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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-08-2606.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Medicine, Pharmacology, and
Pathology, Baylor College of Medicine, Houston, TX; VA Medical Center,
Houston, TX; and Mayo Clinic, Rochester, MN.
Although hyperhomocysteinemia is an independent risk factor for
cardiovascular disease, a direct role for homocysteine (Hcy) in this
disease remains to be shown. Whereas diet-induced hyperhomocysteinemia promotes atherosclerosis in animal models, the effects of Hcy on
atherogenesis in the absence of dietary perturbations is not known. We
have generated double knock-out mice with targeted deletions of the
genes for apolipoprotein E (apoE) and cystathionine Despite advances in our understanding of the causes
of cardiovascular disease (CVD), the established risk factors do
not fully account for its occurrence. Numerous clinical studies
have shown that homocysteine (Hcy) is a significant and independent CVD
risk factor. It is not known whether Hcy is a causative agent or only a marker.
Several dietary animal models of hyperhomocysteinemia have been used to
study Hcy-mediated vascular pathogenesis. Diet-induced hyperhomocysteinemia is associated with vascular dysfunction in the
monkey, probably due to the inhibition of nitric oxide (NO) synthesis,1 linked with vascular structural damage in the
minipig because of elastic lamina fragmentation,2
correlated with increased postinjury intimal hyperplasia,3
and impaired endothelial function and leukocyte-endothelium interaction
in the rat.4
A genetic hyperhomocysteinemia model with the gene deletion of
cystathionine Several genetic models of atherosclerosis have been established and
characterized.10 Mice with a targeted disruption of the
apolipoprotein E (apoE) gene, which mediates the removal of plasma
lipoproteins via the low density lipoprotein (LDL) receptors and other
receptors, are severely hypercholesterolemic and develop spontaneous
aortic atherosclerotic lesions.11,12 These mice develop
lesions at an early age if fed an atherogenic diet. Diet-induced hyperhomocysteinemia in apoE In this study, we created double knock-out (KO) mice with targeted
deletions of the apoE and CBS genes. This animal is a good model of
human hypercholesterolemia and hyperhomocysteinemia and is susceptible
to atherosclerosis. We used this model to determine the effect of Hcy
on atherosclerosis and lipid metabolism in animals fed a regular diet,
an atherogenic high-fat (HF) diet, or an HF plus hyperhomocysteinemic
high-methionine (HF+HM) diet.
Gene-targeted mice and diet
Hcy, nonesterified fatty acid (NEFA), lipid, and lipoprotein
distribution analysis
Analyses of atherosclerotic lesions Mouse aortas were removed 2 mm from the heart and excised from the aortic arch to just beyond the renal artery, cut longitudinally, pinned onto a silicon dish, and stained with saturated Sudan IV in propylene glycol.17 Total area, atherosclerotic lesion area of the aortic arch (2 mm from the heart extending to the left subclavian artery), and lesion area of the whole aorta were measured using the Image Plus program (Media Cybernetics, Sliver Spring, MD).Vessel wall lipid analysis and thin-layer chromatography (TLC) After lesion analysis, equal length of aorta, as measured 2 mm from the heart to the branching point of the renal artery, was sonicated in 0.3 mL of 0.1 M Tris (tris(hydroxymethyl)aminomethane)-HCl, pH 7.4. Lipids were extracted with 0.9 mL Folch reagent (chloroform-methanol [2:1, vol/vol]) 3 times, dried using nitrogen (N2) gas, and dissolved in chloroform. Lipid extracts were separated by TLC in hexane-diethyl ether-acetate (75:35:1, vol/vol/vol) and visualized by exposure to iodine vapor. Spots corresponding to cholesteryl ester (CE), TG, NEFA, free cholesterol (FC), and phospholipid were identified by comparing the positions with those of standards.Mouse peritoneal macrophage culture and intracellular cholesterol esterification Mouse peritoneal macrophages were isolated from mice at 25 to 30 weeks of age, plated on 24-well plates (about 4 × 105 cells per well) in Dulbecco modified Eagle medium (DMEM) containing 10% calf serum (CS) to 90% confluence, cultured with 2 µCi/mL (0.074 MBq) [3H]FC in a [3H]FC-M cyclodextrin (MCD, a cholesterol carrier)
mixture (molar ratio 1:1) containing 1 µg/mL of inhibitor of
acyl coenzyme A-cholesterol acyltransferase (ACAT) (S-58035, gift from
Novartis) in dimethyl sulfoxide (DMSO) (0.1% DMSO final
concentration), and incubated for 24 hours.
[3H]FC-MCD mixture was prepared as
described.18 Cellular lipids were extracted with
hexane/methanol (3:1, vol/vol) and transferred to a dry silica column.
CE or FC was eluted with hexane-diethyl ether (6:1, vol/vol) or pure
ether and analyzed by liquid scintillation counting. Cellular protein
was extracted with 0.2 N NaOH/0.2% sodium dodecyl sulfate
(SDS) and assayed for protein concentration. Cholesterol esterification
was analyzed as [3H]CE counts versus
[3H]CE plus [3H]FC counts per milligram
of protein.
ACAT and triglyceride synthase (TGS) activities in mouse macrophages Mouse peritoneal macrophages were cultured in DMEM with 10% lipoprotein-free serum (LPFS) on 24-well plates (about 4 × 105 cells per well) for 12 hours, changed to normal medium containing 10% CS, and 100 µL [3H]oleate-bovine serum albumin (BSA) per milliliter (final 5 µCi/mL [0.185 MBq], 1 mM oleic acid [OA]) at 37°C for 24 hours. Cellular lipids were extracted with hexane-isopropanol (3:2, vol/vol), counted for the incorporation of [3H]oleate, and analyzed by TLC as described above. The activity of ACAT or TGS was assayed by determining the incorporation of radiolabeled oleic acid into CE and TG. Spots of CE and TG were scraped off for radioactivity counting and normalized for protein. Cellular protein was extracted with 0.2 N NaOH/0.2% SDS and assayed for protein. Relative intracellular enzymatic activity is expressed as percentage of [3H]CE or [3H]TG counts versus total lipid counts per milligram of protein.LDL isolation, acetylation, and radioiodination LDL (density = 1.0919 to 1.063) was isolated by sequential ultracentrifugation of EDTA (ethylenediaminetetraacetic acid)-anticoagulated plasma obtained from healthy normolipidemic volunteers. LDL was dialyzed against saline containing 1 mM EDTA (pH 7.4). Acetylation of LDL was carried out by chemical modification of LDL with acetic anhydride.19 The extent of modification was confirmed by changes in relative electrophoretic mobility on 0.75% agarose gels. LDL was radioiodinated with [125I] according to the iodine monochloride method as described.20 Radioiodinated LDL was assayed for protein, stored at 4°C under N2 in the dark, and used within 3 days.LDL uptake Mouse peritoneal macrophages were cultured in DMEM with 10% LPFS on 24-well plates (about 4 × 105 cells per well) for 12 hours and changed to normal medium containing 10% CS with [125I]acetylated-LDL (Ac-LDL) or [125I]native-LDL (20 µg/mL) for 2 hours. Cellular protein was extracted with 0.2 N NaOH/0.2% SDS, counted for incorporation of [125I]LDL, and assayed for protein and protein-associated [125I] radioactivity.21 LDL uptake activity was expressed as [125I] counts per milligram of protein. The uptake activity of [125I]Ac-LDL in macrophages from CBS /+/ApoE/ mice under HF diet was set as
the control value.
Statistics Statistical comparisons were performed with a Student t test using SigmaStat 2.03 (Chicago, IL).
CBS/apoE double KO mice CBS/apoE double KO mice were produced by crossbreeding CBS/+ females with apoE/ males. The
general health and body weight of
CBS+/+/apoE/ and
CBS/+/apoE/ were not different compared
with normal mice. CBS//apoE/ had a high
incidence of death during the first 3 postnatal weeks. About 5% of
CBS//apoE/ survived to 15 weeks of age,
about 2% to 6 months.
CBS/apoE double KO mice are hyperhomocysteinemic and hypercholesterolemic CBS gene deficiency, on an apoE KO background, resulted in an about 2-fold increase in plasma Hcy levels in CBS heterozygote mice (CBS/+/apoE/) compared with CBS wild-type
animals (CBS+/+/apoE/). Severe
hyperhomocysteinemia was found in CBS homozygotes
(CBS//apoE/) (Table
1). The ratio of plasma Hcy levels
was 1:2.1:54 (CBS+/+/apoE/ to
CBS/+/apoE/ to
CBS//apoE/), which is greater
than that in CBS single KO mice (1:2:40).5 The double KO
mice had significantly increased plasma TC levels, which were similar
to those in apoE single KO mice.12 Hyperhomocysteinemia caused by CBS gene deletion did not change the levels of plasma NEFA
but was associated with significantly increased plasma TC levels and
decreased plasma TG levels in the absence of dietary manipulation.
Plasma very low density lipoprotein (VLDL) and LDL cholesterol, as well
as high density lipoprotein (HDL) and LDL TG, were not markedly changed
in the double KO mice (Table 2). However,
plasma VLDL TG levels were significantly decreased. Although it did not
reach statistical significance, the HDL cholesterol levels exhibited a
trend to decreases in CBS homozygous mice.
Dietary hyperhomocysteinemia increases the concentration of plasma TC but not fatty acid (FA) and TG An HF diet doubled Hcy levels in both CBS+/+/apoE/ and
CBS/+/apoE/. This HF diet also increased
plasma NEFA by 2-fold and TC levels about 3-fold, with no significant
differences between CBS+/+/apoE/ and
CBS/+/apoE/ mice (Table 1). An HF+HM diet
resulted in severe hyperhomocysteinemia in
CBS/+/apoE/ mice, which resembles that of
CBS//apoE/. This severe
hyperhomocysteinemia moderately increased the concentration of plasma
TC but not NEFA and TG.
Hyperhomocysteinemia accelerates aortic lesion in CBS/apoE double KO mice At 15 weeks, atherosclerotic lesions were apparent in apoE/ mice at the branch points of the aortic arch and
at all the ostia of the intercostal arteries (Figure
1A). Hyperhomocysteinemic apoE/ mice had a slightly larger lesion area in the
aortic arch, but this was not statistically significant at this time
point (Figures 1A and 2A). At 6 months, lesions were enhanced in the
aortic arch and significantly increased with CBS gene deletion in a
dose-dependent manner. At 1 year of age, advanced lesions were observed
in all apoE/ mice in the aortic arch; however, lesions
were significantly increased by the coexistence of
hyperhomocysteinemia. Advanced lesions in the aortic arch were also
observed at 5 months of age in
CBS/+/apoE/ mice that had been fed with
HF or HF+HM diet for 3 months (Figure 1B). These lesions were
comparable to those of CBS/+/apoE/ at 1 year of age on regular diet (Figure 1A), readily visible upon exposure
of the aortic arch (Figure 1C). Similar to that seen in
hyperhomocysteinemic mice on a regular diet, dietary
hyperhomocysteinemic mice had a markedly increased lesion area in the
aortic arch and in the whole aorta (Figure
2A-B). Interestingly, extensive lesions were found in the descending aorta in dietary mice, whereas lesions were found mostly in the aortic arch in regular diet-fed mice. In
hyperhomocysteinemic mice, the increases of plasma Hcy levels were
significantly correlated with increases in atherosclerotic lesion area
in the aortic arch (Figure 2C). These results indicate that genetically
induced mild hyperhomocysteinemia enhances atherogenesis in old mice,
whereas both genetic and dietary severe hyperhomocysteinemia significantly increases lesion formation in young mice. Interestingly, there was a trend of increasing lesion size in CBC heterozygous male
compared with female mice fed an HF or HF+HM diet. However, this did
not achieve statistical significance.
Hyperhomocysteinemia increases accumulation of CE and TG in the vessel wall in CBS/ApoE double KO mice We next analyzed lipid composition in the vessel wall. Hyperhomocysteinemia increased vascular CE content in 6-month-old and 1-year-old double KO mice, and this correlated with the dose of CBS gene deletion (Figure 3). TG was found in early lesions at 15 weeks in CBS/+/apoE/
and CBS//apoE/ mice but not in
CBS+/+/apoE/ mice, and this was slightly
increased with aging. As mice aged to 1 year, TG content in
CBS+/+/apoE/ matched those in
CBS/+/apoE/ mice. FC content increased
with aging but independent of hyperhomocysteinemia. Dietary
hyperhomocysteinemia increased lesion CE and FC content without
affecting lesion TG content. These results suggest that mild, moderate,
and severe hyperhomocysteinemia increases CE and TG content in the
vessel wall in an age- and dose-dependent fashion with or without
dietary manipulation.
Hyperhomocysteinemia does not affect [3H]cholesterol esterification in mouse peritoneal macrophages We speculated 2 potential mechanisms that might be responsible for the accumulation of CE and TG in the vessel wall of the double KO mice. One involves alteration in cellular lipid metabolism, while the other relates to increases in cellular LDL uptake. Because enhanced intracellular cholesterol esterification has been considered a major feature of atherosclerosis, we examined the cellular conversion of [3H]FC into [3H]CE in mouse peritoneal macrophages. We found that the relative size of cellular [3H]FC and [3H]CE pools were consistent between CBS+/+/apoE/ and
CBS/+/apoE/ in macrophages incubated with
[3H]FC for 24 hours (Figure
4A), indicating that hyperhomocysteinemia did not change macrophage intracellular cholesterol esterification.
Hyperhomocysteinemia does not affect TG or CE synthesis To better understand the effect of hyperhomocysteinemia on the cellular metabolism of CE and TG, we examined enzymatic activities of ACAT and TGS, which are the major enzymes catalyzing CE and TG metabolism in mouse macrophages. Intracellular activities of ACAT and TGS were not different in the double KO mice (Figure 4B). Similar results were observed in enzymatic assays using microsomes isolated from mouse aorta or peritoneal microphages (data not shown). Taken together, these data suggest that hyperhomocysteinemia does not affect CE and TG intracellular metabolism in the vessels and in peritoneal macrophages from the double KO mice.Hyperhomocysteinemia increases the uptake of Ac-LDL in peritoneal macrophages The second potential mechanism by which CE accumulates in the lesions of the double KO mice is via enhanced LDL uptake. To test this possibility, we incubated mouse macrophages with [125I]Ac-LDL or [125I]native-LDL (20 µg/mL) for 2 hours and then measured protein-associated [125I] radioactivity. As shown in Figure 5, native-LDL uptake was somewhat decreased in macrophages from hyperhomocysteinemic mice. In sharp contrast, hyperhomocysteinemia resulting from CBS gene deletion significantly increased Ac-LDL uptake. A similar pattern was observed in macrophages from dietary hyperhomocysteinemic mice. These data indicate that the uptake of Ac-LDL by macrophages from genetically severe hyperhomocysteinemic mice and by genetically moderate hyperhomocysteinemic mice is increased relative to those from control mice.
In the present study, we found accelerated aortic atherosclerosis
in CBS/apoE double KO mice, a genetic model of hyperhomocysteinemia. Increased lesion formation was observed in a genetically determined mild form of hyperhomocysteinemia in old mice
(CBS Hyperhomocysteinemia is an independent CVD risk factor and does not
correlate with most traditional risk factors, such as hyperlipidemia.
Our identification of increased plasma TC and decreased HDL cholesterol
in genetic hyperhomocysteinemic mice without dietary manipulation
suggests a possible link between hyperhomocysteinemia and altered
hepatic lipid metabolism. This finding is partially consistent with the
report of Werstuck et al9 showing that Hcy increases
cholesterol and TG content of HepG2 cells and that diet-induced
hyperhomocysteinemia increases the accumulation and synthesis of
hepatic cholesterol and TG in mice. The increased lesion formation in
CBS Notably, an HF diet not only elevated NEFA and TC concentrations but
also doubled Hcy levels in both CBS wild-type and heterozygous mice.
However, this may be related, in part, to the higher content of
methionine and lower content of choline in the HF diet. A prior study
using a high-methionine plus low-folate diet in CBS We observed that both genetic and dietary hyperhomocysteinemia
increased aortic lesion formation and neutral lipid (CE and TG) content
in the lesions of apoE Several lines of evidence suggest that atherogenic lipids in lesions
are derived from circulating lipoproteins, particularly LDL. Models for
the mechanism of atherogenic lipid accumulation in vascular lesions
emphasize increased LDL uptake by macrophages into the vessel wall or
increased cholesterol esterification catalyzed by ACAT. TG synthesis
involves several enzymes, which are collectively identified as TGS in
this study. Because there were no significant changes in microsomal and
cellular enzymatic activities of ACAT and TGS, we conclude that altered
cellular lipid metabolism is not responsible for the increased lipid
accumulation in the lesions of hyperhomocysteinemic
apoE In vitro studies have established that LDL can be modified by oxidation, acetylation, glycation, methylation, and other conditions.24,25 During Hcy autooxidation, liberated ROS could initiate lipid peroxidation and lead to impaired endothelial function and the formation of atherogenic LDL.26 Although Hcy and other thiols induce LDL peroxidation in vitro,27,28 no difference in the extent of oxidation of LDL has been found in patients with moderate and severe hyperhomocysteinemia in case-control studies.29,30 It has been proposed that hypomethylation is a specific biochemical mechanism by which Hcy induces vascular injury.31 Hcy can utilize adenosine to form S-adenosylhomocysteine (SAH), a potent inhibitor of cellular methylation. Elevated Hcy levels in patients are linked to increased SAH and impaired erythrocyte membrane protein methylation.32 CBS-deficient mice have increased SAH levels and decreased DNA methylation.8 Hcy arrests endothelial cell (EC) growth and increases cellular SAH in a cell type-specific way.33,34 It is relevant that methylation of LDL abolished its recognition by LDL receptors,35 retarded the degradation of aggregated LDL by macrophages,36 and decreased CE formation in macrophages.37 It is possible that hyperhomocysteinemia may inhibit lipid or protein methylation in LDL, which may result in increased endocytosis of LDL-derived CE in the lesions. In addition, we considered that enhanced uptake of modified LDL may account for the increase in lesion lipid content. Modified LDL stimulates the secretion of cytokines and growth factors from vascular cells and, in contrast to native LDL, is avidly taken up by macrophages in a process that is mediated by interaction with a family of scavenger receptors (SRs).38 Modified LDL binds to SRs (class A and B). SR-A and SR-B are detected in macrophage-rich areas within atherosclerotic lesions of apoE KO mice39 and in human atherosclerotic lesions.40 SR-A is proatherogenic under hyperlipidemic conditions, and both apoE and LDL receptor-deficient mice have reduced atherosclerosis in the absence of SR-A. The interaction of SR-A with ligands induces cellular signaling leading to gene transcription and cytokine release. SR-B1 binds to HDL with high affinity.41 The expression of SR-B family members (SR-B1 and CD-36) is inducible. Unlike LDL receptors (LDLRs), macrophage SRs are not regulated by the cellular cholesterol content; hence, macrophage uptake of modified LDL can contribute to the cellular accumulation of CE and eventually to increased atherosclerosis. Whereas uptake of native LDL by macrophages from hyperhomocysteinemic mice was decreased, uptake of Ac-LDL was higher in the hyperhomocysteinemic mice than in control mice. Thus, the enhanced uptake of a modified LDL by macrophages from the hyperhomocysteinemic mice could account for the observed increase in lesion severity in these mice. In summary, these data support the concept that Hcy causes atherosclerosis and is not merely associated with the disease. Our findings support a model in which hyperhomocysteinemia promotes atherosclerosis by altering hepatic lipid metabolism and increasing the uptake of modified LDL in macrophages, leading to the accumulation of CE and TG in the vessel wall. Results from lipid analyses and the LDL uptake assay suggest that hyperhomocysteinemia increases plasma cholesterol, decreases HDL cholesterol, and increases the number and/or activity of receptors for modified LDL. Additional studies are now underway to closely examine cholesterol and HDL metabolisms and SR regulation by hyperhomocysteinemia in the hyperhomocysteinemic mice. These studies should yield insights into the mechanistic link between hyperhomocysteinemia and atherosclerosis.
We thank Dr Larry Chan for important suggestions and critical review of this manuscript, Drs Mark Entman and David Via for helpful discussions, and Novartis for the ACAT inhibitor S-58035.
Submitted August 26, 2002; accepted December 17, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/ blood-2002-08-2606.
Supported by National Institutes of Health grants HL-67033 (H.W.), HL-62467 and HL-59976 (A.I.S.), HL-56865 and HL-30914 (H.J.P.), and HL-59976 (W.D.); American Heart Association Texas Affiliate grant 0160041y (H.W.); American Heart Association grant 0140067N (W.D.); and American Health Assistance Foundation grant H2001-010 (H.W.). W.D. is an Established Investigator of the American Heart Association. H.W. is an awardee of the Junior Faculty Scholar Award from the American Society of Hematology.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hong Wang, VA Medical Center, Baylor College of Medicine, 2002 Holcombe Blvd 109-129, Houston, TX 77030; e-mail: hongw{at}bcm.tmc.edu.
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-synthase and apolipoprotein E double knock-out mice with and
without dietary perturbation
Top
Abstract
Introduction
P value versus
CBS
P value
versus CBS+/+/ApoE
