(Received for publication, September 26, 1995; and in revised form, November 22, 1995)
From the
Cholesterol esterification within plasma lipoprotein particles
is catalyzed by lecithin:cholesterol acyltransferase (LCAT). The impact
of the overexpression of this enzyme on plasma concentrations of the
different plasma lipoproteins in an animal model expressing cholesteryl
ester transfer protein was evaluated by generating rabbits expressing
human LCAT. A 6.2-kilobase human genomic DNA construct was injected
into the pronuclei of rabbit embryos. Of the 1002 embryos that were
injected, 3 founder rabbits were characterized that expressed the human
LCAT gene. As in mice and humans, the principal sites of mRNA
expression in these rabbits is in the liver and brain, indicating that
the regulatory elements required for tissue-specific expression among
these species are similar. The -LCAT activity correlated with the
number of copies of LCAT that integrated into the rabbit DNA. Compared
with controls, the high expressor LCAT-transgenic rabbits total and
high density lipoprotein (HDL) cholesterol concentrations were
increased 1.5-2.5-fold with a 3.1-fold increase in the plasma
cholesterol esterification rate. Analysis of the plasma lipoproteins by
fast protein liquid chromatography indicates that these changes
reflected an increased concentration of apolipoprotein E-enriched,
HDL
-sized particles, whereas atherogenic apolipoprotein B
particles disappeared from the plasma. The concentrations of plasma HDL
cholesterol were highly correlated with both human LCAT mass (r = 0.93; p = 0.001) and the log LCAT
activity (r = 0.94; p < 0.001) in the
transgenic rabbits. These results indicate that overexpression of LCAT
in the presence of cholesteryl ester transfer protein leads to both
hyperalpha-lipoproteinemia and reduced concentrations of atherogenic
lipoproteins.
The esterification of cholesterol with fatty acid in the plasma
is mediated by the enzyme lecithin:cholesterol acyl transferase
(LCAT)()(1) . Sperry first demonstrated that a
plasma enzyme could both esterify and de-esterify cholesterol in 1935 (2) . However, the physiologic impact of this enzyme in
vivo awaited the first descriptions of LCAT deficiency by Norum
and Gjone in 1967(3) . By evaluating the plasma LCAT activity
in controls and in patients with deficient plasma LCAT activity,
Glomset (1) suggested that the LCAT reaction, occurring on the
surface of HDL particles, was key to the net transport of cholesterol
from peripheral tissues to the liver(4) . This 63-kDa protein
circulates in the plasma bound to lipoprotein particles and converts
cholesterol and phosphatidylcholine to cholesteryl esters and
lysophosphatidylcholine(5) . A great deal has been learned
about the activity, substrate characteristics, and importance of
cofactors of this enzyme using in vitro assays (6, 7, 8, 9) . The physiologic role
of this enzyme in vivo has been inferred from patients lacking
LCAT activity in their plasma(3) . These individuals have
severely depressed concentrations of HDL cholesterol and accumulate
cholesterol in specific tissues(5) . Therefore the
esterification of cholesterol in the plasma by LCAT is important in
both HDL metabolism and in human cholesterol homeostasis.
The recent
cloning of the human LCAT gene (10) has permitted further
understanding of the LCAT enzyme itself as well as the ability to
explore the physiologic and pathophysiologic consequences of differing
degrees of LCAT expression. The characterization of mutations
underlying LCAT deficiency have structure-function implications for
this enzyme(11, 12) . In addition, we have undertaken
the investigation of the impact of LCAT overexpression on plasma
lipoprotein metabolism. Overexpression of human LCAT in transgenic mice
led to elevations in both total and HDL cholesterol
concentrations(13, 14) . The enhanced esterification
of cholesterol in the plasma of transgenic mice led to the accumulation
of large HDL-sized particles with a high correlation
between LCAT activity and HDL cholesterol concentrations in the plasma.
Therefore reduced plasma LCAT activity leads to
hypoalphalipoproteinemia and increased LCAT activity generates
hyperalphalipoproteinemia in vivo.
After the initial esterification of cholesterol by LCAT on HDL particles, the metabolism of cholesteryl ester within HDL is modulated by cholesteryl ester transfer protein (CETP)(15) . This plasma protein leads to the net transfer of cholesteryl ester from HDL particles to apoB particles in exchange with triglycerides that also reside in the hydrophobic particle core. CETP is present in man as well as in animal models that develop atherosclerosis, and it is absent in those animal species resistant to atherogenesis(15) . Within animal species that develop arterial lesions, plasma CETP activity has been correlated with the severity of atherogenesis. The metabolism of HDL cholesteryl ester in man involves both CETP and LCAT. In order to evaluate the potential protection conferred by the hyperalpalipoproteinemia induced by LCAT overexpression, we have extended our studies to include the rabbit, which both expresses CETP and develops diet-induced atherosclerosis.
Rabbits have long been used in lipoprotein and atherosclerosis
research (16) . The methods for generating transgenic mice has
been extended to rabbits. Using the human LCAT gene that was successful
in overexpressing functional enzyme in transgenic mice, we have
developed transgenic rabbits expressing this human gene in the presence
of substantial plasma CETP activity. The overexpression of human LCAT
leads to hyperalphalipoproteinemia, reflecting an increased
concentration of HDL that directly correlates with the
degree of LCAT expression. In addition, in the presence of CETP, LCAT
overexpression led to a reduction in the concentration of
apolipoprotein B particles. These changes in the plasma lipoproteins
may change not only the plasma cholesteryl ester metabolism but may
alter the susceptibility these animals have for the development of
atherosclerosis.
Identification of kits with integration of human LCAT DNA was assessed by Southern blot analysis. After the weaning at 5-7 weeks of age, DNA was isolated from snippets of tails from the potentially transgenic kits. Southern blots were performed using 10 µg of rabbit DNA that had been digested with PstI. Hybridization with a 446-base pair fragment of the LCAT gene was performed as previously outlined (13) . The number of LCAT copies per genome was quantitated by comparing the intensity of the hybridization signal by both direct Betagen scintigraphy and by scanning laser densitometry (LKB, Sweden). The degree of hybridization of transgenic samples was compared with the intensity of the hybridization signal with human LCAT DNA standards.
The sites of
expression of the transgene were identified by detecting the gene in
the RNA of the tissues. After phenobarbital-induced euthanasia, the RNA
species from LCAT transgenic rabbit spleen, liver, kidney, intestine,
heart, brain, muscle, lung, and adrenal gland were isolated. After
separation by 0.7% formaldehyde-agarose gel electrophoresis and
transfer to a nytran membrane, the sample RNA was probed with the same P-labeled 446-base pair LCAT probe and the 412-base pair DraI-RsaI fragment (17) of the human
-actin cDNA(18) . The nytran membrane was autoradiographed
using Kodak XAR-2 film.
Rabbit apoA-I concentrations in plasma
were determined using a mouse monoclonal anti-rabbit antibody in an
enzyme-linked immunosorbent assay. The cholesterol esterification rate
was determined in duplicate 250-µl plasma samples by evaluating the
incorporation of [C]cholesterol into cholesteryl
ester in the presence of control and transgenic rabbit plasma as
originally outlined by Stokke and Norum(20) . LCAT mass was
determined by radioimmunoassay (21) , and
-LCAT activity
was determined using 10 µl of plasma in a proteoliposome
assay(22) .
Plasma CETP activity was determined by assessing
the transfer of C-labeled HDL
cholesteryl
ester to the d < 1.060 g/ml lipoproteins as described by
Albers and co-workers(23) . This was performed after incubation
for 18 h at 37 °C with or without the addition of 5 µl of
plasma as a source of CETP. The HDL
and the d <
1.060 g/ml lipoproteins were separated by heparin-MnCl
precipitation, and the radioactivity in the supernatant
(HDL
) was then determined. The increase in CETP activity
was linearly increased during 18 h of incubation.
The apoB and apoA-I content of the FPLC
fractions were determined by Western blot analyses as described
previously(25) . For apoE content, the FPLC fractions
corresponding to LDL, HDL, and HDL were subjected to
4-22.5% polyacrylamide gel electrophoresis in the presence of SDS
on 1.5-mm-thick vertical slab gels, as described
previously(26) . Briefly, 200 µl of each nondelipidated
fraction were reduced in 30 µl of SDS sample buffer for 10 min at
80 °C. Gels were run at 45 V for approximately 18 h. The proteins
were then electrophoretically transferred to Immobilon-P (Millipore,
Bedford, MA) at 30 V for 24 h. Membranes were blocked for 1 h with a 3%
solution (w/v) of bovine serum albumin in phosphate-buffered saline (pH
7.4) and incubated overnight with a 1:2,000 dilution of monoclonal
anti-rabbit apoA-I for the immunolocalization of apoA-I. For the
detection of apoE, membranes were incubated for 1 h with a 1:2,000
dilution of polyclonal goat anti-human apoE. Membranes were washed with
phosphate-buffered saline and incubated with the appropriate
biotinylated secondary antibody using Vectastain ABC kits (Vector
Laboratories, Burlingame, CA). Color development was catalyzed in a
reaction mixture consisting of 100 ml of phosphate-buffered saline
supplemented with 60 µl of 30% hydrogen peroxide and 60 mg of
4-chloro-1-naphthol dissolved in 20 ml of chilled methanol.
Embryos were harvested from does that had undergone superovulation. Of the 1704 embryos that were harvested, 59% were in the one cell stage, lacked a mucinous coat, and were injectable. Of these, 5% led to live births and 0.5% integrated the human genomic LCAT DNA. Of the five transgenic founder animals, three expressed the human LCAT gene. The three transgenic lines T-1, T-2, and T-3 all integrated human LCAT DNA that lead to the 690-base pair insert (data not shown). Using known concentrations of construct DNA, the number of copies of LCAT DNA that integrated could be calculated with the copy number for these three lines and ranged from 38 to 1436 copies per genome.
The
large variation number of copies of LCAT that integrated into the three
founder LCAT transgenic rabbits varied reflected marked differences in
the degree of LCAT expression in the plasma in these animals (Table 1). The integrated copy number was correlated with the
plasma LCAT mass (r = 0.98),
-LCAT
activity (r
= 0.99), and HDL cholesterol
concentration (r
= 0.86). In addition, the
human LCAT mass correlated well with both the
-LCAT activity (r
= 0.99) and the HDL cholesterol
concentration (r
= 0.76) in these three
founder animals. Although the high expressing transgenic line
integrated an astonishing number of copies, this high copy number is
most likely not required for high levels of expression, because the
high copy number most likely represents serial tandem repeats that do
not function to the same extent.
The sites of expression of human
LCAT in these rabbits was evaluated by Northern blot analysis (Fig. 1). Liver, heart, brain, and muscle all have detectable
quantities of LCAT mRNA. Compared with the -actin mRNA, which
served as a control for message expression for the tissues analyzed,
the liver was the principal site of expression. The brain also had a
considerable degree of expression. This tissue distribution is similar
to that of LCAT transgenic mice (13) . These rabbits appear to
be neurologically intact and have no aberrant behavior.
Figure 1:
Northern blot analysis of RNA isolated
from tissues of the T-1 founder line. A total of 20 µg of total RNA
was separated by electrophoresis in a 1% agarose gel, transferred to
nytran paper, and hybridized with P-labeled LCAT cDNA and
with
P-labeled
-actin cDNA. The RNA from each of the
indicated tissues isolated from transgenic (T) and
nontransgenic control (C) were electrophoresed in alternating
lanes. The autoradiograph for the hybridization with the LCAT cDNA is
shown in the top panel, and the
-actin cDNA hybridization
is represented in the bottom
panel.
The marked
changes in the plasma LCAT activity were paralleled by changes in the
plasma lipid and lipoprotein concentrations in these animals (Table 2). The greater the degree of LCAT expression, the higher
the concentrations of the plasma total (r =
0.92), free (r
= 0.86), and esterified
cholesterol (r
= 0.94) in littermate
controls at the time of weaning. In addition, the ratio of cholesteryl
ester to free cholesterol was higher with greater LCAT mass (r
= 0.98) and LCAT activity (r
= 0.95). Apolipoprotein A-I and plasma
phospholipid concentrations also increased in parallel with the rises
in the plasma cholesterol levels.
The differences in the
concentrations of these plasma lipid constituents reflect changes in
the plasma lipoproteins induced by LCAT overexpression in these
rabbits. Plasma lipoproteins were characterized by Superose-6 column
chromatography (Fig. 2). Nontransgenic NZW rabbits, shown in the solid lines in Fig. 2, have the plasma total, free, and
esterified cholesterol distributed in particles corresponding to VLDL,
IDL + LDL, and HDL (Fig. 2a). The lowest level of
LCAT expression present in T-3 did not result in a significant change
in the FPLC profile compared with nontransgenic controls (data not
shown). In contrast, the highest expressing transgenic founder T-1 has
virtually no detectable cholesterol in VLDL and IDL + LDL. There
is also a virtual absence of cholesterol in particles the size typical
of HDL. The marked elevation in total, free, and esterified cholesterol
in this rabbit is in large HDL particles. The transgenic
rabbit with an intermediate degree of LCAT expression, T-2, also has a
shift of cholesterol to a larger HDL particle species but is
intermediate to that of the high expressor.
Figure 2:
The elution profile from FPLC separation
of plasma isolated from nontransgenic control (solid lines)
and transgenic founder rabbits T-1 (dotted line) and T-2 (dashed lines).Panel a illustrates the total,
esterified, and unesterified cholesterol concentration in each of the
0.5-ml fractions. Panel b depicts the concentrations of
triglycerides and phospholipids in these same fractions. The
concentration of human LCAT mass was determined in T-1 (dotted
lines) and T-2 (dashed lines) by immunoassay. The buoyant
densities (VLDL, IDL + LDL, HDL, and HDL) of the
particles in the different elution fractions are placed for reference
within each panel.
The consistent changes
in the distribution of cholesterol parallels the particle distribution
of phospholipids and triglycerides (Fig. 2b). The high
expressor transgenic has no detectable amount of either of these
analytes outside of an HDL particle distribution, whereas
the intermediate expressor has a shift in the fraction containing
phospholipids and triglycerides to a larger HDL particle. The
distribution of the human LCAT, determined by immunoassay, is identical
to the phospholipid distribution in the transgenic rabbits. The LCAT in
the highest expressor is principally associated with the HDL
particles. However, some of the LCAT is not particle-associated.
The intermediate level expressor has almost all of the human LCAT
present in the large, phospholipid-rich HDL. Therefore human LCAT
overexpression leads to a redistribution of lipids into large HDL-sized
particles.
These changes in the particle distribution of the plasma
lipid is associated with marked changes in the apolipoproteins present
in these particles. Immunoblot analysis of the FPLC column fractions
was performed for the principal rabbit apolipoproteins, apoA-I and apoB (Fig. 3). The major isoproteins of apoB, apoB-48, and apoB-100
were detected in the fractions containing VLDL and IDL + LDL in
the fasted NZW control (Fig. 3, top panel). In
contrast, apoA-I was detected to some extent in almost all of the
column fractions, with most of the apoA-I detected in HDL. The
apolipoprotein distribution in the FPLC fraction from T-1 (Fig. 3, bottom panel) differs from nontransgenic
control for both apoB and apoA-I. No apoB can be detected from the
immunoblot from T-1. These results extend to the progeny of this
rabbit. The plasma apoB concentration is markedly reduced in rabbits
expressing more than 6-fold the LCAT activity shown in control rabbits.
In addition, the apoA-I particle distribution has shifted to the larger
HDL particles.
Figure 3:
Immunoblot analysis of the FPLC elution
fractions for apoB and apoA-I in nontransgenic control (panel
A) and T-1 founder (panel B). Alternate 0.5-ml fractions
from the FPLC separation of the plasma lipoproteins was subjected to
either 6% (apoB) or 15% (apoA-I) NaDoDSO polyacrylamide gel
electrophoresis. After electrotransfer to Immobilon, the samples were
probed with anti-apoA-I or anti-apoB antibodies and then developed.
Nontransgenic control plasma demonstrated both isoforms of apoB,
apoB-48, and apoB-100 in the large VLDL and IDL-LDL particles and
apoA-I in HDL particles. In contrast, LCAT transgenic rabbit T-1 had no
detectable apoB and had apoA-I that was in the fractions representing
HDL
particles.
The changes in the apoA-I and apoB are
paralleled by changes in plasma apoE (Fig. 4). Using sequential
immunoblots for apoA-I and apoE in FPLC fractions containing LDL,
HDL, and HDL particles, a high expressing LCAT transgenic
from the T-1 line has detectable apoE in both LDL and HDL
.
An LCAT transgenic rabbit with low levels of expression has faintly
detectable amounts of apoE, but it is evident only the HDL
particles. In contrast, no detectable apoE is present in any of
these fractions in nontransgenic control rabbits. These changes
indicate that human LCAT overexpression leads to marked changes in
apoB, apoE, and apoA-I particle metabolism.
Figure 4:
Sequential immunoblot for apoE and apoA-I
in the FPLC fractions corresponding to LDL, HDL, and HDL in
a rabbit. A, high levels of LCAT expression (elution volumes:
25, 26.5, and 28 ml, respectively). B, low level of LCAT
expression (elution volumes: 27, 28.5, and 30 ml, respectively). C, nontransgenic control rabbit (elution volumes: 28, 29.5,
and 31 ml, respectively). The far left lane (Std)
contains human apoE
and rabbit apoA-I
standards.
LCAT-T1 is a male and the LCAT gene was expanded into his F1 generation. Because all of the male progeny of this animal expressed human LCAT at a high level, the principal site of integration is into the Y chromosome. The plasma lipid and lipoprotein concentrations of the male offspring of LCAT-Tg1 are summarized in Table 3and compared with age-matched nontransgenic males on a standard rabbit chow diet. As with the original founder, the total, free, and esterified cholesterol concentrations are significantly increased, whereas the ratio of cholesteryl ester to free cholesterol is unchanged. Triglyceride concentrations were unchanged, and the phospholipid and HDL cholesterol concentrations were significantly increased, just as in the founder rabbit. The cholesterol esterification rate and the cholesteryl ester transfer protein activity were also determined in this F1 generation. These animals had a 3-fold increase in the cholesterol esterification rate. As expected, these rabbits had a substantial degree of CETP activity. Overexpression of LCAT did not alter the CETP activity.
The correlations of the plasma LCAT activity and human LCAT mass were evaluated on the F1 generation of LCAT-Tg1. Human LCAT mass and the log LCAT activity were both highly and significantly correlated with total (r = 0.92, p < 0.001; r = 0.94, p < 0.001), free (r = 0.66, p < 0.05; r = 0.73, p < 0.03), and esterified cholesterol (r = 0.91, p < 0.001; r = 0.91, p < 0.001) concentrations. The HDL cholesterol (r = 0.92, p < 0.001) and apoA-I concentrations (r = 0.62, p < 0.04) were also significantly correlated with the log LCAT activity. The ratio of cholesteryl ester to free cholesterol, phospholipids, and CETP were not correlated with the level of LCAT expression. Of interest, the fasting plasma triglyceride concentrations were inversely correlated with the level of log LCAT activity (r = -0.43; p =0.25), although this inverse correlation did not achieve statistical significance.
The generation of transgenic animals overexpressing LCAT was undertaken in order to address several remaining questions regarding this enzyme's role in lipoprotein metabolism. Is this enzyme rate-limiting in the metabolic pathways of HDL, LDL, and plasma cholesterol? Does overexpression of LCAT lead to a recognizable lipoprotein phenotype? What other gene products interact with LCAT in modulating lipoprotein metabolism? Finally, can alteration of plasma LCAT activity affect reverse cholesterol transport and atherogenesis? The overexpression of the LCAT gene in different animal models for lipoprotein metabolism can address these issues(27) . We first overexpressed human LCAT in the mouse(13) . The degree of LCAT overexpression in the mouse correlated with both the size and concentration of the HDL particles in these animals(13, 14) . There are several reasons for extending these studies in LCAT overexpression in mice to the rabbit: 1) Rabbit VLDL are similar in their chemical composition, apolipoprotein content, and electrophoretic mobility with agarose gel electrophoresis to human VLDL(28) . 2) ApoB is evident in rabbit IDL and LDL closely resembling that seen in man(28) . 3) Like man, the rabbit expresses CETP, which not only permits the transfer of HDL-derived cholesteryl ester to apoB-containing lipoprotein particles, it also is likely to play a role in the diet-induced atherosclerosis that rabbits develop(15) . 4) The atherosclerosis in the rabbit not only resembles that of human arterial disease (29) but also the macrovascular lesions that develop in the rabbit can be detected and quantitated in vivo(30) .
As in the mouse, overexpression of LCAT in the rabbit leads to elevated concentrations of HDL cholesterol ( Table 1and Table 3). This hyperalphalipoproteinemia reflected a dose-response relationship with the degree of human LCAT mass and total LCAT activity in the plasma. These particles are larger than typical HDL particles (Fig. 3a) and are associated with phospholipid, LCAT, and apoA-I (Fig. 3b and Table 2). Because rabbits do not express apoA-II(31) , these apoA-I only particles may be particularly effective in removing free cholesterol from cells(32) . The increased concentration of apoA-I and the increased size of these particles would be expected to lead to a reduced removal of apoA-I-containing particles from the circulation(33, 34) .
In contrast to the mouse, the rabbit has very high levels of CETP activity(35) . The substantial CETP activity (17 ± 1%/5 µl/18 h) in the plasma of transgenic rabbits leads to transfer of nascent cholesteryl ester to apoB-containing lipoprotein particles. This may then affect the metabolism of the apoB particle pathway. Immunoblot analysis indicates that the apoB-48 and apoB-100 present in VLDL, IDL, and LDL in nontransgenic rabbits is not evident in the transgenic rabbits (Fig. 4). This reduced apoB particle concentration could be due to enhanced clearance of these particles from the circulation or a reduced production apoB-containing lipoprotein particles. The removal of apoB from the circulation could be modulated by the presence of CETP. In addition to remodeling the apoB particles, the uptake of these particles could be affected by the delivery of cholesterol to the hepatocyte mediated by CETP. Alternatively, LCAT overexpression within the secretory pathway of the hepatocyte could modify production of apoB. The assembly and secretion of nascent apoB requires cofactors for particle assembly, and alteration of this process could enhance the intracellular degradation of nascent apoB. These results indicate that LCAT overexpression not only leads to hyperalphalipoproteinemia, but it also affects the concentration apoB particles in plasma.
The
endogenous LCAT activity in rabbits using the proteoliposome assay is
about one third that of human plasma(36) , yet it was increased
from 5- to 15-fold in the LCAT transgenic compared with nontransgenic
control rabbits. In addition, the cholesterol esterification rate in
the transgenic rabbits, a measure of endogenous LCAT activity in
plasma, was increased more than 3-fold. Yet the increase was not as
striking as the LCAT activity determined by the -proteoliposome
assay. These observations suggest that the in vitro
-proteoliposome assay might not reflect the changes in the
esterification rate in vivo in the transgenic rabbits.
These findings may have direct physiologic and pathologic relevance
to man. Albers and co-workers demonstrated that in adults aged
20-59 years in the Pacific Northwest Bell Telephone Company study
the plasma activity of LCAT ranged 3-fold, from 2.87-8.56
µg/ml(37) . Subgroup analysis in this study indicated that
women and nonsmokers, groups known to have lower risk for developing
cardiovascular disease, had significantly higher LCAT mass
concentrations than men or smokers, respectively. The transgenic
rabbits in this study expressed human LCAT comparable (LCAT-T-2; 4.2
µg/ml) with that of normal human concentrations to 10-fold the
median value for man (LCAT-Tg1; 54.0 µg/ml). As with the data in
the Pacific Northwest Bell Telephone Company study, the degree of human
LCAT gene expression in rabbits was highly correlated with the plasma
cholesterol concentrations (r = 0.85; p < 0.001).
The combined results of this study establish that the overexpression of human LCAT in the rabbit leads to markedly increased concentrations of large HDL particles containing apoE. In addition, the substantial plasma CETP activity in the rabbit markedly reduces the concentrations of the apoB-containing VLDL and IDL + LDL particles. The dose dependence that we observed indicates that LCAT can be rate-limiting in affecting these particle concentrations in vivo.