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(Received for publication, October 12, 1995; and in revised form, January 9, 1996) From the
The concentration of high density lipoproteins (HDL) is
inversely related to the risk of atherosclerosis. The two major protein
components of HDL are apolipoprotein (apo) A-I and apoA-II. To study
the role of apoA-II in lipoprotein metabolism and atherosclerosis, we
have developed three lines of C57BL/6 transgenic mice expressing human
apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed
that human apoA-II mRNA was present only in the liver of transgenic
mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis
demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma
of transgenic mice. After 3 months on a regular chow, the plasma
concentrations of human apoA-II were 21 ± 4 mg/dl in the 25.3
line, 51 ± 6 mg/dl in the 21.5 line, and 74 ± 4 mg/dl in
the 11.1 line. The concentration of cholesterol in plasma was
significantly lower in transgenic mice than in control mice because of
a decrease in HDL cholesterol that was greatest in the line that
expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the
plasma concentration of mouse apoA-I (32 ± 2, 56 ± 9, 91
± 7, and 111 ± 2 mg/dl for lines 11.1, 21.5, 25.3, and
control mice, respectively) that was inversely correlated with the
amount of human apoA-II expressed. Additional changes in plasma
lipid/lipoprotein profile noted in line 11.1 that expressed the highest
level of human apoA-II include elevated triglyceride, increased
proportion of total plasma, and HDL free cholesterol and a marked
(>10-fold) reduction in mouse apoA-II. Total endogenous plasma
lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a
level directly correlated with the degree of increased plasma human
apoA-II in the transgenic lines. LCAT activity toward exogenous
substrate was, however, only slightly decreased. The biochemical
changes in the 11.1 line, which is markedly deficient in plasma apoA-I,
an activator for LCAT, are reminiscent of those in patients with
partial LCAT deficiency. Feeding the transgenic mice a high fat, high
cholesterol diet maintained the mouse apoA-I concentration at a normal
level (69 ± 14 mg/dl in line 11.1 compared with 71 ± 6
mg/dl in nontransgenic controls) and prevented the appearance of HDL
deficiency. All this happened in the presence of a persistently high
plasma human apoA-II (96 ± 14 mg/dl). Paradoxical HDL elevation
by high fat diets has been observed in humans and is reproduced in
human apoA-II overexpressing transgenic mice but not in control mice.
Finally, HDL size and morphology varied substantially in the three
transgenic lines, indicating the importance of apoA-II concentration in
the modulation of HDL formation. The LCAT and HDL deficiencies observed
in this study indicate that apoA-II plays a dynamic role in the
regulation of plasma HDL metabolism.
The concentration of high density lipoprotein (HDL) ( Apolipoprotein (apo) A-I
and apoA-II are the main protein components of HDL. ApoA-I plays a
structural role in HDL and is a cofactor of the enzyme
lecithin:cholesterol acyltransferase
(LCAT)(5, 6, 7) . Human apoA-II has a
cysteine in position 6 and is associated with HDL mainly as
disulfide-linked homodimers (17.4 kDa) and heterodimers with apoD and
apoE (38 and 43 kDa,
respectively)(8, 9, 10) . The function of
apoA-II remains poorly defined. ApoA-II was found to activate or
inhibit hepatic
lipase(11, 12, 13, 14) , inhibit or
not influence the action of the cholesteryl ester transfer
protein(14, 15) , interact with a putative HDL
receptor(16) , and displace apoA-I from HDL
particles(17) . The functional role of apoA-II in vivo is unclear; one individual with complete apoA-II deficiency did
not show any lipoprotein alterations(18) . A role for apoA-II
in the coagulation pathway has also been proposed(19) . Human HDL particles can be divided into those with apoA-I but
without apoA-II (LpA-I) and those with both apoA-I and apoA-II
(LpA-I-A-II) (20) . Some studies found that cholesterol efflux
from cells is induced by LpA-I but not by LpA-I-A-II (for a review see (21) ). A third type of HDL that contains apoA-II but not
apoA-I has been described recently (22) . The concentration of
LpA-I and LpA-I-A-II appear to be regulated by different mechanisms;
although apoA-I levels are regulated primarily by apoA-I catabolism,
those of apoA-II appear to be regulated mainly by apoA-II
production(23) . Transgenic mice overexpressing human
apoA-II were first reported by Schultz et al.(24) .
These animals were found to have smaller HDL particles than control
mice; otherwise, there were no other significant changes in lipoprotein
profile. Transgenic mice overexpressing both human apoA-I and apoA-II
showed no difference in lipoprotein plasma concentrations compared with
transgenic mice expressing human apoA-I alone. Interestingly, although
mice expressing both human apoA-I and apoA-II were more resistant than
nontransgenic animals to diet-induced atherosclerosis, they were more
susceptible to this process than transgenic animals expressing human
apoA-I only(25) . In contrast, mice overexpressing a mouse
apoA-II transgene showed increased plasma triglycerides and HDL
cholesterol (HDLc) in plasma, increased HDL size, and increased
susceptibility to atherosclerosis development compared with
nontransgenic controls(26, 27) . These different
effects of mouse and human apoA-II on lipoprotein metabolism and
atherogenesis may be related to their structural differences. Although
human apoA-II contains a cysteine residue and exists as a dimer, most
mammalian apoA-IIs, including mouse apoA-II, have no cysteine and exist
as monomers(28) . Furthermore, the homodimeric human apoA-II
but not monomeric apoA-II is able to induce the efflux of cholesterol
from different cell types to the extracellular
medium(16, 29, 30) . However, recent results
from transgenic mice of a mutated form of human apoA-II that cannot
form dimers through disulfide linkage are clearly different from those
obtained by the overexpression of mouse apoA-II(31) .
Therefore, dimerization per se is probably not the cause of the
different phenotypic effects observed in transgenic mice overexpressing
human versus mouse apoA-II. The role of human apoA-II in
lipoprotein metabolism and atherosclerosis is thus of considerable
interest. In developing transgenic mice expressing human apoA-II, we
noted heretofore unreported phenotypic effects of transgene expression
on plasma apoA-I and LCAT activity. The functional LCAT deficiency
induced in these animals is associated with a marked HDL deficiency.
Furthermore, the phenotypic effects of human apoA-II overexpression are
modulated by a high fat, high cholesterol diet in a manner similar to
the response of normal human subjects to similar diets.
HDL size was analyzed by nondenaturing gradient gel
electrophoresis of Sudan Black B prestained plasma in 4-27%
polyacrylamide gels (Jule, Inc.)(37) . In some cases, the gels
were blotted onto nitrocellulose filters and probed with antibodies to
human apoA-II, mouse apoA-I, or apoA-II. Electrophoresis of pooled
plasma or isolated lipoproteins prestained with Sudan Black B was also
conducted in a 2-3% discontinuous polyacrylamide gels (Lipofilm,
Sebia). HDL isolated by preparative ultracentrifugation were dialyzed
against ammonium acetate buffer (pH 7.4), and aliquots were stained
with 2% sodium phosphotungstate for evaluation by electron microscopy
according to previously described methods(38) .
Figure 1:
A, representation of
the 3-kilobase pair fragment of genomic DNA containing the exon-intron
structure of the human apoA-II gene. The fragment, which contains a HindIII site close to the 3`-end, was prepared by digestion
with MspI and used for microinjection into C57BL/6 eggs. B, Southern blot after polymerase chain reaction performed as
explained under ``Materials and Methods.'' Lane 1,
DNA from human apoA-II transgenic mice; lanes 2 and 3, DNA from control mice; lane 4, DNA from a human
control. C, Northern blot. Part A, 21.5 transgenic
mice; part B, 11.1 transgenic mice; part C, 25.3
transgenic mice. lane 1, liver; lane 2, spleen; lane 3, small intestine; lane 4, kidney; lane
5, testis; lane 6, heart; lane 7, lung; lane
8, muscle. D, relative mRNA abundance of mouse apoA-I
mRNA (top) and mouse apoA-II mRNA (bottom). Black
bars, liver mRNA; shaded bars, small intestinal
mRNA.
Because mouse apoA-II exists only as a
monomer (28) and human apoA-II exists mainly as a homodimer,
the two proteins can be separated on SDS-PAGE. HDL isolated from plasma
of transgenic mice showed an additional band with the apparent
molecular weight of human apoA-II (17.4 kDa) (Fig. 2A),
which was absent in the HDL of control mice. In the two human apoA-II
high expression lines (11.1 and 21.5) the electrophoretic analysis
revealed a decrease in the amount of mouse apoA-I and other low
molecular weight HDL apoproteins, including mouse apoA-II (Fig. 2A). Western blot analysis using an antibody to
human apoA-II confirmed that the 17.4-kDa band present in the HDL of
the transgenic mice was indeed human apoA-II. The transgene-specific
band also showed the expected oxidation reduction pattern
characteristic of human apoA-II (Fig. 2B).
Immunoreactive human apoA-II was undetectable in VLDL and LDL of
transgenic mice (data not shown).
Figure 2:
A, 15%
SDS-PAGE stained with Coomassie Brilliant Blue R-250 of nonreduced mice
HDL isolated by FPLC. Lane 1, molecular weight standards; lane 2, human HDL isolated by ultracentrifugation; lane
3, control mice; lane 4, 25.3 mice; lane 5, 11.1
mice; lane 6, 21.5 mice. In all cases mouse HDL was isolated
from the same volume of plasma. The migration of human apoA-II and
mouse apoA-I and apoA-II is shown by arrows. B,
Western blot analysis using polyclonal antibodies to human apoA-II of a
15% SDS-PAGE of HDL under nonreduced (NRC) or reduced (RC) conditions. Lane 1, molecular weight standards; lane 2, human HDL; lane 3, 11.1 mice; lane
4, 25.1 mice; lane 5, 23.5 mice; lane 6, control
mice. The migration of dimeric (D) and monomeric (M)
human apoA-II is indicated by arrows.
Figure 3:
Electrophoresis of Sudan Black B
prestained plasma and lipoproteins on a 2-3% discontinuous
acrylamide gel. VLDL, LDL, and HDL from control and transgenic mice
were isolated from 1 ml of plasma by sequential ultracentrifugation and
diluted to the same final volumes in control and 11.1 transgenic mice
(0.45 ml of VLDL, 0.5 ml of LDL, and 0.7 ml of HDL). In all cases, 2.5
liters of sample were applied. Lane 1, plasma of control mice; lane 2, VLDL of control mice; lane 3, LDL of control
mice; lane 4; HDL of control mice; lane 5, plasma of
11.1 mice; lane 6, VLDL of 11.1 mice; lane 7, LDL of
11.1 mice; lane 8, HDL of 11.1 mice. The migration of the
samples as
The lipid and apolipoprotein contents of plasma and
isolated lipoproteins after 3 months of regular chow diet are shown in Table 1. In agreement with the SDS-PAGE analysis, the 11.1 line
showed the highest concentration of human apoA-II (74.1 ± 4
mg/dl), the 25.3 line showed the lowest (21.4 ± 4 mg/dl), and
the 21.5 line showed an intermediate concentration (50.8 ± 6
mg/dl) (Table 1, A). There was no significant difference in the
concentration of human apoA-II among male and female transgenic mice in
individual lines. All transgenic lines had significantly lower
concentrations of mouse apoA-I. The concentrations of mouse apoA-I in
the plasma of lines 11.1, 21.5, and 25.3 were 29, 50, and 82% of that
of nontransgenic controls, respectively. The concentration of mouse
apoA-II in the plasma of lines 21.5 and 25.3 was 75 and 79% that of the
controls, but the difference was not statistically significant. In
contrast, the concentration of mouse apoA-II in the plasma of line 11.1
was significantly lower, being only 9% of that of control.
Total
plasma cholesterol was significantly lower in transgenic mice than in
control animals (Table 1, B). This was caused almost exclusively
by the decreased HDLc in transgenic animals. The degree of
hypocholesterolemia was more severe in lines 11.1 and 21.5 than in line
25.3. The proportion of free cholesterol in plasma was elevated in line
11.1. Plasma triglycerides were elevated 3-fold in line 11.1 compared
with the control group but were unchanged in lines 21.5 and 25.3. VLDL cholesterol and triglycerides were increased 8.6- and 6.3-fold,
respectively, in line 11.1 compared with the control group (Table 1, C). The VLDL lipids in the other two lines were similar
to control except that line 21.5 showed a 2-fold increase in VLDL
triglycerides. LDL lipids were different among the different lines; LDL
cholesterol and triglycerides were significantly lower in lines 25.3
and 21.5 than in the controls but were mildly and insignificantly
elevated in line 11.1. HDL lipids were decreased in apoA-II transgenic
mice compared with control mice, but the decline in HDL triglycerides
in lines 11.1 and 25.1 was not significant.
Also in contrast to the results obtained
while the animals were on a regular chow diet, there was no significant
difference in the percentage of free cholesterol in total plasma
between 11.1 mice and controls (Table 2, B). Plasma
triglycerides, HDL triglycerides, and HDLc were increased in line 25.3
compared with control (Table 2, B and C). In contrast, the high
expression line 11.1 developed decreased plasma cholesterol and VLDL
cholesterol and increased LDL cholesterol. As a result of these
changes, the ratio of VLDL cholesterol + LDL cholesterol/HDLc was
decreased in transgenic mice (especially in line 25.3) compared with
controls.
Figure 4:
Electrophoresis of Sudan Black B
prestained plasma (20 liters) in a 4-27% nondenaturing
polyacrylamide gel. A, plasma of mice on regular chow diet. Lane 1, human HDL isolated by ultracentrifugation; lanes 2 and 3, plasma from control mice; lane 4, plasma
from 21.5 mice; lanes 5 and 6, plasma from 11.1 mice; lane 7, plasma from 25.3 mice. B, plasma of mice on a
high cholesterol high fat diet. Lanes 1 and 2, plasma
from control mice; lane 3, 4, and 5, plasma
from 11.1 mice; lanes 6 and 7, plasma from 25.3 mice.
The size of the molecular weight markers in nm is shown at the left.
HDL size
distributions were generally similar when line 11.1, line 25.3, and
control animals were put on a high fat, high cholesterol diet for 3
months (Fig. 4B). The HDL bands were, however, somewhat
broader and slightly skewed to larger particle size than those seen in
plasma of mice fed a regular chow diet. Occasionally, very poor
staining of HDL lipids was observed when plasma from line 11.1 was
fractionated by nondenaturing gradient gel electrophoresis (Fig. 4B, lane 5).
Plasma LCAT activity was measured using the same plasma pools. The
assay was performed both against exogenous substrates and against
endogenous substrates. The LCAT activity against exogenous substrates
was mildly suppressed in the transgenic mouse lines compared with
controls, being 10.6 ± 0.3 , 8.0 ± 0.8 , and 9.9 ±
0.3 nmol ml
Figure 5:
A, LCAT activity toward endogenous and
exogenous substrates was determined in duplicate using pooled plasma
from groups of mice after 3 months of regular chow diet. B,
Correlation of LCAT endogenous activity with mouse plasma apoA-I
control and transgenic mice. The correlation coefficient calculated
corresponds to the r of Spearman.
This change in LCAT activity was
persistent and reproducible. Similar results were obtained when plasma
was obtained from these animals 6 months later. The decrease in the
endogenous LCAT activity was in direct proportion to the degree of
reduction in plasma apoA-I (Fig. 5B).
Figure 6:
Western blot analysis after nondenaturing
gel gradient electrophoresis of plasma probed with antibodies to human
apoA-II (A), mouse apoA-II (B), and mouse apoA-I (C). In the three panels, lanes 1 and 2,
plasma from 21.5 mice; lanes 3 and 4, plasma from
25.3 mice; lanes 5 and 6, plasma from 11.1 mice; lanes 7 and 8, plasma from control mice. Plasma from
all mice was analyzed after 3 months of regular chow diet. The size of
the molecular weight markers is shown on the left.
In summary, human apoA-II represents
The morphology of HDL from transgenic 25.3
and 11.1 mice and from control mice was further analyzed by electron
microscopy (Fig. 7). Although the morphology of HDL particles
from control and 25.3 mice was similar, only sparse clusters of HDL
particles were seen in 11.1 mice (Fig. 7). The marked
heterogeneity of the size of the 11.1 HDL particles corroborated the
electrophoresis data (Fig. 4A). Images suggesting the
existence of a few discoidal particles were seen only rarely (data not
shown); we did not observe the typical HDL pattern described in fish
eye disease and LCAT deficiency that is characterized by the massive
accumulation of discoidal particles forming rouleaux(45) .
Figure 7:
Representative electron micrographs of HDL
isolated from pooled plasma from control mice (A), 23.5 mice (B), and 11.1 mice (C) on a regular chow diet. The
final magnification shown is
Three lines of transgenic mice overexpressing human apoA-II
were generated by microinjection of a 3-kilobase pair fragment of
cloned human apoA-II gene. The transgenic mice expressed human apoA-II
mRNA only in the liver (Fig. 1), a natural tissue of apoA-II
production in humans(24, 46, 47) . The human
protein was secreted into the plasma compartment and was associated
with mouse HDL. The apparent molecular mass of 17.4 kDa, its
recognition by antibodies, and the oxidation reduction pattern were
those expected for human apoA-II (Fig. 2).
When control and
transgenic mice were fed a high cholesterol, high fat diet, the
lipoprotein profiles between transgenic and control mice became more
similar. Interestingly, the HDL deficiency, characterized by low mouse
apoA-I and apoA-II and low HDLc with increased percentage of free
cholesterol, disappeared when the transgenic mice in the 11.1 line were
fed a high cholesterol, high fat diet. Moreover, the 11.1 mice showed
twice as much HDLc and HDL triglycerides compared with levels observed
with a regular chow diet. A notable increase of HDL, compared with an
actual lowering in HDL in control mice, was also observed in 25.3 mice
fed a high cholesterol, high fat diet ( Table 1and Table 2). These observations are especially interesting because
the production of a similar lipoprotein profile in both control and
transgenic mice fed a high fat diet occurred in the presence of an
equally high, if not higher, plasma human apoA-II, which is 119 and
130% that of control values, respectively, in lines 25.3 and 11.1. The mechanism for the restoration of the plasma mouse apoA-I and
apoA-II and HDL lipids in animals on the high fat, high cholesterol
diet is unclear. It is possible that the increased amounts of lipids in
HDL provided enough lipoprotein surface to accommodate mouse apoA-I and
apoA-II and human apoA-II at the same time. Conversely, the increase in
HDL lipids could be the consequence rather than the cause of the more
``normal'' mouse apoA-I and apoA-II levels. These human
apoA-II transgenic mice represent a useful animal model in which to
study the paradoxical HDL-raising effect of a high cholesterol, high
fat diet observed in humans. An increase in HDL level in plasma after a
high cholesterol, high fat diet was also seen in human apoA-I
transgenic mice and found to be a result of increased HDL transport and
decreased HDL fractional catabolic rate(48) . These data, taken
together, strongly suggest that specific structural characteristics of
both apoA-I and apoA-II may be involved in the increase of HDL in
humans after a high cholesterol, high fat diet. A significant
difference between the HDL of mice that expressed high levels of human
apoA-II (line 11.1) and those that expressed lower levels (line 25.3)
is the presence of small particles that were not found in the latter or
in control mice. The presence of these particles was detected in all
cases in mice on a regular chow diet and in most cases in mice on high
cholesterol, high fat diet, which suggests that a threshold of human
apoA-II may be needed to produce these small HDL particles. The ability
of human apoA-II to induce the formation of small HDL particles had
been observed previously both in vivo and in
vitro(24, 49) . Therefore, we can infer from
these and other data (reviewed in (50) ) that both human apoA-I
and apoA-II can contribute to the size heterogeneity of human HDL.
Even in the case of a greater
susceptibility of apoA-II-containing particles to hepatic lipase
action, that should not in itself cause a marked reduction in the
amount of mouse apoA-I or apoA-II in these particles. An excess of
human apoA-II can displace apoA-I from HDL from several species
(including humans(15, 17) ); it can also displace
human apoA-I in artificial vesicles in vitro(55) . We
speculate that the human apoA-II physically displaced the mouse apoA-I
from these HDL particles. The displaced mouse apoA-I would be
metabolized rapidly, being cleared mainly by the
kidney(56, 57, 58) . We postulate an
increased catabolism instead of decreased synthesis of the mouse apoA-I
protein as the cause of the reduction in mouse apoA-I in these animals.
This hypothesis is supported by the observation that the level of mouse
apoA-I mRNA showed only minor differences between transgenic and
nontransgenic animals (Fig. 1D). The cause of the
almost complete disappearance of mouse apoA-II in the high expressor
line 11.1 is not known. The mechanism could be similar to that proposed
for the decrease of mouse apoA-I, i.e. displacement by human
apoA-II. However, to our knowledge, there is no evidence that human
apoA-II displaces mouse apoA-II from lipoprotein surfaces, although it
is not an unreasonable hypothesis. Interestingly, a similar deficiency
of mouse apoA-I in human apoA-I transgenic mice was described, and the
mechanism was found to be mediated at a post-transcriptional
level(5) . The metabolic effects, if any, of mouse apoA-II
deficiency are unclear. In contrast, the displacement of apoA-I by
human apoA-II has been shown to sharply decrease LCAT
activity(59) . Unlike apoA-I, apoA-II-lipid complexes cannot
serve as substrates for LCAT (60, 61) even though they
can bind LCAT(62) . Moreover, mice genetically modified to be
deficient in apoA-I are also LCAT-deficient(63) .
Interestingly, compared with controls, mice heterozygous for apoA-I
deficiency have similar LCAT activity toward exogenous substrates but
only half their endogenous LCAT activity(63) . These findings
are very similar to those for 11.1 mice (Fig. 5). Thus, it is
reasonable to conclude that the low apoA-I concentration of 11.1 mice
results in its low endogenous LCAT activity and in an increased
proportion of HDL free cholesterol. On the other hand, the much higher
LCAT exogenous activity found in 11.1 mice is consistent with the idea
that LCAT can associate with apoA-II-containing HDL particles. The
lipoprotein phenotype of 11.1 mice is similar in certain respects
(increased free cholesterol in plasma and HDL and hypertriglyceridemia
that could be caused by a low LCAT activity) to that of fish eye
disease. This disease is characterized by partial LCAT deficiency
caused usually by LCAT gene mutations (64, 65) and
rarely by apoA-I mutations(66) . It is noteworthy that, as in
11.1 mice, a significant improvement of the HDL deficiency also occurs
in patients with LCAT deficiency fed a high fat diet(67) .
However, the 11.1 transgenic mouse HDL did not show the rouleau
formation characteristic of the accumulation of discoidal particles in
fish eye disease HDL(45) . It may be that the concentration of
11.1 HDL on the mesh grid is below the threshold necessary for forming
rouleaux, or alternatively, apoA-II-containing particles may have lower
tendency to form discs than apoA-I-containing particles(68) . The lipoprotein changes observed in the human apoA-II transgenic
mice on a regular chow diet in this study are much more profound than
those reported by Schultz et al.(24) , who noted
mainly the appearance of small HDL particles. The reasons for these
differences are unclear, although the substantially higher human
apoA-II expressed in this study (about one-third higher than the
highest overexpressor lines in the previous study) could be a factor.
It is noteworthy that the analysis of the progeny of the same human
apoA-II transgenic mice generated by Schultz et al.(24) demonstrated lower cholesterol and HDLc
concentrations and higher triglycerides in transgenic mice than
controls(14) . Moreover, a preliminary communication in
abstract form from another group also reported the existence of
hypocholesterolemia and possible LCAT impairment in human apoA-II
transgenic mice(69) . Further studies on these genetically
modified animals should allow a better understanding of the role of
human apoA-II in lipoprotein metabolism and atherosclerosis. While
this manuscript was in preparation, Francone et al.(70) presented evidence that transgenic mice expressing
human LCAT showed preference for HDL particles that contain only
apoA-I, an observation that complements the findings in this study.
Portions of this work
have been presented at the 1994 Meeting of the United States and
Canadian Academy of Pathology and at the Xth International Symposium on
Atherosclerosis.
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 6720-6728
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)cholesterol in plasma is inversely correlated with the
risk of atherosclerosis(1, 2, 3) . However,
the mechanism by which HDL exerts its anti-atherogenic action is poorly
understood. Reverse cholesterol transport is one HDL function that may
be important in this respect(4) .
Animals and Preparation of Nucleic Acids
The
fertilized eggs used in the microinjection were from the matings of
C57BL/6 mice. The microinjected human apoA-II gene was a 3-kilobase
pair fragment isolated from human genomic DNA prepared by digestion
with the enzyme MspI and was subcloned into the AccI
site of Bluescript KS + phagemid (Stratagene). It contains 1
kilobase pair in the 5`-flanking region. The human apoA-II gene
fragment was removed from the plasmid by double digestion with ClaI and XhoI, fractionated on agarose gels, and
purified with a Qiaex kit (QIAGEN) before microinjection. The procedure
for the identification of transgenic animals consisted of a combination
of polymerase chain reaction and Southern blotting of tail DNA. 2
µg of genomic DNA was amplified using as primers oligonucleotides
with the sequences 3`-CCTCATCGTGTCAACGACGC-5` and
3`-TTCCTCGGTACACACCTCTC-5`. One-fifth of the polymerase chain reaction
products (total volume, 50 µl) was electrophoresed on a 2% agarose
gel, transferred to a nylon membrane, and hybridized to a 5`-end
P-labeled oligonucleotide with the sequence
3`-GACAGAGGCTTGGTTT-5`, specific for the human apoA-II gene. From 2000
embryos implanted, 145 pups were born. Four of these were transgenic
and designated 24.2, 21.5, 25.3, and 11.1. The 24.2 founder died before
breeding. Three different lines of transgenic mice were developed from
the other 3 founders. The mode of transmission of the transgene in the
11.1 line suggested integration in chromosome X and the other two lines
were autosomal. Transgenic and control mice used for the RNA,
apolipoprotein, and lipoprotein studies were between 2 and 3 months old
at the beginning of the studies. They were fed ad libitum a
regular chow diet (5001, The Richmond Standard, PMI Feeds, Inc.,
Richmond, IN) or a high fat, high cholesterol diet (75% mouse chow
5015, 7.5% cocoa butter, 1.25% cholesterol, 0.5% sodium cholate; TD
88051, Harlan Teklad, Medium WI) and had free access to water. Total
RNA was isolated from different tissues as described previously by
Chomczynski and Sacchi(32) . 5 µg of RNA was
electrophoresed in 2% agarose gels, blotted to nylon membranes, and
probed with cloned cDNAs labeled with [
P]dCTP by
nick translation (human apoA-II cDNA was used to probe the human
apoA-II mRNA, whereas mouse apoA-II and rat apoA-I cDNAs were used to
probe the mouse apoA-II and apoA-I mRNAs, respectively). The intensity
of the x-ray bands was quantified by a LKB Bromma VitroScan XL enhance
laser densitometer with the Gel Scan XL software.
Lipids, Lipoproteins, and Apolipoproteins
Blood
was obtained from the tail veins of mice and collected in tubes
containing EDTA and sodium azide(33) . Total cholesterol, free
cholesterol, and triglycerides were measured colorimetrically using
commercial kits adapted to an autoanalyzer RA-XT
(Technicon)(34) . Triglyceride determinations were corrected
for the free glycerol present in plasma (Sigma). Plasma lipoproteins
were fractionated by FPLC; 0.2 ml of plasma was loaded on a Superose 6
HR column (Pharmacia Biotech Inc.) as described
previously(35) . In some cases, plasma was pooled from two
siblings of the same sex that were kept in the same cage before FPLC
analysis. Twenty-five 1-ml fractions were collected, and samples
corresponding to very low density lipoproteins (VLDL) (3 ml), low
density lipoproteins (LDL) (3 ml), and HDL (6 ml) were pooled
individually. When large quantities of isolated lipoproteins were
required for analysis, sequential ultracentrifugation was used (36) .Apolipoprotein Isolation and Generation of
Antibodies
ApoHDL was prepared by delipidation of HDL with
diethyl ether-ethanol 3:1 (v/v)(39) . Mouse apoA-I was isolated
by gel filtration chromatography (Sephacryl S-200, 1200 
1.5 cm)
from apoHDL (40) . Antiserum to mouse apoA-I was produced by
subcutaneous immunization of a rabbit with the purified apolipoprotein.
Antiserum to mouse apoA-II was kindly provided by Drs. R. T. Kitchens
and Gustav Schonfeld (Department of Internal Medicine, Washington
School of Medicine, St. Louis, MO). Antiserum to human apoA-II was
purchased from Boehringer Mannheim. Plasma human apoA-II concentration
was measured using a commercial immunoassay kit (Immuno AG).
Concentrations of plasma mouse apoA-I and apoA-II were determined by a
radial immunodiffusion assay(41) .LCAT Assays
Plasma LCAT activity against
endogenous substrate was measured by using the lipoproteins of the
whole plasma as substrate(42, 43) . Results were
expressed as cholesterol molar esterification rate. Plasma LCAT
activity against exogenous substrate was determined by using model
reassembled HDL that contained
1-palmitoyl-2-oleoyl-sn-3-phosphocholine,
[
H]cholesterol and apoA-I as a
substrate(44) .
Expression of the Human Apolipoprotein A-II in
Transgenic Mice
Independent human apoA-II transgenic mouse lines
were bred from the three founder animals; these lines were designated
25.3, 21.5, and 11.1 (Fig. 1, A and B). By
quantitative dot-blot analysis (data not shown), the 11.1 and 21.5
lines each contained 20 copies, and the 25.3 line contained
40 copies of the human apoA-II gene. The tissue distribution of
transgene mRNA expression was determined by Northern blot analysis (Fig. 1C). All three lines expressed human apoA-II mRNA
in the liver only, with no detectable expression in the small
intestine, spleen, kidney, testis, heart, lung, or muscle. Finally,
despite the different levels of expression of human apoA-II mRNA, the
amount of mouse apoA-II and apoA-I mRNAs only showed minor differences (Fig. 1D).
Lipoprotein Profile of Transgenic Mice on a Regular Chow
Diet
We first analyzed the plasma and lipoprotein fractions by
native polyacrylamide gels. Lipoprotein fractions were isolated by
sequential ultracentrifugation. They were prestained with Sudan Black B
and fractionated by 2-3% polyacrylamide nondenaturing gradient
gel electrophoresis. The electrophoretic pattern of total plasma from
the 21.5 and 25.3 lines was similar to that of control mice, with HDL
appearing as the main lipoprotein band, which was slightly less intense
in staining than the HDL band from control mice. In contrast, the total
plasma of the 11.1 line showed a clearly different pattern. VLDL was
the major lipoprotein band in 11.1 plasma, and no HDL band was detected (Fig. 3, lane 5). When HDL isolated by ultracentrifugal
flotation from this line was analyzed by this method, it was detectable
albeit at markedly lower concentrations (Fig. 3, lane
8).
, pre-
, or -lipoproteins is indicated by arrows.
Lipoprotein Profile of Transgenic Mice on High
Cholesterol, High Fat Diet
We studied the effect of a high fat,
high cholesterol diet on the plasma lipoproteins in the apoA-II
transgenic animals. Control mice and transgenic mouse lines 11.1 and
25.3 were put on a diet containing 15.75% fat, 1.25% cholesterol, and
0.5% sodium cholate for 3 months. The results of the quantitative
analysis of apolipoproteins and lipids in whole plasma and isolated
lipoproteins are shown in Table 2. The concentrations of mouse
apoA-I and apoA-II were not significantly different among the three
groups (Table 2, A). This is especially noteworthy for line 11.1
because the mouse apoA-I and apoA-II levels were markedly reduced
compared with controls when they were fed a regular chow. The
normalization of the plasma mouse apoA-I and apoA-II in line 11.1 was
not a result of any change in the transgene-specific human apoA-II
concentration, which remained largely unchanged by the diet. We also
noted a greater variability in the concentration of plasma apoA-I
compared with the results obtained when they were fed a regular chow
diet. This was especially true in line 11.1, where the apoA-1 level
varied from 28 to 124 mg/dl, when these animals were on a high fat,
high cholesterol diet.
HDL Particle Size in the Plasma of Transgenic
Mice
The size of the HDL particles was analyzed by nondenaturing
4-27% gradient polyacrylamide gel electrophoresis of prestained
plasma from mice on a regular diet (Fig. 4A) and from
those on a high cholesterol, high fat diet (Fig. 4B).
In the first immunoblot, human HDL isolated by ultracentrifugation was
used as a control. It migrated in a polydisperse pattern with particles
ranging in size from 6.7 to 12.2 nm (Fig. 4A). All
three transgenic lines on a regular diet showed substantially reduced
lipid staining of their HDL particles compared with controls. In
keeping with the results of lipid and lipoprotein composition, this
decrease in lipid staining was especially marked in the HDL of line
11.1. There was no difference in the HDL size distribution of the 25.3
line compared with controls, both showing a single peak with a diameter
of 10.2 nm. In contrast to the relatively homogeneous HDL size found in
control mice, HDL particles from the plasma of line 21.5 and 11.1 were
heterogeneous in size, containing two main populations having peak
diameters of 7.9 and 10 nm, the former marking the predominant HDL
species in the plasma of these two transgenic lines.
Percentage of Free Cholesterol in Lipoproteins and LCAT
Activities in the Plasma of Transgenic Mice on Regular Chow
Diet
In order to confirm that there is increased percentage of
free cholesterol found in the plasma of 11.1 mice and to know if this
parameter was elevated in the individual lipoprotein fractions, we
pooled plasma from control mice and individual transgenic lines.
Lipoproteins from the plasma pools were isolated by ultracentrifugation
and total and free cholesterol was measured (Table 3). The
percentage of free cholesterol in plasma was elevated in 11.1 and 21.5
mice compared with control mice. This was due to an increased
proportion of free cholesterol in LDL and HDL in 11.1 mice, whereas the
proportion of free cholesterol was increased also in the LDL in 21.5
and 25.3 mice. It is noteworthy that a considerable amount of
cholesterol was detected in d>1.21 g/ml plasma fraction in
transgenic and control mice, mostly free cholesterol. This was not due
to a delay in preparing the lipoproteins because ultracentrifugation
was initiated within a few hours after the collection of plasma.
h
in lines 25.3, 21.5,
and 11.1, respectively, and 14.8 ± 0.3 nmol
ml
h
in controls (Fig. 5A). When the assay was performed against
endogenous substrates, there was a substantially greater decrease in
LCAT activity in transgenic animals compared with controls. The
activities were: 76 ± 0.6, 50 ± 10.2, and 18.4 ±
2.8 nmol ml
h
in lines 25.3, 21.5,
and 11.1, respectively, compared with a value of 92.7 ± 2.0 nmol
ml
h
in controls (Fig. 5A). Therefore, compared with controls, the LCAT
activities in transgenics were: for exogenous substrates, 72, 54, and
66.9% and for endogenous substrates, 82, 54, and 20% for lines 25.3,
21.5, and 11.1, respectively.
Apolipoprotein Composition and Electron Microscopy of HDL
of Control and Control Mice
The reduction in LCAT activity in
transgenic mice was much more pronounced when endogenous substrates
were used instead of exogenous substrates. We decided to examine in
greater detail the major endogenous substrate, HDL, in these animals.
The relative content of human apoA-II, mouse apoA-II, and mouse apoA-I
in HDL samples from transgenic and control mice were investigated by
Western blot analysis after fractionation on nondenaturing
polyacrylamide gradient gel electrophoresis. No human apoA-II was
detected in control HDL (Fig. 6A). It was present only
in the 10.2-nm region of line 25.3 mice, the line with the lowest level
of expression. In the two high expressor lines, 21.5 and 11.1, it was
detected in HDL particles in the 10-, 7.9-, and 7.6-nm regions. The
distribution of immunoreactive mouse apoA-II was somewhat different.
Both control and 25.3 mouse HDL showed a major band at 10.2 nm. In the
21.5 and 11.1 lines, a weakly staining diffuse pattern spanning from 10
to 7.3 nm was observed (Fig. 6B). Major changes in
mouse apoA-I immunoreactivity were also seen (Fig. 6C).
In control and the lower expressor 25.3 line, an intensely stained band
was detected at 10.2 nm. The immunoreactivity in the two high expressor
lines 21.5 and 11.5 was diffuse and spanned from 10 to 7.9 nm. There
was also a major difference between these two lines in that the one
with the highest human apoA-II expression (line 11.1) had very faint,
barely detectable immunoreactive apoA-II in its HDL (Fig. 6C).
40% of the entire apoprotein mass in the HDL of the 21.5 line and
accounts for almost the entire complement of apolipoproteins in the HDL
of the highest human apoA-II expressor line 11.1. In other words, the
vast majority of HDL particles in this line, and to a lower extent in
line 21.5, contain only human apolipoprotein A-II and little or no
mouse apoA-I or apoA-II.
180,000.
Changes in the Lipoprotein Profile of Transgenic
Mice
The concentration of human apoA-II in lines 23.5, 21.5, and
11.1 on a regular chow diet was 62, 147, and 215% of that found in
normal human plasma, respectively(30) . All transgenic mouse
lines showed a significant reduction of plasma cholesterol due to
decreased HDLc concentration (Table 1). The decrease in these
lipid values was correlated with the degree of overexpression of human
apoA-II. In the animals (line 11.1) that showed the highest human
apoA-II expression, there was also an increased proportion of free
cholesterol in both plasma and HDL, an increase in triglycerides and a
marked decrease in the concentration of plasma mouse apoA-I and
apoA-II. Thus, there were substantial quantitative and qualitative
differences in the lipoprotein profiles of the different transgenic
lines. The magnitude of the changes was related to the concentration of
human apoA-II in plasma (see Table 1), and the changes are likely
the direct result of human apoA-II overexpression.
Possible Mechanisms Underlying the Lipoprotein Changes
Found in the Transgenic Mice Fed a Regular Chow Diet
There are a
number of possible mechanisms that are responsible for the changes
found in the lipoprotein profiles in the transgenic mice. Human apoA-II
associated with VLDL has been postulated to be an inhibitor of
lipoprotein lipase (51) . However, no human apoA-II was
detected in the triglyceride-rich particles in the transgenic mice when
analyzed by SDS-PAGE. Increased apoA-II has also been correlated with
elevated plasma free fatty acids (52) , which could in turn
stimulate VLDL synthesis by the liver. However, we did not find any
correlation between free fatty acid and human apoA-II concentrations in
plasma in these animals (data not shown). ApoA-II-containing human HDL
particles have been found to be better substrates for hepatic lipase
than those without apoA-II(11, 12) . Hepatic lipase is
thought to mediate the conversion of HDL
to the smaller
HDL particles, liberating lipids and apoA-I during the
process(53) . Transgenic mice overexpressing human hepatic
lipase displayed HDL particles that are smaller, containing 34% less
HDLc than HDL from nontransgenic controls but having normal apoA-I
content(54) . It is possible that the more effective action of
hepatic lipase on the human apoA-II-containing HDL particles
contributed to the lower HDLc in the transgenic animals (see Table 1). This interpretation is supported by the appearance of
smaller human apoA-II-containing HDL particles in transgenic lines 21.5
and 11.1 that expressed high levels of human apoA-II but not in line
25.3, which expressed lower levels of the protein. In contrast, another
study using human apoA-II transgenic mice proposed that human apoA-II
could inhibit the action of hepatic lipase(14) , a conclusion
corroborated by other studies(13) . Any possible
interrelationship between apoA-II and hepatic lipase action must be
considered unproven at this point.
)
We thank Dr. Attila Pethö-Schramm
for advice in the cloning strategy, Gerri Hanten for microinjection,
Sally Tobola and Linda Phillips for secretarial assistance, and Dr.
Jordi Ordóñez-Llanos for
support.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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