From the Molecular Disease Branch, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892, the
Laboratory
of Immunology, NEI, National Institutes of Health, Bethesda, Maryland
20892, the ** Jackson Laboratory, Bar Harbor, Maine 04609, the
Renal Cell Biology Laboratory, University
of Miami School of Medicine, Miami, Florida 33101, the
§§ Lipid Cell Biology Laboratory, NIDDK,
National Institutes of Health, Bethesda, Maryland 20892, the
¶¶ Clinical Pathology Department, Clinical Center,
National Institutes of Health, Bethesda, Maryland 20892, and the
Laboratory of Receptor Biology and Gene Expression, NCI,
National Institutes of Health, Bethesda, Maryland 20892
Received for publication, September 15, 2000, and in revised form, December 22, 2000
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ABSTRACT |
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To evaluate the biochemical and molecular
mechanisms leading to glomerulosclerosis and the variable development
of atherosclerosis in patients with familial lecithin cholesterol acyl
transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and
cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein
receptor (LDLr) KO, and cholesteryl ester transfer protein
transgenic mice. LCAT-KO mice had normochromic normocytic anemia with
increased reticulocyte and target cell counts as well as decreased red
blood cell osmotic fragility. A subset of LCAT-KO mice accumulated
lipoprotein X and developed proteinuria and glomerulosclerosis
characterized by mesangial cell proliferation, sclerosis, lipid
accumulation, and deposition of electron dense material throughout
the glomeruli. LCAT deficiency reduced the plasma high density
lipoprotein (HDL) cholesterol ( As the key enzyme responsible for the esterification of free
cholesterol present in circulating lipoproteins,
LCAT1 plays a major
role in HDL metabolism (1). Several lines of evidence that include
epidemiological data, transgenic animal studies, and, more recently,
prospective human clinical trials (2-6) indicate that increased plasma
HDL levels protect against the development of atherosclerosis. One of
several proposed functions of HDL as an anti-atherogenic lipoprotein is
to facilitate reverse cholesterol transport, a process by which
cholesterol is transported from peripheral cells to the liver for
removal from the body (7, 8). LCAT may play a major role in this
process by maintaining a free cholesterol gradient between peripheral
cells and the HDL particle surface, thus promoting free cholesterol
efflux (1). The newly generated cholesteryl esters (CE) accumulating in
the HDL core may be transferred directly to the liver via whole
particle and/or selective uptake (9, 10). Alternatively HDL-CE may be
transferred to apoB-containing lipoproteins as a result of the activity
of cholesterol ester transfer protein (CETP) (8).
The important role that LCAT plays in HDL metabolism has been
established by the identification and characterization of patients with
LCAT deficiency. Mutations in the human LCAT gene result either in
familial LCAT deficiency (FLD) or in fish eye disease (FED). FED
patients have a partial deficiency of LCAT function leading to
hypoalphalipoproteinemia and the development of corneal opacities (11,
12). FLD patients have a total deficiency of LCAT function (12, 13). In
addition to hypoalphalipoproteinemia and corneal opacities, many FLD
patients also develop a mild normochromic, normocytic anemia,
proteinuria, and/or renal dysfunction (14-16). The mild hemolytic
anemia in FLD appears to be secondary to changes in red blood cell
membrane lipids resulting from altered cholesterol metabolism (17, 18).
Glomerulosclerosis, the major cause of morbidity and mortality in FLD,
may ultimately lead to renal failure in the fourth or fifth decade of
life (19, 20), but its pathogenesis is poorly understood. Likewise, the
role of LCAT in modulating the development of atherosclerosis in these
patients remains unclear. Despite reduced plasma HDL, many patients
with FLD or FED do not appear to have an increased risk for developing
cardiovascular disease (21). Nevertheless, premature coronary heart
disease has been documented angiographically and clinically in a subset of these patients (22-27). LCAT overexpression has been shown to enhance aortic atherosclerosis in LCAT transgenic mice (28, 29) but
decrease atherogenesis in LCAT transgenic rabbits (30). The mechanisms
by which LCAT modulates the development of glomerulosclerosis and
atherosclerosis in patients with FLD remain unknown.
We (31) and others (32) have previously described the generation of
LCAT-KO mice. On a regular chow diet these animals have markedly
reduced total cholesterol, HDL cholesterol, and apoA-I levels (31, 32).
In the present study we characterize the lipid, ophthalmologic,
hematological, renal, and cardiovascular organ systems of LCAT-KO mice
on both regular and high fat/high cholesterol (HF/HC) diets.
LCAT-KO mice develop a clinical phenotype similar to human patients
with FLD. In addition to severe hypoalphalipoproteinemia, LCAT-KO
mice present with normochromic normocytic anemia and
glomerulosclerosis. Our findings indicate that the induction of LpX by
the HF/HC diet is associated with the development of glomerulosclerosis
in these mice. We also report that, in addition to decreased HDL, LCAT deficiency reduces the plasma levels of apoB-containing lipoproteins by
up-regulating LDL receptor gene expression. Despite the low plasma HDL levels, these changes in the plasma lipid profile lead to
markedly reduced aortic atherosclerosis in mice with LCAT deficiency. These combined findings provide important new insights into the role
that LCAT plays in the development of glomerulosclerosis and
atherosclerosis in LCAT-deficient states.
Animals and Diet--
Homozygous LCAT knockout mice (31) were
backcrossed to strain C57BL/6 for eight generations. LCAT-KO mice were
cross-bred with apoE-KO mice (33), LDL receptor KO mice (34), and
CETP-Tg mice (35) with C57BL/6 background. Control C57BL/6 mice were obtained from Charles River (Wilmington, MA). The animals were 25-30 g
at the beginning of the study. Mice first maintained on a regular
NIH-07 chow diet containing 5% fat (Ziegler Brothers Inc., Gardners,
PA) were put on a HF/HC diet containing 15% fat (ratio of
polyunsaturated to saturated fatty acids = 0.69), 1.25% cholesterol, and 0.5% cholic acid (36).
Measurement of Plasma Lipids, Lipoproteins, Apolipoproteins, LCAT
Activity, and Cholesterol Esterification Rate--
Total cholesterol
and triglycerides (Sigma), as well as free cholesterol and
phospholipids (Wako, Osaka, Japan) concentrations were measured in a
12-µl aliquot of plasma using commercial kits and the Hitachi 911 automated chemistry analyzer (Roche Molecular Biochemicals). HDL
cholesterol was determined after dextran sulfate-Mg2+
(Ciba-Corning, Oberlin, OH) or heparin (Sigma) CaCl2
precipitation of apoB containing lipoproteins (37, 38). Mouse apoA-I,
A-II, and apoB were quantitated by a sandwich enzyme-linked
immunosorbent assay (31, 39). Plasma lipoproteins were analyzed by gel
filtration on two Superose 6 columns in series (fast protein liquid
chromatography, Amersham Pharmacia Biotech) at 0.3 ml/min in
phosphate-buffered saline containing 0.1 mM EDTA and 0.02%
sodium-azide. The LCAT activity was determined as previously described
(40), using 40 µl of mouse plasma. The cholesterol esterification
rate was assessed by determining the rate of esterification of
[14C]cholesterol using 125 µl of mouse plasma (41).
Characterization of Lipoprotein Fractions--
Lipoproteins were
obtained from 2 ml of pooled plasma by sequential ultracentrifugation
(42), and their morphology was assessed by negative staining electron
microscopy (43). Plasma lipoprotein electrophoresis as well as
immunofixation electrophoresis were carried out in agarose gels (REP
Vis Cholesterol kit, Helena Laboratories, Beaumont, TX). Rabbit
anti-mouse apoC-I, C-II, and C-III goat anti-human (Biodesign
International, Kennebunk, ME), and sheep anti-mouse albumin-IgG
fraction (The Binding Site Inc., San Diego, CA) were used for
immunofixation electrophoresis. Patterns were developed by staining
lipids (Fat Red 7B, Sigma) and/or proteins (Amido Black 10B; Paragon
Blue, Beckman Coulter Inc., Brea, CA). LpX in mouse plasma was
specifically analyzed by the polyanion precipitation method (44).
Briefly, after electrophoresis of plasma samples in a 0.5% agar gel
(Agar Noble, Difco Labs, Detroit, MI), lipoproteins were precipitated
in a solution containing heparin and MgCl2. Gels were
evaluated by visually observing the development of white precipitates,
by staining for lipids with Fat Red 7B, and by immunofixation using the
above antibodies.
Eye Examination--
Mice were killed, and the eyes were
enucleated immediately. The right eye was fixed in 4% glutaraldehyde
for 30 min and then transferred into 10% buffered formalin for at
least 24 h before processing. The tissue was embedded in
methacrylate and sectioned through the central-vertical plane. The
sections of the corneas were stained with hematoxylin/eosin, periodic
acid-Schiff, or oil red-O (Sigma) before examination.
Urine, Hematology, and Blood Chemistry Tests--
Urine samples
were obtained from the mice immediately prior to sacrifice and kept
frozen at Light and Electron Microscopy Analysis of Renal Lesions--
The
kidneys were perfusion fixed as previously described (45). Briefly, the
aorta was cannulated below the renal arteries, and the kidneys were
perfused with buffered 2% paraformaldehyde. For light microscopy
coronal sections were embedded in methacrylate, and sections were
stained with periodic acid Schiff. Alternatively the sections were
embedded in OCT compound, frozen, and stained with oil red-O. A subset
of kidneys were fixed in 3% paraformaldehyde, 0.2% glutaraldehyde,
0.2 M sodium cacodylate, pH 7.4, and sections were stained
in 0.05% filipin (Sigma). Slides were analyzed with a Zeiss
microscope, using excitation filter BP 360 for filipin, without the
observer being aware of the animal grouping. Lesions were scored on a
1-4+ grading scale (45). For electron microscopy, cortical regions
containing glomeruli were sectioned, and photographs were obtained of
at least three glomeruli in each mouse.
Histological Analysis of Aortic Lesions--
Hearts and upper
aortas were dissected and placed in 0.9% saline, followed by fixation
in 4% formaldehyde for 24 h and then 10% formaldehyde for 5 days. The mean aortic lesion areas were quantitated from five
cross-sections/mouse as previously described (46).
Preparation of Labeled Targets for Hybridization--
Mice
livers (~0.5 g) were harvested, submerged in RNA later solution
(Ambion, Austin, TX), and frozen at Array Hybridization--
A set of four oligonucleotide arrays
(Mu6500 GeneChip, Affymetrix) were used for hybridization. The oligo
array cartridges were prehybridized with 200 µl of 1× hybridization
buffer (100 mM MES, 1 M NaCl, 20 mM
EDTA, and 0.01% Tween 20 (all reagents, Sigma)) for 10 min at
45 °C/60 rpm on a rotisserie. Fragmented cRNA was brought to 1 ml in
1× hybridization buffer with 0.1 mg/ml herring sperm DNA (Promega,
Madison, WI), 0.5 mg/ml acetylated bovine serum albumin (Life
Technologies, Inc.), as well as 50 pM B2, 1.5 pM BioB, 5 pM BioC, 25 pM BioD, and
100 pM Cre control oligonucleotides (Affymetrix). The
mixture was incubated for 5 min at 99 °C and centrifuged to pellet
debris. Prehybridization solution was removed from the cartridges, and
200 µl of the fragmented RNA solution was added and hybridized for
16 h at 45 °C/60 rpm. After hybridization, the samples were
removed, and the cartridges were washed, stained with Streptavidin
Phycoerythrin, and rinsed on a fluidix station using the EukGE-WS1
protocol. The arrays were scanned on a Hewlett Packard confocal laser
scanner and visualized using the GeneChip 3.3 software (Affymetrix) to
determine the expression of each gene and their differential expression
between mice. Comparison analysis between three untreated LCAT-KO mice and three controls (i.e. a total of nine comparisons) was
performed. The genes differentially expressed (i.e. more
than 2-fold change) in at least seven comparisons were listed.
Western Blot Analysis--
Mouse livers (~0.5 g) were cut into
small pieces and transferred in 5 ml of phosphate-buffered saline 1×
containing a protease inhibitor mixture P2714 (Sigma). The suspensions
were homogenized and centrifuged at 10,000 rpm for 10 min at 4 °C.
The supernatants were centrifuged in a TI-60 rotor at 40,000 rpm using
a L8-70M ultracentrifuge (Beckman, Palo Alto, CA) for 1 h at
4 °C. Pellets were resuspended in lauryl dodecyl sulfate
sample buffer (1× final concentration). Liver proteins (10 µg) were
resolved on Nu-PAGE 4-12% Bis-Tris gels in MES-SDS buffer (Novex, San
Diego, CA) under nonreducing conditions. Proteins were transferred onto
an Immobilon membrane (Millipore, Bedford, MA), probed with polyclonal
anti-human-LDLr-IgG (RDI, Flanders, NJ) using Vectastain PK6101
(Vector, Burlingame, CA). ApoA-I, apoA-II, and apoE levels of LCAT-KO
and control mice plasma samples (0.1 µl) were similarly also analyzed
by Western blot using anti mouse apoA-I, apoA-II, and apoE IgG
(Biodesign, Saco, ME).
Statistical Analysis--
Data are expressed as the means ± S.E. The statistical significance of the differences of the mean
between two groups was evaluated using the Student's t
test. Differences in mean aortic lesion areas were evaluated using the
Mann-Whitney test (Instat, GraphPad Software Inc., San Diego, CA).
Plasma LCAT Activity, Lipids, Apolipoproteins, and
Lipoproteins--
The changes in the lipid profile and apolipoproteins
in the two study groups of mice before and 16 weeks after a HF/HC diet are summarized in Table I. Compared with
controls, the plasma LCAT activity was reduced by 99% in LCAT-KO mice.
On a regular chow diet the plasma levels of total cholesterol,
phospholipids, CE, HDL cholesterol, apoA-I, and apoA-II of LCAT-KO mice
were decreased to 25, 54, 7, 14, 10, and 23% of controls,
respectively, whereas the plasma triglycerides levels increased by 50%
(p < 0.01, all). The non HDL cholesterol and apoB
levels were similar in both study groups. On the HF/HC diet, the plasma
levels of a total cholesterol, phospholipids, CE, HDL cholesterol,
non-HDL cholesterol, apoA-I, apoA-II, and apoB of LCAT-KO mice were
decreased to 45, 48, 44, 6, 52, 17, 50, and 45% of controls,
respectively (p < 0.01; all), whereas the plasma
triglycerides levels remained unchanged. The plasma CE levels
dramatically increased as a result of the HF/HC diet in both controls
and LCAT-KO mice. Of particular interest was the increase in the
CE/total cholesterol ratio from 21 to 76%, indicating an alternate
pathway for the esterification of cholesterol in LCAT-KO mice. Fig.
1 illustrates the fast protein liquid
chromatography elution profile of the plasma lipoproteins in control
and LCAT-KO mice maintained 16 weeks on a HF/HC diet. Most of the
cholesterol eluted in the very low/intermediate density lipoproteins
(VLDL and IDL) and LDL fractions in both study groups. However,
the VLDL cholesterol, IDL cholesterol as well as HDL cholesterol were
significantly reduced in LCAT-KO mice. Electron microscopy evaluation
of negatively stained lipoprotein fractions revealed discoidal
particles forming rouleaux structures (i.e. pre- Eyes--
Sections of the corneas from LCAT-KO and control
mice fed a HF/HC diet for 16 weeks were stained with hematoxylin and
eosin, periodic acid Schiff, or oil red-O. No ocular abnormalities were found in the LCAT knockout mice compared with controls using these three histological techniques (data not shown).
Blood--
Blood samples from LCAT-KO and control mice were
analyzed for cell counts, hemoglobin, hematocrit, and total bilirubin
levels. LCAT knockout mice had anemia with decreased red blood
cell count (9.6 ± 0.1 versus 10.4 ± 0.1 106/µl), hematocrit (42.4 ± 0.4 versus
46.0 ± 0.5%), hemoglobin levels (13.5 ± 0.1 versus 14.9 ± 0.1 g/dl) as well as white blood
cell count (3.63 ± 0.35 versus 5.84 ± 0.61 103/µl) compared with controls (p < 0.005, all). The platelet count, the red blood cell indices (mean
corpuscular volume and mean corpuscular hemoglobin), and the plasma
bilirubin levels were normal in both study groups (data not shown).
Dietary status did not alter blood counts, hemoglobin, hematocrit, and
bilirubin levels in either mouse model (data not shown). Despite the
absence of hyperbilirubinemia, the proportion of target cells
(11.2 ± 2.6 versus 2.3 ± 0.5%, p < 0.01) as well as reticulocytes (13.5 ± 4.1 versus 3.2 ± 1.4%, p < 0.04) was
increased in LCAT-KO mice compared with controls, indicating increased
red blood cell hemolysis. As described in FLD patients (17, 18, 48),
the osmotic fragility tests showed that red blood cells from LCAT-KO
mice were more resistant to hemolysis than those of controls (5.3 ± 1.5 versus 43.7 ± 3.1% hemoglobin leakage in 0.5%
NaCl and 86.3 ± 3.5 versus 97.7 ± 2.1% in 0.4%
NaCl; p < 0.01, all).
Renal Function--
Plasma samples from LCAT-KO and control mice
were collected before and after the HF/HC challenge and analyzed for
albumin, blood urea nitrogen, and creatinine. On either diet no
significant difference between the two groups was detected (data not
shown). However, after a HF/HC diet, proteinuria was detected by
electrophoresis and immunofixation electrophoresis in 10 of 11 LCAT-KO
mice urine samples, whereas the urine samples from all 11 control mice
were negative (data not shown).
Kidneys--
Histologic lesions were only present in a subset of
LCAT-KO mice on the HF/HC diet (i.e. those presenting with
LpX). The kidneys of the other mice were normal. The lesions consisted
of an increase in mesangial matrix (Fig.
3, A and B), which
involved the glomeruli irregularly, within and between individual
glomeruli. Occasional vacuoles were observed in the cellular cytoplasm
of cells, most prominent near the vascular pole. There were no tubular
or interstitial lesions. Occasional preglomerular arterioles contained
periodic acid Schiff positive droplets. There were no lesions of larger blood vessels. Fig. 3 (C and D) shows
representative kidney sections from LCAT-KO and control mice after the
16 weeks HF/HC challenge analyzed by electron microscopy. The glomeruli
from control animals were unremarkable. In LCAT-KO mice, the
endothelial cytoplasm and the vascular spaces were smaller than in
controls. Depositions of electron dense material in the capillary lumen
and mesangial regions were evident. The mesangial regions contained an
increased amount of extracellular matrix, and there were irregular but
prominent zones containing lucent lacunae. Occasional cells resembling
macrophages were present within the mesangium. No changes were seen in
either the peripheral basement membrane or the podocytes. In
particular, the pedicels were intact. Unlike controls, a subset of
LCAT-KO mice on a HF/HC diet accumulated lipids within their glomeruli, which were detected by oil red-O staining (Fig. 3, E and
F). The glomeruli of these LCAT-KO mice had increased free
cholesterol content compared with those of controls, as shown on
filipin fluorescence-stained sections (Fig. 3, G and
H). Renal lesions were only present in LCAT-KO mice in which
LpX was detected in plasma. No renal lesions were detected in
heterozygous LCAT-KO mice.
Effect of LCAT Deficiency on the Plasma Cholesterol Levels
and Atherosclerosis in apoE-KO, LDLr-KO, and CETP-Tg Mice--
LCAT
deficiency reduced the plasma levels of TC, CE, and non-HDL cholesterol
(Fig. 4) in apoE-KO, LDLr-KO, and CETP-Tg
mice (p < 0.05; all). The mean aortic lesion area
of apoE-KO and LCAT-KO × apoE-KO mice maintained on a regular
chow diet and that of LCAT-KO, LDLr-KO, LCAT-KO × LDLr-KO,
CETP-Tg, LCAT-KO × CETP-Tg, and control mice maintained 16 weeks
on a HF/HC diet was quantitated from five cross- sections of the
proximal aortas (Fig. 5). LCAT deficiency significantly reduced the mean aortic lesion area in C57BL/6 (-85%), apoE-KO (-51%), LDLr-KO (-35%), and CETP-Tg (-99%) mice.
Analysis of Differentially Expressed Hepatic Genes in LCAT-KO
Mice--
Table II summarizes the
changes in hepatic gene expression induced by LCAT deficiency in mice.
Of 6500 possible transcripts, the expressions of 51 transcripts were
altered by greater than 2-fold in LCAT-KO compared with control mice.
Besides LCAT mRNA, the transcripts of Cyp 7 In the present study we have investigated potential mechanisms
leading to renal disease and altered atherogenic risk in LCAT-KO and
LCAT-KO mice crossed with atherosclerosis-susceptible mouse models.
LCAT-KO mice present with a clinical phenotype similar to patients with
FLD, including normochromic normocytic anemia, glomerulosclerosis,
hypoalphalipoproteinemia, and hypertriglyceridemia. In addition, LCAT
deficiency alters the plasma levels of the pro-atherogenic apoB
containing lipoproteins by modulating the hepatic expression of the
LDLr and apoE, which, despite decreased HDL levels, leads to reduced
atherogenic risk.
LCAT deficiency in mice markedly reduces total cholesterol, HDL
cholesterol, and apoA-I levels as well as increases pre- Lipoprotein disorders are frequently associated with alterations in the
morphology and lipid composition of the cornea. Corneal opacities are
found in several syndromes associated with HDL deficiency including
Tangier disease (58), apoA-I deficiency (59, 60) as well as FED and FLD
(12, 61). However, analysis of the cornea of LCAT-KO mice with
hematoxylin and eosin, periodic acid-Schiff, or oil red-O failed to
reveal similar changes.
LCAT-KO mice developed anemia characterized by normochromic normocytic
indices, increased target cells, and reticulocyte count findings
consulted with hemolysis. The mild hemolytic anemia appears to be
secondary to altered red blood cell membrane lipids resulting from
changes in plasma cholesterol metabolism. The red blood cell membrane
fragility was decreased in LCAT-KO compared with controls, consistent
with altered red blood cell membrane lipids as well as an increased
proportion of target cells that are more resistant to hemolysis
(62).
We further examined the consequences of LCAT deficiency on the
morphology and function of the kidneys from mice maintained either on
regular or HF/HC diets. Glomerulosclerosis was not detected in LCAT-KO
mice on a regular chow diet. In contrast, on the HF/HC diet, a subset
of LCAT-KO mice developed renal lesions similar to those previously
described in patients with FLD (19, 20, 63-65). Histologically, these
lesions were characterized by significant reductions in the vascular
space, expanded mesangial region with increased number of mesangial
cells and mesangial sclerosis, and increased extracellular matrix with
accumulation of lipid droplets and macrophages. Filipin and oil
red-O staining demonstrated the accumulation of free cholesterol
and polar lipids in the glomeruli. Ultrastructurally, the
endothelial cytoplasm and the vascular spaces were smaller in LCAT-KO
than in controls, and the mesangial regions contained an increased
amount of extracellular matrix. Although significant, the glomerular
lesions in LCAT-KO mice fall within the milder spectrum of lesions
described in FLD patients. However, most of the tissue analysis has
been performed in patients with end stage renal disease. The absence of
lesions on a regular chow diet, and the mildness of the lesions after
the HF/HC diet, in the LCAT-KO mice, are both consistent with previous
observations that the C57Bl/6 mouse strain is resistant to the
induction of glomerulosclerosis (66). The plasma albumin, total
protein, blood urea nitrogen, and creatinine levels were normal in all study animals, even in LCAT-KO mice found to have renal lesions. In
patients with FLD these plasma indicators of renal function remain
normal until the development of severe renal disease. Proteinuria, one
of the earliest findings in patients with FLD (12, 13, 62) was also
detected in 10 of 11 LCAT-KO mice on the HF/HC diet. Thus, the renal
lesions in LCAT-KO mice lead to abnormal renal function.
The pathogenesis of renal injury in FLD is poorly understood. Some
investigators have suggested that the renal damages observed in FLD
may be immune complex- and complement-mediated (20, 65) and may be
further exacerbated by the accumulation of oxidized phospholipids in
the glomeruli (67). It has also been suggested that large LDL particles
as well as LpX trapped in capillary loops induce endothelial damage and
vascular injury in the kidneys of FLD patients (51, 54, 63). However,
these abnormal lipoproteins are not always detected in FLD (20). It is
of interest that LCAT-KO mice developed glomerulosclerosis only while
on the HF/HC diet, when the plasma levels of apoB-containing
lipoproteins were increased. Both large LDL and LpX were present in the
plasma of LCAT-KO mice only on the HF/HC diet. Furthermore, renal
lesions were only detected in the group of mice that accumulated LpX. These findings provide strong support for the importance of large LDL and LpX in the development of renal disease in FLD (68).
In patients with LCAT deficiency the atherogenic risk is variable. The
mechanisms leading to altered atherosclerosis are poorly understood.
Despite low HDL, many FLD and FED patients do not appear to be at
increased risk of developing premature coronary artery disease (21).
However, some patients presenting with severe coronary artery disease
have been reported (22-27, 69). To investigate potential mechanisms
leading to this variable atherogenic risk, we examined the consequences
of LCAT deficiency on the development of atherosclerosis in LCAT-KO and
LCAT-KO mice crossed with atherosclerosis-susceptible mouse models.
Despite decreased HDL, LCAT-KO, LCAT-KO × apoE-KO, LCAT-KO × LDLr-KO, and LCAT-KO × CETP-Tg mice had a significant reduction in the mean aortic lesion area compared with their respective controls. Analysis of LCAT-induced changes in other plasma lipoproteins provided some insights into these unexpected findings. In addition to
lowering HDL, LCAT deficiency reduced the plasma levels of the
pro-atherogenic apoB containing lipoproteins by enhancing the hepatic
expression of LDLr and apoE. The marked reduction in the plasma levels
of the anti-atherogenic HDL may be offset by a reduction in the
plasma levels of the proatherogenic apoB-containing lipoproteins, thus
resulting in reduced atherosclerosis despite decreased HDL.
Decreased plasma LDL levels have been reported in most (12, 13, 49-53)
but not all (23, 27) patients with LCAT deficiency, reflecting
different genetic backgrounds. This same variability in apoB containing
lipoproteins levels can be observed in apoE-KO, LDLr-KO, and CETP-Tg
mice crossed with LCAT-KO mice, where the apoB levels vary according to
genetic background. Interestingly, in contrast to LCAT-deficient
patients with normal or reduced apoB levels, patients with increased
triglycerides and apoB levels (23, 27) have been shown to have
increased cardiac risk. These patients have delayed catabolism of LDL
(70). These combined data suggest that, in LCAT-deficient states, the
plasma levels of the proatherogenic apoB-containing lipoproteins,
rather than anti-atherogenic HDL, modulate atherogenic risk.
Additionally, in contrast to patients with Tangier disease, who cannot
generate pre- In summary, we have characterized the lipid, ophthalmologic,
hematological, renal, and cardiovascular organ systems of LCAT-KO mice,
a new animal model for FLD. Like human patients with FLD, LCAT
deficiency in mice leads to marked hypoalphalipoproteinemia, anemia,
and glomerulosclerosis. Our studies provide strong support for the role
of LpX in the development of glomerulosclerosis in LCAT deficiency. We
also report for the first time that despite decreased HDL, LCAT
deficiency protects against the development of diet-induced aortic
atherosclerosis in mice. The increased plasma levels of pre-70 to
94%) and non-HDL cholesterol
(
48 to
85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl
ester transfer protein-Tg mice. Transcriptome and Western blot analysis
demonstrated up-regulation of hepatic LDLr and apoE expression in
LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was
significantly reduced (
35% to
99%) in all mouse models with LCAT
deficiency. Our studies indicate (i) that the plasma levels of apoB
containing lipoproteins rather than HDL may determine the atherogenic
risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit
future evaluation of LCAT gene transfer as a possible treatment for
glomerulosclerosis in LCAT-deficient states.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C prior to analysis. Electrophoresis of the urine
samples was done in 1% agarose gel (Paragon SPE kit, Beckman Coulter
Inc., CA), followed by Amido Black 10B staining. Protein fractions were
quantified by scanning the gels with an EDC densitometer (Helena Labs).
Complete blood count was determined on an Abbot Cell Dyn 3500 automated
analyzer (Santa Clara, CA). Erythrocyte osmotic fragility was
determined using the Unopette kit (Becton Dickinson, Franklin Lakes,
NJ). Erythrocytes were stained with methylene blue (Sigma), and the
cells containing stained RNA precipitates (i.e.
reticulocytes) were counted manually under microscope. Target cells
were counted manually under microscope. Serum total bilirubin, albumin,
blood urea nitrogen, and creatinine levels were measured using
commercial kits and the Hitachi 917 automated analyzer (Roche Molecular Biochemicals).
70 °C. Total RNA was isolated
with Trizol (Life Technologies, Inc.). Poly (A)+ RNA was
purified using oligo(dT) conjugated latex beads (Qiagen, Valencia, CA),
and 5 µg was denaturated with 100 pmol of
GGCCAGTGAATTGTAATACGACGACTCACTAT- AGGGAGGCGG-d(T)24
primer at 70 °C for 10 min. Reverse transcription and second strand
cDNA synthesis were performed using the Superscript Choice System
(Life Technologies, Inc.). The resulting cDNA was extracted with
phenol:chloroform:isoamylalcohol (25:24:1) (Ambion). In
vitro transcription was performed using the
BioArray-HighYield-RNA-transcript-labeling kit (Enzo, Farmingdale, NY)
with 1 µg of cDNA as a template. The resulting biotinilated RNA
(cRNA) was purified with Rneasy columns (Qiagen), and 50 µg cRNA was
fragmented in 40 mM Tris-HCl, pH 8.0, 100 mM
KOAc, 30 mM MgOAc (Sigma) for 35 min at 95 °C in 75 µl. The quality of total RNA, poly(A)+ RNA, cDNA,
cRNA, and fragmented cRNA was judged by running aliquots on a
Tris borate/EDTA/urea 6% polyacrylamide gel (Novex, San Diego, CA), followed by SYBRGold (Molecular Probes, Eugene, OR) staining. The
efficiency of the in vitro transcription was ascertained on a Test1 oligonucleotide array (Affymetrix, Santa Clara, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HDL)
only in the HDL fraction of LCAT-KO mice (Fig. 1, inset), as
previously described (32). On the HF/HC, the morphology of HDL from
control mice did not change, whereas there was an increase in the mean
diameter of the small spherical particles in the HDL size range of the
LCAT-KO mice (10.9 ± 0.4 nm on chow versus 14.5 ± 0.4 nm on HF/HC diet). In addition the proportion of discoidal particles in the HDL fraction from LCAT-KO mice decreased from 14 to
4% after the HF/HC diet (p < 0.05). LDL of LCAT-KO
mice were polydisperse consisting of smaller 19.6 ± 1.0 nm as
well as normal 33.1 ± 1.4 nm spherical lipoproteins and some
larger structures 66.7 ± 1.8 nm in size, whereas those of control
mice consisted in particles of 28.0 ± 1.8 nm in diameter. The
sizes of the particles present in the LDL fraction of both control, and
LCAT-KO mice did not significantly change after the HF/HC challenge.
Lipoprotein electrophoresis (Fig.
2A, middle panel) showed a virtual absence of
-lipoproteins in the plasma of LCAT-KO mice and increased staining at the application site (indicated by
arrows) after a HF/HC diet in both control and LCAT-KO mice (Fig.
2A). On agarose gel, an LpX-like lipoprotein (Fig.
2A, middle panel) migrated in the
-globulin
fraction (Fig. 2A, left panel). Like human LpX
(47), the mouse LpX was shown to contain apoC-I, C-II, and C-III (Fig.
2A, right panel) and albumin (data not shown) by
immunofixation. On agar gel, mouse LpX, which migrated anodally (Fig.
2B), was detected only in a subset of LCAT-KO mice (8 of 12)
after 16 weeks on the HF/HC diet. LpX was not present in the plasma of
LCAT-KO mice on a regular chow diet (6 mice) or control mice on a
regular chow (6 mice) or a HF/HC diet (11 mice).
Plasma lipids, lipoproteins, apolipoproteins, and LCAT activities in
control and LCAT-knockout mice on a regular chow diet as well as a high
fat/high cholesterol diet
View larger version (37K):
[in a new window]
Fig. 1.
Elution profile of control ( ) and LCAT
knockout (
) mice plasma on fast protein liquid chromatography after
16 weeks HF/HC diet. 50 µl of pooled mouse plasma was applied on
two Superose 6 columns in series. The concentration of phospholipids,
triglycerides, and cholesterol in each fraction are indicated in
the y axis. Inset, left panel, low
magnification of HDL fraction (1.063 < d < 1.21)
from LCAT KO mouse showing numerous discs present in the fraction.
Bar, 10 nm). Right panel, high magnification of
HDL fraction from LCAT-KO mouse showing a rouleau of discs associated
with HDL particle. Bar, 5nm).
View larger version (61K):
[in a new window]
Fig. 2.
Electrophoresis analysis of LCAT-KO and
control mice plasma lipoproteins. A, plasma
lipoproteins were separated in agarose gels. Protein staining
(left panel), lipoprotein staining (middle
panel), and immunofixation with anti-apoCI, CII and CIII
antibodies (right panel) are shown. B, plasma
lipoproteins were resolved in agar gel followed by precipitation with
heparin/MgCl2. ApoC-containing LpX is present only in the
plasma of LCAT-KO mice after 16 weeks HF/HC diet. Arrows
indicate the point of application.
View larger version (100K):
[in a new window]
Fig. 3.
Analysis of renal sections from control
(A, C, E, and
G) and LCAT-KO (B,
D, F, and H) mice
maintained on a HF/HC diet for 16 weeks. A and
B, light microscopy of periodic acid Schiff stained sections
embedded in methacrylate. C and D, electron
microscopy of frozen sections. E and F,
light microscopy of oil red-O stained sections embedded in OCT
compound. G and H, fluorescent light microscopy
of filipin stained sections embedded in OCT compound.
View larger version (23K):
[in a new window]
Fig. 4.
Plasma cholesterol levels in LCAT +/+ and
LCAT /
mice on the C57BL, CETP transgenic, apoE knockout, and LDL
receptor knockout backgrounds. The feeding status of the animals
is indicated in the legend of Fig. 5. *, p < 0.05; **,
p < 0.001 versus LCAT +/+ mice of the same
genetic background.
View larger version (22K):
[in a new window]
Fig. 5.
Extent of atherosclerosis in the proximal
aorta of LCAT +/+ ( ) and LCAT
/
(
) mice on the C57BL
(A), CETP transgenic (B), apoE
knockout (C), and LDL receptor knockout
(D) backgrounds. The animals (A,
B, and D) were maintained for 16 weeks on a HF/HC
diet, whereas apoE-knockout mice (C) were fed a regular chow
diet. All mice were females and 3 months old at the beginning of the
study.
hydroxylase, the
rate-limiting enzyme for bile synthesis as well as squalene epoxydase,
involved in the cholesterol biosynthetic pathway, were down-regulated
in LCAT-KO mice livers. Of particular interest is the up-regulation of
the LDL receptor and apoE, whose increased expressions were also
confirmed by Western blot hybridization analysis (Fig.
6). Despite no differences in their
mRNA levels, apoA-I and apoA-II plasma levels were reduced in
LCAT-KO mice.
Differentially expressed genes and ESTs in LCAT knockout mice
View larger version (41K):
[in a new window]
Fig. 6.
Immunoblot of hepatic LDLr and
plasma apolipoproteins in control (lanes 1 and
2) and LCAT-KO (lanes 3 and
4) mice.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
HDL and
triglyceride levels (31, 32). Surprisingly, on the HF/HC diet LCAT-KO
mice had significantly lower plasma levels of the proatherogenic
apoB-containing lipoproteins. Decreased plasma LDL levels have been
reported in some (12, 13, 49-53) but not all (23, 27) patients with
LCAT deficiency reflecting genetic variability absent in LCAT-KO mice.
Despite the absence of LCAT activity, LCAT-KO mice had a dramatic
increase in their plasma CE levels on the atherogenic diet, suggesting
enhanced contribution by the enzyme acylCoA:cholesterol acyltransferase responsible for the intracellular esterification of cholesterol. Further analysis of the plasma lipoproteins in LCAT-KO mice revealed that the VLDL density fractions were triglyceride-enriched. LDL were
polydisperse consisting of normal spherical lipoproteins as well as
large structures of 66.7 nm in diameter previously designated as large
molecular weight LDL (54). The two major HDL particles described in FLD
(i.e. discoidal particles forming rouleaux and spherical
particles) (12) were also present in LCAT knock-out mice with a
decreased proportion of discoidal particles and an increase in the mean
diameter of the spherical particles after the HF/HC diet. LpX was
detected by electrophoresis and immunofixation electrophoresis on
agarose and agar gels in a subset of LCAT-KO mice fed a HF/HC diet for
16 weeks. LpX, first described in patients with primary biliary
obstruction (55), appear to be bilayer vesicles with diameters of
30-70 nm (47, 56) that contain primarily free cholesterol,
phosphatidylcholine, albumin, and apoCs. These particles also
accumulate in FLD patients as a result of the high plasma
concentrations of free cholesterol and phosphatidylcholine observed in
LCAT deficiency (47, 57).
HDL (71), patients with LCAT deficiency have
increased plasma levels of this nascent lipoprotein particle, the most
effective plasma cholesterol acceptor. Thus, despite reduced HDL, the
process of reverse cholesterol transport may still function in LCAT deficiency.
HDL as
well as the decreased proatherogenic apoB-containing lipoproteins play
a pivotal role in determining the atherogenic risk in patients with
hypoalphalipoproteinemia due to LCAT deficiency. These combined
findings provide new insights into the role that LCAT plays in the
development of glomerulosclerosis and atherosclerosis in LCAT-deficient
states. The identification of LCAT-KO mice as a mouse model in FLD will
permit future evaluation of LCAT gene transfer as a possible treatment
for glomerulosclerosis in patients with LCAT deficiency.
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ACKNOWLEDGEMENTS |
---|
We thank Natalie Murray for excellent technical assistance as well as Donna James for helping in the preparation of this manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Molecular Disease Branch, NHLBI, NIH, Bldg. 10, Rm. 7N115, 10 Center Dr., Bethesda, MD 20892-1666. Tel.: 301-496-6750; Fax: 301-402-0190; E-mail: gl68i@nih.gov.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M008466200
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ABBREVIATIONS |
---|
The abbreviations used are: LCAT, lecithin cholesterol acyl transferase; HDL, high density lipoprotein; LDL, low density lipoprotein; LDLr, low density lipoprotein receptor; CE, cholesteryl ester(s); CETP, cholesteryl ester transfer protein; FLD, familial LCAT deficiency; FED, fish eye disease; apo, apolipoprotein; HF/HC, high fat/high cholesterol; KO, knockout; LpX, lipoprotein X; MES, 4-morpholineethanesulfonic acid; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein.
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