setting of severe dyslipidemia.
A number of mechanisms by which macrophage LPL may be proatherogenic have been proposed. Local hydrolysis of lipoproteins in the artery wall with release of free fatty acids may increase the local production of atherogenic remnant lipoproteins (34). Lipolysis mediated by macrophage LPL results in the local release of free fatty acid, which could serve as potential ligands for PPARs. Recent evidence points to a role for PPAR␥ in foam cell formation (35,36). Therefore, we propose that LPL might promote foam cell formation by enriching the FIG. 3. Immunocytochemical detection of macrophages and LPL in the proximal aorta of LPL ؉/؉ 3 LDLR ؊/؊ (A and C) and LPL ؊/؊ 3 LDLR ؊/؊ (B and D) mice after 19 weeks of Western diet. Macrophages are stained with rat monoclonal antibody, MOMA-2, or with a chicken antibody to recombinant human LPL followed by biotinylated goat anti-rat or anti-chicken IgGs. The sections were treated with avidin-biotin complex labeled with alkaline phosphatase, and the enzymatic activity was visualized with Fast Red TR/Naphthol AS-NX substrate. As a negative control, the primary antibody was omitted during the incubation of some sections and resulted in no staining (data not shown). Note LPL expression in control mice is co-localized with macrophages, whereas macrophages in LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice do not stain for LPL (ϫ 40). Note mRNA signal appears as black grains located over macrophage-rich area on bright field (A) and as white dots on the dark field of the same section (C). The sense probe did not show specific hybridization signal (B and D) under the same conditions (ϫ 20). microenvironment with free fatty acids that stimulate PPARs. Noncatalytic functions of LPL proposed to be proatherogenic include: (a) binding of both lipoproteins and cell proteoglycans, causing the retention of lipoproteins and facilitating cell lipid uptake (5, 15); (b) direct effect of LPL as a ligand for the receptors promoting cell uptake of lipoproteins (14,16); and (c) promotion of the oxidation of LDL cholesterol (37). Mild oxida-tion of LDL and VLDL increases their affinity for binding to LPL, compared with native LDL and VLDL or extensively oxidized LDL (38). In vitro studies have demonstrated that macrophage uptake of oxidized VLDL or oxidized VLDL remnants does not require catalytically active LPL (39), whereas macrophage binding and uptake of hypertriglyceridemic VLDL and VLDL remnants requires catalytically active LPL (40).  Fig. 5. Data are represented as the average mean lesion area in cross-sections of the proximal aorta (A), and the percentage of lesion area in the aorta by en face analysis for each mouse and the mean for each group (B). The extent of atherosclerosis is significantly reduced in LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ mice and LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice compared with LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice by the Student's t test (p ϭ 0.014 and p Ͻ 0.0001, respectively). The extent of atherosclerosis in the LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice is also significantly reduced compared with LPLϮ 3 LDLR Ϫ/Ϫ mice by the Student's t test (p ϭ 0.004). C shows the lack of correlation between the extent of atherosclerosis in the aortic tree by en face analysis and in cross-sections of the proximal aorta. Individual values shown are LDLR Ϫ/Ϫ mice transplanted with LPL ϩ/ϩ (closed circles), LPL ϩ/Ϫ (gray circles), and LPL Ϫ/Ϫ (open circles) FLC.
If catalytically active macrophage LPL is required for the ability of LPL to promote lipid accumulation by the macrophage, the amount of LPL produced by LPL ϩ/Ϫ macrophages might be adequate to serve this role. In contrast, a gene dosage effect might be more likely to be seen if bridging or other noncatalytic functions of LPL were the dominant mechanism. In the mice fed the Western diet for 19 weeks, the aortic lesion area by en face analysis was significantly different between the LPL ϩ/ϩ 3 LDLR Ϫ/Ϫ mice and the LPL ϩ/Ϫ 3 LDLR Ϫ/Ϫ mice suggesting that noncatalytic functions of LPL may be important in this model. Contributions of the catalytic function and the noncatalytic functions of LPL are not mutually exclusive and may both contribute to foam cell formation.
In summary, LDLR Ϫ/Ϫ mice chimeric for macrophage LPL gene expression were created by transplanting lethally irradiated female LDLR Ϫ/Ϫ mice with fetal liver cells from day 14 LPL Ϫ/Ϫ , LPL ϩ/Ϫ , or LPL ϩ/ϩ fetuses. When challenged with an atherogenic diet for 8 or 19 weeks, LDLR Ϫ/Ϫ mice reconstituted with LPL Ϫ/Ϫ macrophages developed significantly less atherosclerosis by en face analysis of the pinned out aortae than LDLR Ϫ/Ϫ mice reconstituted with LPL ϩ/Ϫ or LPL ϩ/ϩ macrophages, in the absence of significant differences in serum lipids or lipoprotein profiles. Analysis of the extent of atherosclerosis by analysis of cross-sections of the proximal aorta revealed a similar decrease in atherosclerosis in the LPL Ϫ/Ϫ 3 LDLR Ϫ/Ϫ mice after 8 weeks on the Western diet, but the difference in the proximal aorta was not present after 19 weeks on the diet when lesions were extremely complex. Therefore, we conclude that, under atherogenic conditions, macrophage LPL expression promotes foam cell formation and atherosclerosis in vivo, but the impact of macrophage LPL on the extent of atherosclerosis may be lost in complex atherosclerotic lesions.