© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
LCAT Modulates Atherogenic Plasma Lipoproteins and the Extent of Atherosclerosis Only in the Presence of Normal LDL Receptors in Transgenic Rabbits
From the Molecular Disease Branch, National Heart, Lung, and Blood Institute (M.E.B., R.D.K., S.J.D., W.E., S.S-F., H.B.B., J.M.H.), National Institutes of Health, Bethesda, Md; the Biomedical Engineering Center (E.E.H.), Ohio State University, Columbus; and the Northwest Lipid Research Laboratories (S.M.), University of Washington School of Medicine, Seattle.
Correspondence to Margaret E. Brousseau, JM-USDA/HNRCA at Tufts University, Lipid Metabolism Laboratory, 711 Washington St, Boston, MA 02111. E-mail mbrousseau{at}hnrc.tufts.edu
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Abstract |
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Abstract—Elevated low density lipoprotein cholesterol (LDL-C) and reduced high density lipoprotein cholesterol (HDL-C) concentrations are independent risk factors for coronary heart disease. We have previously demonstrated that overexpression of an enzyme with a well established role in HDL metabolism, lecithin:cholesterol acyltransferase (LCAT), in New Zealand White rabbits not only raises HDL-C concentrations but reduces those of LDL-C as well, ultimately preventing diet-induced atherosclerosis. In the present study, the human LCAT gene (hLCAT) was introduced into LDL receptor (LDLr)–deficient (Watanabe heritable hyperlipidemic) rabbits to (1) investigate the role of the LDLr pathway in the hLCAT-mediated reductions of LDL-C and (2) determine the influence of hLCAT overexpression on atherosclerosis susceptibility in an animal model of familial hypercholesterolemia. Heterozygosity or homozygosity for the LDLr defect was determined by polymerase chain reaction, and 3 groups of hLCAT-transgenic (hLCAT+) rabbits that differed in LDLr status were established: (1) LDLr wild-type (LDLr+/+), (2) LDLr heterozygotes (LDLr+/-), and (3) LDLr homozygotes (LDLr-/-). Data for hLCAT+ rabbits were compared with those of nontransgenic (hLCAT-) rabbits of the same LDLr status. Plasma HDL-C concentrations were significantly elevated in the hLCAT+ animals of each LDLr status. However, LDL-C levels were significantly reduced only in hLCAT+/LDLr+/+ and hLCAT+/LDLr+/- rabbits but not in hLCAT+/LDLr-/- rabbits (405±14 versus 392±31 mg/dL). Metabolic studies revealed that the fractional catabolic rate (FCR, d-1) of LDL apolipoprotein (apo) B-100 was increased in hLCAT+/LDLr+/+ (26±4 versus 5±0) and hLCAT+/LDLr+/- (4±1 versus 1±0) rabbits, whereas the FCR of LDL apoB-100 in both groups of LDLr-/- rabbits was nearly identical (0.16±0.02 versus 0.15±0.02). Consistently, neither aortic lipid concentrations nor the extent of aortic atherosclerosis was significantly different between hLCAT+/LDLr-/- and hLCAT-/LDLr-/- rabbits. Significant correlations were observed between the percent of aortic atherosclerosis and both LDL-C (r=0.985) and LDL apoB-100 FCR (-0.745), as well as between LDL-C and LDL apoB-100 FCR (-0.866). These data are the first to establish that LCAT modulates LDL metabolism via the LDLr pathway, ultimately influencing atherosclerosis susceptibility. Moreover, LCAT’s antiatherogenic effect requires only a single functional LDLr allele, identifying LCAT as an attractive gene therapy candidate for the majority of dyslipoproteinemic patients.
Key Words: familial hypercholesterolemia • metabolism • gene therapy • WHHL rabbits • low density lipoproteins
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Introduction |
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Elevated LDL cholesterol (LDL-C) and reduced HDL cholesterol (HDL-C) concentrations are independent risk factors for coronary heart disease (CHD),1 2 3 creating the potential for synergistic effects on disease pathophysiology. An example of this synergism is observed in patients with the autosomal codominant disease familial hypercholesterolemia (FH). FH heterozygotes and homozygotes not only have LDL-C levels that are elevated as much as 2- and 6-fold,4 respectively, but also have significant reductions in HDL-C. Thus, an ideal therapeutic intervention for FH patients should decrease LDL-C concentrations while simultaneously increasing those of HDL-C. We have previously reported that, when overexpressed in New Zealand White (NZW) rabbits, human lecithin:cholesterol acyltransferase (hLCAT) has precisely these effects, reducing LDL-C and raising HDL-C,5 6 consequently preventing diet-induced atherosclerosis.7
As the major enzyme responsible for the esterification of free
cholesterol (FC) present in plasma lipoproteins,
LCAT,8 9 a 63-kDa glycoprotein, plays a
critical role in the metabolism of HDL and in reverse
cholesterol transport,10 a process that
involves the movement of cholesterol from
peripheral cells to the liver for catabolism. Although the
role of LCAT in HDL metabolism has long been known, our
finding that hLCAT overexpression also reduced LDL-C concentrations in
both chow- and cholesterol-fed NZW rabbits was unexpected
and has led to the generation of several hypotheses as to the
mechanism(s) involved in this regulation. Kinetic studies conducted in
the cholesterol-fed animals indicated that LCAT reduced
LDL-C concentrations by accelerating LDL clearance from the plasma
compartment, rather than by decreasing the synthesis of apolipoprotein
(apo) B–containing lipoproteins.6 In view of the fact
that 
60% to 80% of LDL is cleared via hepatic LDL receptor
(LDLr)–mediated endocytosis in the normal rabbit,11 12 we
hypothesized that the significantly reduced concentrations of LDL
observed in our hLCAT-transgenic NZW rabbits might be due to
upregulation of the LDLr pathway. If correct, LCAT would be an
attractive treatment modality for heterozygous FH patients, whereas it
would likely prove less beneficial for homozygous FH patients who lack
normal LDLrs.
The present study was designed with 2 goals in mind: (1) to establish the involvement of the LDLr pathway in the LCAT-mediated reductions of LDL and (2) to determine the influence of hLCAT overexpression on atherosclerosis susceptibility in an animal model of FH, namely the Watanabe heritable hyperlipidemic (WHHL) rabbit. hLCAT-transgenic rabbits, either heterozygous or homozygous for a 4–amino acid deletion in the ligand-binding domain of the LDLr,13 were generated by selective breeding, and LDL kinetics and aortic atherosclerosis were determined. hLCAT-transgenic and control LDLr+/+ (NZW) rabbits were also studied, thus allowing for assessment of the effects of the presence or absence of hLCAT and LDLr status on LDL metabolism. Our work revealed that hLCAT overexpression neither reduced LDL-C concentrations nor protected against atherogenesis in homozygous LDLr-deficient rabbits, whereas it did reduce LDL-C concentrations in normal and LDLr heterozygous rabbits by accelerating LDL catabolism and was therefore antiatherogenic. These data are the first to establish that LCAT modulates LDL metabolism via the LDLr pathway, ultimately influencing atherosclerosis susceptibility. Moreover, LCAT’s antiatherogenic effect requires only a single functional LDLr allele, identifying LCAT as an attractive gene therapy candidate for the majority of dyslipoproteinemic patients.
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Methods |
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Animals and LDLr Status
The hLCAT gene was introduced into LDLr-deficient rabbits by selective breeding. The generation of transgenic rabbits was approved by the Animal Care and Use Committee of the National Heart, Lung, and Blood Institute, as was the experimental protocol. Rabbits were determined to be transgenic for human LCAT by Southern blot analysis. Six experimental groups were defined by the presence or absence of the hLCAT gene, as well as by LDLr status. For the LDL kinetic studies, each group comprised 3 age-matched males with the exception of the hLCAT-/LDLr+/- group, which consisted of 2 females and 1 male. For the atherosclerosis experiments, 19 LDLr+/- rabbits (14 hLCAT+, 5 hLCAT-) and 16 LDLr-/- rabbits (8 hLCAT+, 8 hLCAT-),
22 months of age, were studied. The data in
Figure 5A
were generated from 19 LDLr+/+ rabbits (9 hLCAT+, 10
hLCAT-) and have been previously reported.7
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Heterozygosity or homozygosity for the LDLr defect in the present
study was established by polymerase chain reaction (PCR) by using
genomic DNA that was isolated from whole blood. The mutation in WHHL
rabbits involves a 12-bp in-frame deletion that removes 4 amino acids
from the fourth ligand-binding repeat of the LDLr. As shown in Figure 1, a 306-bp portion of exon 4 of the
rabbit LDLr gene, including the 12-bp mutant region from 369 to 380
that contains a BglI restriction site, was amplified by PCR.
PCR products were applied to a 20% Tris-borate-EDTA
polyacrylamide gel (Novex) and electrophoresed for 2
hours at 120 V. Gel-purified PCR products were digested with
BglI at 37°C overnight, applied to a 20% Tris-borate-EDTA
polyacrylamide gel, and stained with ethidium bromide.
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Plasma LCAT Activity, Mass, and Lipids
Samples were collected from the central ear artery of each
rabbit at each designated time point and were added to tubes containing
tripotassium EDTA. Plasma was isolated by
centrifugation at 2500 rpm for 30 minutes at 4°C.
Plasma 
-LCAT activity was determined in duplicate by using a
proteoliposome assay, as previously described,14 and LCAT
mass was determined by radioimmunoassay.15 Total
cholesterol (TC) and triglyceride (TG)
concentrations (Sigma Chemical Co) were measured with a Hitachi 911
autoanalyzer (Hitachi USA) with the use of enzymic reagents.
All lipid determinations were performed on plasma from rabbits that had
been fasted overnight. Plasma HDL-C was determined after dextran
sulfate–Mg2+ precipitation of VLDL and
LDL.16
Gel Filtration Chromatography
Two hundred microliters of rabbit plasma was applied to a fast
protein liquid chromatography (FPLC) system consisting
of 2 Superose 6 columns connected in series (Pharmacia Biotech, Inc).
Lipoproteins were eluted at 0.3 mL/min with PBS containing 1
mmol/L EDTA and 0.02% (wt/wt)
NaN3.17 After the initial 10 mL was
eluted, the next 30 mL was collected in 0.5-mL fractions. TC content
and LCAT mass distribution were determined for each fraction as
described above.
LDL Composition
LDL composition was determined subsequent to sequential
ultracentrifugation (d=1.019 to 1.063 g/mL).
TC and TG were analyzed as above, and FC and phospholipids (PL)
(Wako Chemicals) in the LDL fraction were also determined with a
Hitachi 911 autoanalyzer by using enzymic reagents. Cholesteryl
ester (CE) was calculated by subtracting FC from TC. Total protein
(Pro) content was measured with the bicinchoninic acid protein
assay (Pierce Chemical Co). LDL apoB-100 concentrations were determined
by a competitive ELISA assay utilizing a chicken polyclonal antiserum
directed against rabbit LDL apoB-100 (Covance Laboratories, Inc).
In Vivo Metabolic Studies
Narrow-cut LDL (d=1.030 to 1.050 g/mL) for
metabolic studies was isolated from the plasma of fasting
LDLr-deficient rabbits by sequential
ultracentrifugation. We chose this tracer because it
provided us with the best means of isolating a large quantity of LDL
apoB that was not significantly contaminated with other apolipoproteins
and, furthermore, provided for consistency among
experiments. After isolation, the LDL was dialyzed against PBS/EDTA to
remove excess KBr. Agarose gel electrophoresis and FPLC
analysis confirmed that the isolated LDL was pure and not
contaminated with other lipoprotein classes.
The dialyzed LDL was next radiolabeled with 131I (Dupont/NEN) by using a modification of the ICl method previously described.18 19 Immediately before injection, the 131I-labeled LDL preparation was filter-sterilized (0.22-µm Millex-GV filters, Millipore), and a 25-µCi infusate dose was prepared for each rabbit. Infusates were injected into the marginal ear vein of each rabbit, and blood samples were obtained at 5 and 30 minutes and at 1, 3, 6, 9, 24, 32, 48, 72, and 144 hours after injection. LDL (d=1.019 to 1.063 g/mL) was isolated from each time point of each rabbit by sequential ultracentrifugation. The resultant LDL fractions for each rabbit were then subjected to SDS–polyacrylamide gel electrophoresis on 4% to 22.5% gradient gels,20 and LDL apoB-100 bands were excised and analyzed for their radioactive content with a Packard Cobra gamma counter (Packard Instrument Co).
Residence time (RT) was determined from the area under each radioactivity decay curve by using a multiexponential computer curve-fitting program (SAAM31).21 Fractional catabolic rate (FCR) was calculated as the reciprocal of the RT. LDL apoB pool sizes were derived from the formula: [plasma volume (dL)xLDL apoB concentration (mg/dL)]/body weight (kg). Plasma volume was estimated at 3.28% of body weight.22 Production rate (PR) was calculated as the product of FCR and pool size.
Analysis of Aortic Lesions
Rabbits were killed by using intravenous sodium
pentobarbital. Aortas were harvested and sliced longitudinally. One
half was stained with Sudan IV, and the percentage of the surface area
stained was determined by planimetry of the digitized
image.23 The remaining half of the aorta, specifically the
arch and upper thoracic down to the left subclavian artery ostia, was
used for lipid extraction. Each slice of aorta was weighed and
extracted in 20 mL of chloroform/methanol (2:1, vol/vol), according to
the method of Folch and coworkers.24 After the organic
phase was dried under N2, lipid content was
determined gravimetrically, and the lipids were resolubilized in
isopropanol. Pro, lipid, TC, and unesterified cholesterol
contents were determined as previously described.7
Statistical Analysis
Data for hLCAT-transgenic rabbits were assessed for significance
relative to those of nontransgenic littermate control rabbits of the
same LDLr status by using Student’s nonpaired, 2-tailed t
test. Correlation coefficients were determined by the method of
Pearson. In all cases, statistical significance was set at
P<0.05. Data presented in the text, tables, and
figures represent mean±SEM.
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Results |
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Determination of LDLr Status
Heterozygosity or homozygosity for the 12-bp deletion in exon 4 of the rabbit LDLr gene was established by PCR, as described in Methods. Figure 1
shows the gel-purified PCR products of LDLr+/+,
LDLr+/-, and LDLr-/- rabbits with the exception of lane 2, which
shows the undigested PCR product of an LDLr+/+ animal before gel
purification. The additional high-molecular-weight band was present
owing to the lack of gel purification. Subsequent to gel purification
and BglI digestion, PCR products from LDLr+/+ rabbits
generated 2 bands of 212 and 94 bp (lane 3). In the case of LDLr+/-
rabbits, 2 bands at 306 and 294 bp were generated before digestion
(lane 4), whereas 3 product bands were observed in LDLr+/-
animals after incubation with BglI (lane 5) because of the
presence of 1 normal and 1 mutant allele. In contrast, LDLr-/-
rabbits generated only 1 band at 294 bp, both before (lane 6) and after
(lane 7) incubation with BglI.
Plasma LCAT Activity, Lipids, and LDL ApoB-100 Levels
Table 1 summarizes the mean plasma
LCAT activity, lipid, and LDL apoB data for hLCAT-transgenic and
nontransgenic rabbits of each LDLr status. As expected, plasma LCAT
activity was significantly increased in transgenic animals relative to
controls, with at least 3-fold increases in activity observed for each
group. Similarly, plasma HDL-C concentrations were significantly
elevated in hLCAT-transgenic animals, independent of LDLr status.
hLCAT+/LDLr+/+ rabbits had the highest mean plasma LCAT activity and
consequently, the highest levels of HDL-C compared with their
nontransgenic littermate controls. The mean plasma HDL-C concentration
of the hLCAT+/LDLr+/- group was 3 times greater than that of the
hLCAT-/LDLr+/- group, whereas that of the hLCAT+/LDLr-/- group was
5 times greater than that of its control group. With regard to LDL-C
and apoB concentrations, only in the LDLr+/+ and LDLr+/- groups, and
not in the LDLr-/- group, did hLCAT-transgenic rabbits have
significant reductions relative to controls. Unexpectedly, plasma TGs
were reduced by 66% in hLCAT+/LDLr+/- rabbits and by 56% in
hLCAT+/LDLr-/- rabbits compared with those of their respective
control groups. Preliminary studies in these animals have shown that
this reduction is in part due to accelerated VLDL apoB-100 catabolism,
suggesting that hLCAT overexpression may influence both VLDL and
LDLr-mediated pathways. No differences were noted in plasma TGs between
transgenic and nontransgenic LDLr+/+ rabbits.
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Distribution of Cholesterol and hLCAT Mass Among
Lipoproteins and LDL Composition
Figure 2 illustrates the
distribution of TC in the plasma of representative
hLCAT+ and hLCAT- LDLr+/+, LDLr+/-, and LDLr-/- rabbits, as
assessed by gel filtration chromatography. The elution
profile of the radiolabeled rabbit LDL tracer is also provided for
comparative purposes. As discussed above, plasma HDL-C content was
greater in the hLCAT+ animals of each LDLr status. The enrichment of
HDL with CE resulted in an increased particle size in all 3 groups of
transgenic rabbits relative to their controls, with hLCAT+ wild-type
rabbits having the largest HDL size and hLCAT+/LDLr-/- rabbits the
smallest. However, only in the LDLr+/+ and LDLr+/- groups was HDL
rather than LDL the predominant lipoprotein class in the plasma of
hLCAT-transgenic animals. Conversely, apoB-containing lipoproteins were
prevalent in the plasma of LDLr-/- rabbits, independent of the degree
of LCAT expression. The distribution of hLCAT among the lipoprotein
fractions of transgenic rabbits of each LDLr status is shown in Figure 3. hLCAT was principally associated with
HDL1 in LDLr+/+ rabbits, whereas its distribution
was more heterogeneous in transgenic LDLr+/- rabbits,
with hLCAT mass detected in IDL-LDL, HDL1 and HDL
particles. Interestingly, in transgenic LDLr-/- rabbits, the majority
of hLCAT was associated with LDL rather than HDL.
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LDL composition data for hLCAT-transgenic and nontransgenic
rabbits of each LDLr status are provided in Table 2. No differences were noted in LDL
composition between hLCAT+ and hLCAT- LDLr+/+ rabbits, with the Pro,
CE, and TG components accounting for 29%, 26%, and 22% of LDL,
respectively. Similarly, the LDLs of both transgenic LDLr+/- and
LDLr-/- rabbits were not significantly different from those of their
respective controls. In LDLr+/- rabbits, Pro was the principal
component of LDL, accounting for 35% of the mass. Conversely, CE
was the principal constituent of LDL in LDLr-/- rabbits. The relative
increase in CE content along with reduced TG content
represented the greatest differences in LDL composition
between the 3 groups, as distinguished by LDLr status. With regard to
LDL apoprotein content, apoB-100 levels were lower in both
hLCAT-transgenic LDLr+/+ and LDLr+/- rabbits relative to their
respective controls, whereas those of the 2 groups of LDLr-/- rabbits
did not differ. LDL apoE levels were similar in transgenic and
nontransgenic LDLr+/+ and LDLr+/- rabbits, with slightly elevated
levels observed in hLCAT+/LDLr-/- rabbits versus controls (data not
shown).
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Metabolic Parameters of LDL
ApoB-100
The kinetic parameters of LDL apoB-100 are shown in
Table 3. Within the LDLr+/+ and LDLr+/-
groups, the mean LDL apoB pool size was significantly decreased in the
hLCAT-transgenic rabbits relative to their respective controls.
Specifically, hLCAT+/LDLr+/+ rabbits had a mean LDL apoB pool size that
was 72% lower than that of hLCAT-/LDLr+/+ rabbits, whereas
hLCAT+/LDLr+/- rabbits had a 70% reduction in LDL apoB pool size.
LDL apoB pool size (in mg/kg) was not significantly different when LDLr
homozygotes were compared, with values of 82 and 85 for hLCAT+ and
hLCAT- animals, respectively.
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As illustrated in Figures 4A and 4B, the
reductions in LDL apoB pool size observed in both hLCAT+/LDLr+/+ and
hLCAT+/LDLr+/- rabbits were due to enhanced catabolism of LDL
apoB-100 relative to nontransgenic controls. The decline in
131I-LDL apoB-100–associated radioactivity was
very rapid in hLCAT+/LDLr+/+ rabbits, such that only 1% of the initial
dose of 131I-LDL remained 6 hours after injection
relative to >10% in that of the control group. In hLCAT+/LDLr+/-
rabbits, <1% of the initial dose of 131I-LDL
remained 48 hours after injection, whereas >20% remained associated
with the LDL apoB-100 of hLCAT-/LDLr+/- rabbits. The preceding data
translated into a mean LDL apoB-100 FCR (d-1)
of 5.3±0.2 for hLCAT-/LDLr+/+ rabbits, which was 80% lower than that
of 26.2±4.1 for hLCAT+/LDLr+/+ rabbits. Similarly, the LDL apoB-100
FCR of hLCAT-/LDLr+/- rabbits (1.2±0.2) was reduced by 71%
relative to that of hLCAT+/LDLr+/- rabbits (4.1±0.5). In contrast,
Figure 4C shows that the catabolism of LDL apoB-100 in LDLr
homozygotes was independent of LCAT overexpression, with hLCAT+ and
hLCAT- animals having nearly identical radioactivity decay curves and,
thus, LDL apoB-100 FCRs. Also of note was the fact that LCAT
overexpression improved the LDL catabolic defect in LDLr heterozygotes
to a value that was not significantly different from that of
nontransgenic LDLr+/+ rabbits. No significant differences were observed
in LDL apoB PR between the hLCAT+ and hLCAT- rabbits of each LDLr
group.
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Analysis of Aortic Lesions
To further evaluate the role of the LDLr pathway in LCAT’s
ability to reduce LDL-C concentrations and ultimately prevent
atherosclerosis, we assessed spontaneous
atherosclerosis in hLCAT-transgenic and control
LDLr+/- and LDLr-/- rabbits. Aortic lipid concentrations as well as
the percent surface area covered by plaque are provided in Table 4. No significant differences were noted
in either total lipid, FC, or esterified cholesterol
content when transgenic and control LDLr+/- rabbits were compared,
nor was spontaneous atherosclerosis evident in the
aortas of these animals. This was likely due to the fact that, although
the mean LDL-C concentration of hLCAT-/LDLr+/- rabbits was
significantly greater than that of hLCAT+/LDLr+/- rabbits (47 versus
10 mg/dL), this concentration of LDL was not sufficient to promote
spontaneous atherosclerosis in the former group. In
contrast, significant atherosclerosis was present
in both hLCAT- and hLCAT+ LDLr homozygotes, with both groups having
similar aortic cholesterol content and 84±3% of the
aortic surface covered by plaque. This is illustrated in Figure 5, wherein the probability map for aortic
lesion development in LDLr-/- rabbits shows substantial staining,
indicative of severe atherosclerosis, independent of
the degree of hLCAT expression. A comparison of the extent of aortic
lesions in cholesterol-fed hLCAT-transgenic and control
LDLr+/+ rabbits was included in Figure 5A to illustrate the
protective effect of LCAT in the presence of functional LDL receptors.
Only 5% of the aortic surface was covered by plaque in hLCAT+/LDLr+/+
rabbits versus 35% in the hLCAT-/LDLr+/+ group. The data in Figure 5 suggest that LCAT only protects against atherogenesis when
normal LDL receptors are present. Consistent with this,
significant correlations were observed between the percent of aortic
atherosclerosis and LDL-C concentration
(r=0.985, P<0.001) and LDL FCR
(r=-0.745, P<0.035). Moreover, both aortic FC
(r=-0.705) and esterified (r=-0.724)
cholesterol concentrations were significantly
(P<0.05) associated with LDL FCR.
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Relationships Between LCAT Activity, LDL-C, and LDL Metabolic
Parameters
The results of linear regression analyses are
provided in Table 5. LDL-C concentrations
were highly correlated with LDL apoB concentrations. LDL apoB
concentrations were inversely associated with plasma LCAT activity.
Moreover, plasma LCAT activity was significantly correlated with LDL
apoB-100 FCR, but not with LDL apoB PR. This finding, taken together
with the fact that LDL apoB concentrations were correlated with FCR
only, indicates that LCAT-mediated alterations in LDL apoB-100 FCR were
responsible for the observed reductions of LDL in our transgenic
rabbits with functional LDL receptors.
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Discussion |
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We have previously reported that the overexpression of hLCAT in NZW rabbits prevents diet-induced atherosclerosis.7 In addition to having elevated HDL-C levels, the concentrations of proatherogenic apoB-containing lipoproteins were substantially lower in cholesterol-fed, hLCAT-transgenic rabbits relative to nontransgenic littermate controls because of the accelerated catabolism of LDL.6 Although the observed effects on HDL were expected, due to LCAT’s well-established role in HDL metabolism, those on LDL were not. The present study was undertaken to evaluate the role of the LDLr pathway in the LCAT-induced reductions of LDL, as well as to assess LCAT as a potential candidate gene for prevention of atherosclerosis in FH patients.
To investigate the role of the LDLr pathway in the LCAT-mediated reductions of plasma LDL concentrations, we employed an in vivo system, namely the WHHL rabbit, an animal model of FH. hLCAT-transgenic WHHL rabbits, either heterozygous or homozygous for a 4–amino acid deletion in the cysteine-rich ligand-binding domain of the LDLr protein, were generated by selective breeding. hLCAT-transgenic and nontransgenic LDLr+/+ (NZW) rabbits were also studied, allowing for assessment of the interrelationships between the presence or absence of LCAT and LDLr status. We observed that the overexpression of hLCAT resulted in significant reductions of plasma LDL-C and apoB concentrations only in animals with functional LDL receptors. Hence, in both hLCAT+/LDLr+/+ and hLCAT+/LDLr+/- rabbits, HDL rather than LDL was the predominant lipoprotein class present in plasma, whereas apoB-containing lipoproteins were prevalent in the plasma of LDLr homozygotes, independent of the degree of LCAT expression. Metabolic experiments with radiolabeled rabbit LDL revealed that the reductions in LDL observed in the hLCAT+/LDLr+/+ and hLCAT+/LDLr+/- rabbits were due to enhanced catabolism of LDL apoB-100 relative to nontransgenic littermate controls, with no significant differences noted in LDL PRs. Conversely, hLCAT+ and hLCAT- LDLr homozygotes had nearly identical rates of LDL apoB-100 catabolism. Additionally, LCAT overexpression significantly improved the catabolic defect in LDLr heterozygotes such that the LDL apoB-100 FCR of this group was not dramatically different from that of the hLCAT-/LDLr+/+ group. Taken together, the preceding data demonstrate the pivotal role of the LDLr pathway in the LCAT-induced reductions of LDL concentrations.
The precise mechanism(s) responsible for the accelerated catabolism of LDL apoB-100 in hLCAT-transgenic rabbits with functional LDL receptors remains to be defined. However, 1 potential mechanism may be eliminated on the basis of the results of the present study, namely, the role of lipoprotein composition in determining metabolic fate. It is well documented that both the lipid and apolipoprotein content of LDL can influence LDL catabolism in rabbits.25 26 In general, LDL clearance from the plasma compartment is more rapid when LDLs are TG enriched and/or increased in apoE content. When the LDL of hLCAT-transgenic and nontransgenic rabbits of each LDLr status were compared in our study, no significant differences were found in either lipid or apoE content, decreasing the likelihood that LCAT accelerated LDL catabolism by altering the composition of the LDL particles themselves and, ultimately, their interaction with receptor surfaces. Also of note, our LDL composition data in nontransgenic NZW and homozygous WHHL rabbits were not substantially different from those previously reported by Havel and colleagues.27 Because it is difficult to completely resolve LDL and HDL1 particles by FPLC, we cannot entirely rule out the possibility that LDL-associated hLCAT may have facilitated LDL clearance in both LDLr+/+ and LDLr+/- rabbits. However, we hypothesize that rather than altering LDL composition, LCAT overexpression, the highest levels of which were in the liver, may have directly influenced LDLr activity by altering intracellular membrane composition, thus increasing membrane fluidity. In support of this hypothesis, increased membrane fluidity due to enrichment with polyunsaturated fatty acids has been shown to accelerate catabolism of LDL via receptor-dependent pathways in nonhuman primates.28 LCAT may have induced such changes in hepatic membranes by altering the PL content, as it has been shown to alter HDL PL content in an analogous manner.29 30 Alternatively, LCAT may have influenced the LDLr pathway by differentially modulating the synthesis of cholesterolgenic enzymes or by affecting intracellular regulatory pools of cholesterol, in turn mediating the transcription of genes encoding the LDLr as well as enzymes involved in cholesterol biosynthesis.31 Downregulation of the gene encoding the LDLr is precisely the mechanism by which CE transfer protein, another enzyme with a pivotal role in lipoprotein metabolism, has been shown to modulate the concentrations of apoB-containing lipoproteins in transgenic mice.32
Elevated LDL-C and reduced HDL-C concentrations are independent risk factors for CHD.1 2 3 We and others have previously reported that the loss of LDLr function in homozygous FH patients not only increases LDL-C concentrations but decreases those of HDL-C as well,4 33 the latter due in part to a 30% reduction in LCAT activity relative to age- and sex-matched controls.33 Thus, a goal of the present study was to evaluate LCAT as a potential gene therapy candidate for FH patients, who are at increased risk for CHD,4 owing to their unfavorable lipoprotein profile. Neither the LCAT activity (nmol · mL-1 · h-1) values for homozygous FH patients (77±4) nor those of human controls (110±5) were very different from those reported for our nontransgenic LDLr-/- (62±4) and LDLr+/+ (125±6) groups, respectively, allowing us to cautiously extrapolate the clinical relevance of our findings.
In the present study, we assessed spontaneous atherosclerosis in heterozygous and homozygous LDLr-deficient, hLCAT-transgenic, and control rabbits. LCAT did not protect against atherogenesis in homozygous FH rabbits nor, as mentioned earlier, did it alter LDL metabolism. This lack of protection in hLCAT-transgenic LDLr homozygotes, despite a 5-fold elevation in HDL-C levels, was likely due to the overwhelming presence of apoB-containing lipoproteins in the plasma. The LDL-C–to–HDL-C ratio in hLCAT+/LDLr-/- rabbits remained high at 15. This would suggest that simply raising HDL-C levels in FH homozygotes without simultaneously reducing those of LDL-C is not sufficient to prevent atherogenesis. Unfortunately, our conclusions for the LDLr heterozygotes were limited by the lack of spontaneous atherosclerosis in the control group. However, hLCAT overexpression clearly improved the LDL catabolic defect in these animals. This, taken together with the fact that hLCAT increased HDL-C in LDLr+/- rabbits,34 identifies LCAT as an attractive target gene for treatment of heterozygous FH patients, as well as for the majority of dyslipoproteinemic patients who have at least 1 functional LDLr allele.
In conclusion, our data are the first to establish that the overexpression of an enzyme with a well known role in HDL metabolism, LCAT, also modulates LDL metabolism via the LDLr pathway, ultimately influencing atherosclerosis susceptibility. We propose that a significant interaction exists between the LCAT and LDLr genes, located on chromosomes 16 and 19, respectively, which may contribute to the variable phenotypic expression observed in FH patients.35 The results of this study emphasize the important role of epigenetic pathways in our comprehension of complex genetic interrelationships and, most notably, in the formulation of novel therapies.
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Acknowledgments |
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This work is dedicated to the memory of my beloved mentor, Jeff Hoeg. The authors are grateful to Santana Flores, Darlene Allen, Susan Meyn, and Glenda Talley for technical assistance, as well as to Tom Meyers of the Northwest Lipid Research Laboratories, University of Washington School of Medicine, for the hLCAT mass determinations.
Received August 20, 1998; accepted June 30, 1999.
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References |
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