(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) ()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) .
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 , pre-
, or
-lipoproteins is indicated by arrows.
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 mlh
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.
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
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.
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
180,000.
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.