(Received for publication, January 2, 1997)
From the Molecular Disease Branch and the § Laboratory of Animal Medicine and Surgery, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1666
We have established a mouse model for human LCAT
deficiency by performing targeted disruption of the LCAT gene in mouse
embryonic stem cells. Homozygous LCAT-deficient mice were healthy at
birth and fertile. Compared with age-matched wild-type littermates, the
LCAT activity in heterozygous and homozygous knockout mice was reduced
by 30 and 99%, respectively. LCAT deficiency resulted in significant
reductions in the plasma concentrations of total cholesterol, HDL
cholesterol, and apoA-I in both LCAT /
mice (25, 7, and 12%;
p < 0.001 of normal) and LCAT +/
mice (65 and 59%;
p < 0.001 and 81%; not significant,
p = 0.17 of normal). In addition, plasma triglycerides
were significantly higher (212% of normal; p < 0.01)
in male homozygous knockout mice compared with wild-type animals but
remained normal in female knockout LCAT mice. Analyses of plasma
lipoproteins by fast protein liquid chromatography and two-dimensional
gel electrophoresis demonstrated the presence of heterogenous
pre
-migrating HDL, as well as triglyceride-enriched very low density
lipoprotein. After 3 weeks on a high-fat high-cholesterol diet, LCAT
/
mice had significantly lower plasma concentrations of total
cholesterol, reflecting reduced levels of both proatherogenic apoB-containing lipoproteins as well as HDL, compared with controls. Thus, we demonstrate for the first time that the absence of LCAT attenuates the rise of apoB-containing lipoproteins in response to
dietary cholesterol. No evidence of corneal opacities or renal insufficiency was detected in 4-month-old homozygous knockout mice. The
availability of a homozygous animal model for human LCAT deficiency
states will permit further evaluation of the role that LCAT plays in
atherosclerosis as well as the feasibility of performing gene transfer
in human LCAT deficiency states.
Lecithin:cholesterol acyltransferase (LCAT)1 is a 63-kDa glycoprotein synthesized primarily by the liver, which plays a major role in the metabolism of HDL (1). In plasma, LCAT is preferentially associated with HDL (2) but may also interact with low density lipoproteins (3), where it catalyzes the transfer of a fatty acid from the sn-2 position of phosphatidylcholine to the 3-hydroxyl group of cholesterol, generating cholesteryl esters and lysolecithin. The newly formed cholesteryl esters (CE) are then transferred to the core of the HDL lipoprotein particle, a process that results in the formation and maturation of spherical HDL (4). LCAT-mediated esterification of free cholesterol in HDL helps maintain a concentration gradient for efflux of cholesterol from peripheral cells to the HDL particle surface for ultimate transport to the liver (4, 5). Thus, together with hepatic lipase and cholesteryl ester transfer protein LCAT appears to be essential for the process of reverse cholesterol transport, one of several proposed mechanisms by which HDL may protect against atherosclerosis (6, 7).
The important role that LCAT plays in HDL metabolism has been established by the identification and characterization of patients with a deficiency of LCAT who present with marked hypoalphalipoproteinemia and moderate hypertriglyceridemia (1, 8). In man, LCAT deficiency leads to the expression of two phenotypically distinct clinical syndromes: classic LCAT deficiency (CLD) and fish eye disease (FED) (1, 8). Although both groups of patients present with corneal opacities and hypoalphalipoproteinemia, CLD, but not FED, is, in addition, associated with the development of anemia and progressive renal disease. The mechanisms leading to these two different clinical manifestations resulting from a functional deficiency of LCAT are poorly understood. However, recent studies indicate that the residual plasma LCAT activity, rather than the location of this activity within different plasma lipoproteins, may in fact modulate the phenotypic expression of LCAT deficiency (9, 10).
Recently, our laboratory has described the generation of transgenic mice and rabbits that overexpress the human LCAT gene (11, 12). Overexpression of human LCAT in these two different animal models leads to alterations in the concentration, composition, and size of HDL. Thus, both mice and rabbits transgenic for human LCAT have marked increases in plasma HDL cholesterol and apolipoprotein (apo) A-I levels (11, 12). Although LCAT expression reduces aortic atherosclerosis by 86% (13) in rabbits, overexpression of the same transgene in mice results in enhanced diet-induced aortic atherosclerosis (14). In man, LCAT deficiency does not appear to be associated with an increased risk of premature coronary artery disease despite markedly reduced plasma concentrations of HDL cholesterol.
To further evaluate the role that LCAT plays in modulating HDL metabolism, reverse cholesterol transport, and atherosclerosis, we have used gene targeting to create mice that are homozygous for LCAT deficiency. We report that homozygous LCAT-deficient mice are healthy and fertile and like their human counterparts have markedly reduced plasma concentrations of total cholesterol, HDL cholesterol, apoA-I and apoA-II. In addition, we describe for the first time that the absence of LCAT activity significantly attenuates the rise of apoB-containing lipoproteins in response to a high-fat high-cholesterol diet. The availability of a homozygous animal model for human LCAT deficiency states will permit further evaluation of the role of LCAT in atherosclerosis as well as the feasibility of performing gene transfer in human LCAT deficiency states.
A mouse genomic library
(129SV Mouse Genomic Library in the Lambda FIXII Vector, Stratagene, La
Jolla, CA) was screened for the mouse LCAT gene using a full-length
human LCAT cDNA as a probe. A sequence replacement vector spanning
exons 1-6 was constructed (Fig. 1), using a 4-kb
BglII-XbaI fragment and a 3-kb
BamHI-NotI fragment of the LCAT gene. These two
fragments were cloned into BclI and XbaI, and
BamHI and NotI restriction enzyme sites,
respectively, of the pPL61 (a generous gift from Dr. Michael Lenardo,
NIDDS, National Institutes of Health, Bethesda, MD). The targeting
plasmid was amplified and purified using CsCl gradient
ultracentrifugation (Lofstrand Laboratories Ltd., Gaithersburg, MD) and
linearized with NotI prior to electroporation.
ES Cell Culture and Generation of Chimeric Mice
Mouse ES
cells (RW-4) derived from 129/SvJ mice and neomycin-resistant mouse
embryo fibroblasts were purchased from Genome System Inc. (St Louis,
MO). Culture and electroporation of ES cells were conducted as reported
previously (15). For Southern blot analysis, 10 µg of DNA isolated
from G418 (350 µg/ml) and 1-(2-deoxy-2
fluoro-
-D-arabinofuranosyl)-5-iodouracil
(2 µM)-resistant clones was digested with KpnI
and probed with a 516-base pair PCR product 2 kb downstream of the
termination codon in the 3
-untranslated region of the LCAT gene (Fig.
1). ES cells from four independently targeted clones were microinjected
into C57BL/6J blastocysts followed by implantation into pseudopregnant
CB6F1 female mice. Male chimeric mice generated from two out of four
targeted clones transmitted ES cell genome to their offspring.
Genotyping was performed by Southern blot analysis of
KpnI-digested genomic DNA (15-20 µg). All
characterizations were performed in the F2 generation descendants, which were hybrids between C57BL/6J and 129/SvJ mice.
Total RNA was isolated from the liver from 2-3-month-old mice using the guanidium thiocyanate/CsCl method (16). Analysis of liver RNA was performed by fractionation on a 1% formaldehyde-agarose gel, transfer to a nylon membrane, and hybridization with the full-length human LCAT cDNA (17) as a probe.
Measurement of Plasma Lipids, Lipoproteins, Apolipoproteins, LCAT Activity, and Cholesterol Esterification Rate (CER)Plasma total
cholesterol and triglycerides (Sigma) as well as free
cholesterol and phospholipids (Wako, Osaka, Japan) concentrations were
measured in 10-µl aliquot of plasma using commercial kits and the
Cobas Mira Plus automated chemistry analyzer (Roche Diagnostic Systems,
Inc., Branchburg, NJ). CE values were calculated by subtracting free
cholesterol from total cholesterol concentrations. Plasma HDL
cholesterol was determined after dextran sulfate-Mg2+
(Ciba-Corning, Oberlin, OH) precipitation of apoB-containing lipoproteins (18). Mouse apoA-I and apoA-II were quantitated by a
sandwich enzyme-linked immunosorbent assay using microtiter immunoassay
plates (Immunlon 1, Dynatech Labs, Chantilly, VA) in which a rabbit
anti-mouse apoA-I or apoA-II polyclonal antibody was used for capture
and a horseradish peroxidase-conjugated rabbit anti-mouse polyclonal
antibody was used for detection. ApoA-I and apoA-II purified from mouse
plasma were used to generate a standard curve. Plasma lipoproteins were
analyzed by a fast protein liquid chromatography (FPLC) system
consisting of two Superose 6 columns connected in series (Pharmacia
Biotech Inc.) (19). The HDL-associated -LCAT activity in plasma was
determined as described previously (20) using 4 µl of mouse plasma.
CER was quantitated by determining the rate of esterification of
[14C]cholesterol using 125 µl of mouse plasma (21).
Plasma lipoproteins were analyzed by two-dimensional electrophoresis according to Asztalos et al. (22). After electrophoresis, lipoproteins were transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA) and probed with rabbit anti-mouse apoA-I polyclonal antibody, followed by the visualization using a Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA).
Statistical AnalysisThe paired or unpaired Student's t test (two-tailed) was used to determine statistical difference at the p < 0.05 level of significance.
Fig. 1A illustrates the strategy
utilized to generate the LCAT knockout mice. A sequence replacement
vector was constructed using two regions of homology which spanned
bases 3500 to +500 and +2200 to +5200 relative to the transcriptional
start site of the endogenous mouse LCAT gene. Homologous recombination
of the targeting vector results in the replacement of exons 2-5 of the
mouse LCAT gene (1.7 kb) by the neo gene and the deletion of
the residues 28-216 from the amino-terminal of the mouse LCAT gene.
The deleted locus encodes for serine 181 which has been proposed to be
the catalytically active serine involved in the initial hydrolysis of
phosphatidylcholine (23). Thus, elimination of exons 2-5 is predicted
to inactivate the mouse LCAT gene.
Southern blot hybridization analysis of KpnI-digested ES
cell genomic DNA utilizing the probe illustrated in Fig. 1A
demonstrated that 14 out of 79 ES clones (18%) were appropriately
targeted, as shown by the presence of a 7.4-kb fragment after digestion with KpnI (data not shown). Four targeted clones were
injected into blastocysts, with two resulting in germline transmission. Mice were then genotyped by Southern blot hybridization analysis of
KpnI-digested tail genomic DNA (Fig. 1B). A total
of 15 matings between LCAT +/ mice yielded 113 offsprings, comprising
22 +/+, 64 +/
, and 27
/
mice identified by Southern blot
hybridization analysis. Fig. 1B illustrates a representative
genomic Southern blot that demonstrates the presence of the diagnostic
bands for the wild-type (5.8 kb) as well as the disrupted LCAT gene
(7.4 kb) after digestion with KpnI.
Evaluation of both LCAT +/ and
/
mice from the F2 progeny
revealed healthy, fertile animals. Northern blot hybridization analysis
failed to detect mouse LCAT mRNA in homozygous knockout mice (Fig.
1C). Accordingly, plasma LCAT activity was reduced by
approximately 30% (male: 30 ± 2 nmol/h/ml: female: 34 ± 4 nmol/h/ml) in heterozygotes and 99% (male: 0.3 ± 0.1; female:
0.3 ± 0.1 nmol/h/mol) in homozygotes, compared with controls
(male: 44 ± 3; female: 49 ± 4 nmol/h/mol) (Table
I). Plasma CER, a more physiologic measurement of
in vivo LCAT activity, was similarly decreased in LCAT-KO
heterozygous (male: 47 ± 1 nmol/h/ml, female: 40 ± 2 nmol/h/ml) and homozygous (male: 0.0; female: 0.1 ± 0.1 nmol/h/mol) mice compared with controls (male: 70 ± 7; female:
46 ± 2). Thus, homozygous LCAT-deficient mice had a complete
deficiency of both
- and
-LCAT activities in plasma, indicating
that biochemically these LCAT deficient mice were most similar to
patients with CLD. Further evaluation of both LCAT
/
and LCAT +/
mice at age 2-3 months by slit lamp exam of the cornea and analysis of
plasma albumin, blood urea nitrogen and creatinine levels revealed no evidence of corneal opacities or renal insufficiency which are characteristic of patients with CLD (data not shown). In man, however,
the age of onset of these clinical manifestations of CLD is variable
(1, 24). Thus, it is possible that with increasing age homozygous
LCAT-deficient mice may develop these abnormal physical findings.
|
The physiological consequences of LCAT deficiency on the plasma lipids,
lipoproteins, and apolipoproteins in the F2 litters derived from the
mating of F1 LCAT +/ mice are summarized in Table I. The plasma
concentrations of total cholesterol, HDL cholesterol, and apoA-I of
fasted LCAT
/
mice were reduced to approximately 24, 7, and 13% of
the normal male and female levels, respectively. The concentrations of
total cholesterol, HDL cholesterol, and apoA-I in +/
mice were also
significantly less than those in wild-type mice (p < 0.05). In males, plasma triglyceride concentrations were 49%
(p < 0.01) and 112% (p < 0.01)
higher than that of wild-type mice in LCAT +/
and LCAT
/
mice,
respectively. However, triglyceride levels in both female LCAT +/
and
/
mice did not statistically differ from those of wild-type mice
(p = 0.21 and p = 0.13, respectively), although female LCAT
/
mice demonstrated marked variability in
plasma triglyceride concentrations, which ranged from 58 to 322 mg/dl.
The cholesteryl ester/total cholesterol ratio in LCAT
/
, +/
, and
+/+ mice were 34-52, 78-83, and 79-81%, respectively, reflecting
the absence of LCAT-mediated cholesterol esterification in the plasma
of homozygous mice. The residual cholesteryl esters still present in
LCAT
/
mice plasma may be originated from intracellular pool formed
by acylcoenzyme A:cholesterol acyltransferase as shown by the analysis
of the fatty acid pattern of plasma cholesteryl esters in human LCAT
deficiency (1). Thus, the changes in the plasma lipids and lipoproteins
in both +/
and
/
LCAT-deficient mice are similar to those
described for patients with FED or CLD (1, 8). Like the homozygous LCAT
knockout mice, not all patients with functional LCAT deficiency present
with hypertriglyceridemia (1, 25), indicating that this clinical
manifestation may be modulated by variation at other genetic loci.
Characterization of the mouse plasma lipoproteins by FPLC (Fig.
2) demonstrated a moderate to severe reduction in the
apoA-I-containing HDL particles present in plasma for LCAT +/ and
/
mice, respectively. In addition, the very low density lipoprotein
fractions from both LCAT +/
and
/
mice appeared to be
triglyceride-enriched, compared with that of control animals. LCAT
/
mice accumulated a smaller, cholesterol-poor apoA-I-containing
particle (see inset, elution volumes 32-33) whose major
lipids consisted of phospholipids.
Analysis of plasma lipoproteins by two-dimensional gel electrophoresis
followed by immunoblotting with mouse apoA-I antibody (Fig.
3) demonstrated a significant increase in
apoA-I-containing pre-migrating lipoproteins present in the plasma
of LCAT
/
mice. These pre
-HDL lipoproteins were very
heterogeneous in size, forming several subspecies visible after
immunoblotting reminiscent of the small disc-like HDL lipoproteins
described in patients presenting with CLD (26). In addition, the size
and levels of the
-migrating lipoproteins in LCAT
/
mice were
significantly reduced compared with those in LCAT +/+ mice.
To investigate the impact of the absence of LCAT on the lipoprotein
response to cholesterol feeding, three groups of female mice, aged 2-3
months, were fed a high-fat high-cholesterol diet which contained 15%
fat (ratio of polyunsaturated to saturated fatty acids = 0.69),
1.25% cholesterol, and 0.5% cholic acid as described previously (27).
Table II summarizes the plasma lipid, lipoprotein, and
apolipoprotein concentrations in the three groups of mice. After being
fed a high-fat high-cholesterol diet for 3 weeks, all three groups of
animals demonstrated significant increases in their base-line plasma
total cholesterol, free cholesterol, cholesteryl ester, and non-HDL
cholesterol concentrations. ApoB levels also significantly increased in
control and homozygote groups. However, compared with control animals,
LCAT /
mice had significantly lower plasma levels of total
cholesterol, free cholesterol, cholesteryl ester, non-HDL cholesterol,
and apoB. Further analysis of the plasma lipoproteins by FPLC (Fig.
4) confirmed that the apoB-containing lipoproteins
increased in a dose-dependent manner with increasing LCAT
activity. Thus, we demonstrate that LCAT modulates the mouse lipid
response to a high-fat high-cholesterol diet.
|
In the present paper we describe the generation of an animal model of
human LCAT deficiency. Like patients presenting with CLD and FED,
homozygous LCAT knockout mice have severe hypoalphalipoproteinemia, variable hypertriglyceridemia, and accumulation of heterogeneous pre
HDL. In addition, we demonstrate that the increase in apoB-containing lipoproteins in response to dietary cholesterol is attenuated in LCAT
/
mice, thus indicating that LCAT modulates the lipoprotein response to dietary cholesterol. The availability of a viable, homozygous animal model for human LCAT deficiency states will permit
further investigation of the mechanisms by which a defect in LCAT can
result in the expression of two distinct clinical syndromes, CLD and
FED. In addition, evaluation of the processes of reverse cholesterol
transport and atherosclerosis in homozygous LCAT knockout mice will
provide further insights into the role that LCAT plays in HDL
metabolism and the development of premature coronary artery
disease.
We thank Alexander Ginberg, Li Yin, Edsel Kim, Alisa Kim, Michael Lenardo, and Louis Staudt for their excellent scientific discussion as well as Judith Burk for her assistance in the preparation of the manuscript.