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【摘要】
Objective- Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is also expressed by macrophage-derived foam cells in human atherosclerotic lesions, but the exact role of macrophage PLTP in atherosclerosis is unknown.
Methods and Results- To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP -/- ) mice into irradiated low-density lipoprotein receptor knockout mice. Selective deficiency of macrophage PLTP (PLTP -M/-M ) resulted in a 29% ( P <0.01 for difference in lesion area) reduction in aortic root lesion area as compared with mice possessing functional macrophage PLTP (384±36*10 3 µm 2 in the PLTP -M/-M group (n=10), as compared with 539±35*10 3 µm 2 in the PLTP +M/+M group (n=14)) after 9 weeks of Western-type diet feeding. The decreased lesion size in the PLTP -M/-M group coincided with significantly lower serum total cholesterol, free cholesterol, and triglyceride levels in these mice. Furthermore, plasma PLTP activity in the PLTP -M/-M group was 2-fold ( P <0.001) lower than that in the PLTP +M/+M group.
Conclusion- Macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.
Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is also expressed by macrophage-derived foam cells in human atherosclerotic lesions, but the exact role of macrophage PLTP in atherosclerosis is unknown. To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP -/- ) mice into irradiated low-density lipoprotein receptor knockout mice. Macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.
【关键词】 apolipoproteins atherosclerosis lipid transfer proteins macrophages mouse models
Introduction
Phospholipid transfer protein (PLTP) is a lipid transfer protein with a wide tissue distribution. 1 In human plasma, 2 forms of PLTP can be distinguished, one with high and one with a low active form. 2,3 PLTP transfers surface phospholipids from chylomicrons and very low-density lipoproteins (VLDL) to high-density lipoprotein (HDL) during lipolysis 4 and also participates in HDL conversion. 5,6 PLTP modifies HDL 3 particles and concomitantly generates large particles resembling HDL 2 and a population of small poorly lipidated preß-HDL particles, which are efficient acceptors of cholesterol from peripheral cells. 7,8
The function of PLTP in the development of atherosclerosis is far from resolved, as PLTP has been reported to be a pro-atherogenic factor, but also anti-atherogenic properties have been associated with PLTP. 9 Increased PLTP activity in plasma is a risk factor for coronary heart disease, 10 whereas serum total PLTP mass protects against coronary heart disease in humans. 11 In studies using genetically modified mice, primarily pro-atherogenic effects of PLTP on atherosclerosis have been reported. Mice overexpressing human PLTP displayed decreased plasma HDL levels, 12-15 increased VLDL levels, 13,16 and elevated susceptibility to atherosclerosis. 13,17,18 Conversely, PLTP deficiency is associated with decreased apolipoprotein B secretion from mouse hepatocytes 19,20 with a concomitant decrease in atherosclerotic lesion size. 20
The role of PLTP in atherosclerosis is complex and functions that affect its atherogenicity include: (1) enhancement of cholesterol efflux via generation of preß-HDL particles; 12,14,15 (2) determination of plasma HDL levels 21,22 by mediating the transfer of post-lipolytic surface remnants of chylomicrons and VLDL into HDL; (3) influencing the production of apoB-containing lipoproteins by the liver; 19,20 (4) influencing the accumulation of anti-oxidative vitamin E in low-density lipoprotein (LDL) and VLDL; 23 (5) transferring lipopolysaccharide from HDL to LDL. 24
Recently, PLTP was demonstrated in lipid-laden macrophage-derived foam cells in human atherosclerotic lesions. 25-27 This observation raised the question whether PLTP has a direct role in cholesterol retention or removal from foam cells present in atherosclerotic lesions. The expression of PLTP in macrophages is upregulated by liver X receptor and retinoid X receptor agonists. 26,28 Furthermore, in vitro studies have demonstrated that PLTP mRNA and protein expression as well as activity is increased on cholesterol loading of macrophages, 25,27 and that exogenously added PLTP promotes cholesterol and phospholipid removal from murine macrophages via an ATP-binding cassette transporter A1-mediated pathway. 29 Currently, however, the function of PLTP production by macrophages in atherosclerosis in vivo is unknown.
Macrophages, present in atherosclerotic lesions, primarily depend on infiltration from bone marrow-derived monocytes into the arterial wall. Therefore, to clarify the role of macrophage PLTP in atherogenesis, we created a mouse model with selective deficiency of PLTP in hematopoietic cells, including macrophages, by using the bone marrow transplantation technique. Our results demonstrate that macrophage PLTP is a significant modulator of plasma PLTP activity and that PLTP deficiency in macrophages leads to lowered atherosclerotic lesion development in LDL receptor knockout (LDLr -/- ) mice on Western-type diet (WTD).
Materials and Methods
For detailed methodology, please see http://atvb.ahajournals.org.
Chimeric mice with a selective deficiency of PLTP in hematopoietic cells, including macrophages, were generated by using the bone marrow transplantation technique. Female LDLr -/- mice (C57Bl/6J strain; N5) were exposed to a single dose of 9 gray (Gy) (0.19 Gy/min, 200 kV, 4 mA) X-ray total body irradiation, using an Andrex Smart 225 Röntgen source (YXLON International, Copenhagen, Denmark) 1 day before transplantation. Irradiated recipients were transplanted by intravenous injection of 0.5 x 10 7 bone marrow cells, isolated from male wild-type C57Bl/6J PLTP +/+ mice or male PLTP -/- mice on the C57Bl/6J background. 21 After that, transplanted LDLr -/- mice (from now indicated as PLTP +M/+M and PLTP -M/-M mice, respectively) were maintained on sterilized regular chow diet (RM3; Special Diet Services, Witham, UK) for 8 weeks to allow the mice to recover from the bone marrow transplantation. To induce the development of atherosclerosis, the mice were fed WTD, containing 15% (w/w) total fat and 0.25% (w/w) cholesterol (Diet W; Special Diet Services) for 9 weeks, after which the mice were euthanized and atherosclerotic lesion development and the composition of the lesions was quantified.
At 8 weeks posttransplantation when the mice were on regular chow diet and at 17 weeks posttransplantation when the animals were fed WTD, blood was drawn after an overnight fasting period for determination of serum cholesterol, triglycerides, and phospholipids. In addition, the distribution of lipids between the different lipoproteins in serum was determined. Preß-HDL and -HDL levels, 12 mouse apolipoprotein (apo)A-I 12, PLTP activity, 5,30 hepatic lipase activity, 31 and lecithin-cholesterol acyltransferase activity 32 were determined as previously described.
PLTP mRNA expression was determined in whole livers of transplanted mice at 17 weeks posttransplantation and in parenchymal, endothelial, and Kupffer cells isolated from livers of wild-type C57Bl/6J mice 33 using real time-quantitative polymerase chain reaction. Furthermore, PLTP protein levels were determined immunohistochemically in livers and lungs of the transplanted mice at 17 weeks posttransplantation.
Animal experiments were performed at the Gorlaeus Laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with the National Laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of Leiden University.
Statistical analyses were performed utilizing the unpaired Student t test (Instat GraphPad software, San Diego, Calif).
Results
Generation of LDLr -/- Mice With a Specific Disruption of PLTP in Bone Marrow-Derived Cells
To evaluate the role of macrophage PLTP in lipoprotein metabolism and in the development of atherosclerotic lesions, PLTP gene expression was specifically disrupted in cells of hematopoietic origin by transplantation of bone marrow from C57Bl/6J mice lacking functional PLTP 21 into LDLr -/- mice (supplemental Figures I and II, available online at http://atvb.ahajournals.org).
Macrophage PLTP Deficiency Decreases Atherosclerosis in LDLr -/- Mice
To induce atherosclerotic lesion development, the transplanted mice were fed WTD, containing 0.25% cholesterol and 15% fat, starting at 8 weeks after transplantation. After 9 weeks of WTD feeding, lesion development was analyzed in the aortic root of the PLTP +M/+M and PLTP -M/-M mice. As shown in Figure 1 A, macrophage PLTP deficiency leads to a 29% ( P <0.01) decrease in the mean atherosclerotic lesion area (PLTP +M/+M, 539±35 x 10 3 µm 2, n=14 versus PLTP -M/-M, 384±36 x 10 3 µm 2, n=10). The relative macrophage content of the lesions of WT LDLr -/- mice was 32±3%, whereas the collagen content was 7±1% ( Figure 1B and 1 C). No significant effect of macrophage PLTP deficiency was observed on the relative macrophage content of the lesions (35±3%). However, a trend to a reduced collagen content was observed (3±1%, P =0.07) in absence of macrophage PLTP production ( Figure 1 C). Analysis of the average thickness of the caps of the lesions showed that the caps were smaller in animals transplanted with PLTP -/- bone marrow (7±2 µm 2, P <0.01) as compared with control transplanted animals (21±4 µm 2 ). The trend to reduced collagen content as well as the smaller cap thickness observed in PLTP -M/-M mice is most likely a direct effect of the smaller and thus less advanced lesions observed in these animals.
Figure 1. Atherosclerotic lesion formation in LDLr -/- mice with macrophage PLTP deficiency. Formation of atherosclerotic lesions was determined at 17 weeks posttransplantation, ie, after 9 weeks WTD, in PLTP +M/+M and PLTP -M/-M mice. A, The mean lesion area (µm 2 ) was calculated from 10 oil red O-stained sections of the aortic root at the level of the tricuspid valves. B, Immunohistochemical quantification of lesion macrophages using MOMA-2. The macrophage content of the lesions is indicated as % of the total lesion area. C, Masson trichrome staining; blue staining indicates collagen. The collagen content of the lesions is indicated as % of the total lesion area. Values represent the means±SEM calculated from 14 PLTP +M/+M mice (white column) and 10 PLTP -M/-M mice (black column). Statistically significant differences between PLTP +M/+M and PLTP -M/-M mice are indicated. ** P <0.01. Original magnification x 50.
Macrophage PLTP Deficiency Influences Serum Lipid Levels and Lipoprotein Distribution
During the course of the experiment, the effects of PLTP deficiency in hematopoietic cells on serum lipid, lipoprotein, and apoA-I concentrations were determined. At 8 weeks posttransplantation, when the mice had been on a chow diet, serum total cholesterol (TC) and free cholesterol (FC) concentrations were similar in both groups ( Table 1 ). No significant differences were observed between groups in VLDL and LDL cholesterol levels ( Figure 2 A). However, HDL cholesterol was significantly higher in the PLTP -M/-M group ( P <0.01). On challenging the mice with WTD, the concentrations of TC (PLTP +M/+M from 7.14 mmol/L to 29.13 mmol/L, PLTP -M/-M from 7.59 mmol/L to 21.67 mmol/L) and FC (PLTP +M/+M from 1.44 mmol/L to 8.42 mmol/L, PLTP -M/-M from 1.64 mmol/L to 6.71 mmol/L) increased dramatically in both groups ( Table 1 ). However, in the PLTP -M/-M group, TC and FC levels were significantly lower than in PLTP +M/+M group ( P <0.05 for both TC and FC). The increases in FC and TC were the result of a marked increase in VLDL and LDL cholesterol, which was significantly lower ( P <0.05) for the PLTP -M/-M group ( Figure 2 B). Under these feeding conditions, HDL cholesterol was decreased in both groups but there was no significant difference between the groups.
TABLE 1. Effect of Macrophage PLTP Deficiency on Serum Lipids and ApoA-I Concentrations
Figure 2. The effect of macrophage PLTP deficiency on plasma cholesterol and phospholipid distribution. Blood samples were drawn after 8 weeks feeding regular chow diet and at 17 weeks posttransplantation after 9 weeks feeding of WTD. Serum from individual mice was fractionated on a Superose 6 PC column and fractions 2 to 14 represent VLDL and LDL, and fractions 15 to 22 represent HDL. The distribution of cholesterol (A, B) and phospholipids (C, D) between the different lipoproteins of PLTP +M/+M and PLTP -M/-M mice was determined. Values shown are the means of 10 to 14 mice. Statistically significant differences between PLTP +M/+M and PLTP -M/-M mice are indicated. * P <0.05 and ** P <0.01.
WTD feeding increased triglycerides (TG) in the PLTP +M/+M group, whereas TG levels in the PLTP -M/-M group were decreased ( Table 1 ). This resulted in significantly lower levels of TG observed in mice transplanted with PLTP -/- bone marrow after 9 weeks WTD feeding (PLTP +M/+M, 2.08 mmol/L versus PLTP -M/-M 1.16 mmol/L, P <0.01). Size-exclusion chromatography analysis showed a trend to lower levels of TG in the VLDL and LDL fractions of PLTP -M/-M mice (data not shown).
The concentration of total phospholipids (PL) was higher in the PLTP -M/-M group after 8 weeks on chow diet ( P <0.01) ( Table 1 ), however HDL phospholipids did not differ significantly between the groups ( Figure 2 C). After 9 weeks on WTD, no differences in total PL were observed, although HDL-associated PL was higher in PLTP -M/-M mice ( P <0.01) ( Figure 2 D).
In addition, the effects of disruption of PLTP in bone marrow-derived cells on serum apoA-I levels were determined both on chow diet and after feeding WTD ( Table 1 ). In PLTP +M/+M mice WTD feeding resulted in a significant reduction in the apoA-I concentration compared with the values on chow diet (from 1.44 g/L to 0.98 g/L, P <0.001), whereas in PLTP -M/-M mice a trend to increased ( P =0.09) apoA-I levels was observed (from 1.30 g/L to 1.63 g/L). As a result, on WTD the serum apoA-I concentration was significantly higher in the PLTP -M/-M group (PLTP +M/+M, 0.98 g/L versus PLTP -M/-M, 1.63 g/L; P <0.001).
As PLTP acts as an important factor in the production of preß-HDL particles, it was also of interest to study preß-HDL levels in this experimental setting. At 8 weeks posttransplantation, no significant effect of macrophage PLTP deficiency was observed on circulating preß-HDL levels. However, after 9 weeks of WTD feeding, the preß-HDL levels were 24% lower in PLTP -M/-M mice as compared with PLTP +M/+M mice (PLTP +M/+M, 20.7% versus PLTP -M/-M, 15.7%; P <0.05).
Macrophage PLTP Is an Important Contributor to Plasma PLTP Activity
Size-exclusion chromatographic analysis demonstrated that PLTP activity was almost exclusively associated with HDL lipoproteins with a similar distribution pattern in both groups (supplemental Figure III). On chow diet, plasma PLTP activity was 1.4-fold lower in the PLTP -M/-M group as compared with the PLTP +M/+M group (PLTP +M/+M, 19.5 µmol/mL per hour versus PLTP -M/-M, 14.3 µmol/mL per hour; P <0.01) ( Table 2 ). WTD feeding increased plasma PLTP activities in both groups of mice and were 2.5-fold higher in PLTP +M/+M mice and only 1.7-fold higher in PLTP -M/-M mice after 9 weeks on WTD as compared with the values on chow diet. As a consequence, plasma PLTP activity in mice of the PLTP -M/-M group was 2-fold lower as compared with the activity in mice of the PLTP +M/+M group (PLTP +M/+M, 48.3 µmol/mL per hour versus PLTP -M/-M 24.0 µmol/mL per hour; P <0.001). These results demonstrate that macrophage PLTP is an important contributor to plasma total PLTP activity. The activity of hepatic lipase and lecithin-cholesterol acyltransferase in plasma did not differ between the 2 groups ( Table 2 ).
TABLE 2. Effect of Macrophage PLTP Deficiency on Plasma PLTP, HL, and LCAT Activities
PLTP is a ubiquitously expressed protein with a moderate level of expression in liver. 1 However, liver as a large organ can contribute to a relatively high level to circulating PLTP. Therefore, PLTP mRNA expression was determined in livers of the transplanted mice ( Figure 3 A). Interestingly, a trend to reduced PLTP expression was evident in livers from PLTP -M/-M mice (0.007±0.002) as compared with livers from PLTP +M/+M animals (0.010±0.003). In addition, we performed experiments in which the PLTP activity levels were analyzed in liver homogenates of the transplanted PLTP +M/+M and PLTP -M/-M animals. In accordance with the mRNA data, PLTP activity levels in the liver homogenates were 26% lower in PLTP -M/-M mice (31±2 nmol/mg protein versus 42±2 nmol/mg; PLTP -M/-M versus PLTP +M/+M; P =0.0019), confirming the importance of PLTP production by bone marrow-derived cells for hepatic PLTP activity.
Figure 3. PLTP mRNA expression and activity in liver samples. A, Left, PLTP mRNA expression in whole liver tissue samples taken from PLTP +M/+M (white column, n=8) and PLTP -M/-M (black column, n=9) mice at 17 weeks posttransplantation. Right, PLTP activities in liver homogenates of the transplanted PLTP +M/+M (white column, n=14) and PLTP -M/-M (black column, n=11) mice at 17 weeks posttransplantatioin. B, Left, relative expression of PLTP mRNA in parenchymal cells (PCs; n=12), endothelial cells (ECs; n=6), and Kupffer cells (KCs; n=6) isolated from livers of wild-type C57Bl/6J mice. Right, Absolute contribution of different cell types to hepatic PLTP mRNA expression calculated based on the protein contribution of the different cell types in livers of C57Bl/6J mice. Data represent the means±SEM. Statistically significant differences are indicated. * P <0.05 and ** P <0.01.
The liver contains several different types of cells which all have their specific localization and function. The majority of the liver consists of parenchymal cells, which contribute 92.5% to the total liver protein mass. In addition, the liver contains endothelial and Kupffer cells that account for 3.3% and 2.5% of the liver protein mass, respectively. 33,34 Because Kupffer cells are of hematopoietic origin, it is likely that the observed reduction in hepatic PLTP activity of the PLTP -M/-M group is attributable to the reconstitution with Kupffer cells derived from the PLTP -/- donor bone marrow. To confirm that Kupffer cells are an important source of PLTP in the liver, the PLTP mRNA expression was determined in purified parenchymal cells, Kupffer cells, and endothelial cells isolated from livers of C57Bl/6J mice on normal chow diet ( Figure 3 B). The expression of PLTP mRNA was 6-fold ( P <0.01) higher in Kupffer cells as compared with parenchymal cells, whereas endothelial cells produced only a minor amount of PLTP mRNA. Thus, although Kupffer cells only contribute to 2.5% of the total liver protein, they do contain 13.8% of the total liver PLTP expression, as compared with 85.7% and 0.5% for parenchymal cells and endothelial cells, respectively. Immunohistochemical localization of PLTP protein in livers of the transplanted PLTP +M/+M and PLTP -M/-M mice also clearly demonstrated a reduction in hepatic PLTP protein expression attributable to disruption of PLTP in bone marrow-derived cells ( Figure 4 A). In addition to liver, PLTP is highly expressed in lung. 1 Therefore also the expression of PLTP protein in lungs of PLTP +M/+M and PLTP -M/-M mice were compared ( Figure 4 B). Interestingly, disruption of macrophage PLTP production also resulted in a drastic decrease in PLTP expression in the bronchioles of the lung, where the highest concentration of F4/80 positive macrophages is localized.
Figure 4. Immunohistochemical detection of PLTP protein in liver and lung samples. For detection of PLTP protein, cryostat sections of liver (A) and lung (B) of the transplanted PLTP +M/+M (left) and PLTP -M/-M mice (middle) were stained immunohistochemically for PLTP (red) and nuclei (blue). Arrows indicate PLTP positive staining. For comparison localization of macrophage F4/80 staining in liver and lung is shown (right). Note the reduced expression of PLTP protein in both livers and lungs of PLTP -M/-M mice.
Thus, the significant contribution of Kupffer cells to the hepatic PLTP production and lung macrophages to PLTP expression in the lung, combined with that of other resident macrophages, may explain the large effects of PLTP deficiency in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis.
Discussion
In the present study we show that selective deficiency of PLTP in macrophages in LDLr -/- mice: (1) decreased the size of atherosclerotic lesions; (2) decreased the relative preß-HDL levels; (3) decreased serum cholesterol; and (4) lowered plasma PLTP activity. We also demonstrate for the first time that hepatic Kupffer cells express higher PLTP mRNA levels than parenchymal cells and endothelial cells in livers of wild-type C57Bl/6J mice.
How can the observed changes explain the anti-atherogenic effect caused by selective PLTP deficiency in macrophages? An important process during the early steps of atherosclerotic lesion formation is the accumulation of cholesterol, derived from modified LDL and/or (ß-)VLDL, in arterial macrophages transforming them into lipid-laden foam cells. The opposite event, reverse cholesterol transport, removes excess cholesterol from macrophages in the artery wall and transports it to the liver for excretion, 35 thereby preventing excessive cholesterol accumulation. On WTD feeding of LDLr -/- mice, the balance between the cholesterol influx and efflux in macrophages is severely compromised leading to excessive accumulation of lipid. Recently, several groups have reported that macrophages in atherosclerotic lesions express PLTP, 25-27 but its relation to atherosclerotic lesion formation was unknown. In the current study we show that disruption of PLTP in macrophages reduces atherosclerosis in LDLr -/- mice.
To get a better understanding of the various potential mechanisms contributing to this beneficial effect of macrophage PLTP deficiency, it is important to recognize the plasma factors affecting lesion formation in the arterial intima, as well as local macrophage-derived arterial effects. In vitro, both wild-type and PLTP -/- macrophages can be converted into foam cells on incubation with acetylated LDL. No differences in the overall cholesterol uptake between wild-type and PLTP -/- macrophages were observed as recently demonstrated by Lee-Rueckert et al. 36 Thus, PLTP deficiency in the macrophage does not influence cholesterol uptake and deposition once challenged with modified LDL. Exogenous PLTP increases HDL-induced phospholipid and cholesterol removal from macrophage foam cells; 29 therefore, via enhancing reverse cholesterol transport it may be anti-atherogenic. One of the best characterized lipid exporters from macrophages is ATP-binding cassette transporter A1, which mediates cholesterol efflux to lipid-poor apolipoproteins, including apoA-I and apoE. 37 In addition, ABCG1 38,39 and scavenger receptor BI 40,41 mediate the efflux of cholesterol to mature HDL. Recently, we have shown that absence of endogenous PLTP impairs ATP-binding cassette transporter A1-dependent efflux from macrophage foam cells in vitro. 36 Macrophages also synthesize and secrete apoE, which can induce cellular cholesterol efflux and protect against the development of atherosclerosis. 42 Interestingly, apoE interacts with human plasma PLTP and activates the low-activity form of PLTP. 43 Furthermore, apoE as well as PLTP are under positive control of liver X receptor agonists and during cholesterol loading expression levels of both are increased, 25-28,44 suggesting that both apoE and PLTP may facilitate cholesterol efflux. 45 Here we show that disruption of macrophage PLTP in LDLr -/- mice reduces atherosclerotic lesion development and that macrophage PLTP is pro-atherogenic in LDLr -/- mice. Thus, in vivo apparently other pro-atherogenic properties of PLTP, probably related to the observed changes in plasma lipoproteins, override the potential anti-atherogenic function of macrophage PLTP in mediating cholesterol efflux.
Macrophage PLTP deficiency significantly reduced plasma PLTP activity. Furthermore, PLTP activity in PLTP -M/-M liver homogenates was significantly reduced as compared with livers obtained from PLTP +M/+M animals depicting that hepatic bone marrow-derived cells can provide active PLTP. Kupffer cells, resident macrophages of the liver, significantly contributed to the hepatic PLTP production and it is thus conceivable that the reduced plasma PLTP activity measured in PLTP -M/-M mice is a direct effect of the absence of PLTP production by Kupffer cells of the liver and other resident macrophages in lung, adipose tissue, and spleen. Replacement of Kupffer cells after bone marrow transplantation was assessed by transplantation of LDLr -/- mice with bone marrow from enhanced green fluorescent protein (EGFP) expressing mice. Already at 8 weeks after transplantation a significant amount of Kupffer cells were EGFP-positive and of donor origin (unpublished data). These findings are in agreement with a previous study from Paradis et al 46 who showed that already at 21 days after bone marrow transplantation Kupffer cells were predominantly of donor bone marrow origin. Immunohistochemical localization also confirmed the reduction in PLTP protein expression in livers of PLTP -M/-M mice. In addition, PLTP protein expression was drastically reduced in lungs, one of the organs with the highest expression of PLTP. 1
The effect of macrophage PLTP deficiency on total plasma PLTP activity significantly influenced lipoprotein metabolism in the PLTP -M/-M mice. On WTD, preß-HDL levels were lower in mice lacking PLTP in macrophages. Preß-HDL is highly efficient in the removal of cholesterol from cells. However, despite the decreased levels of preß-HDL, lesion development was reduced, implicating that the pro-atherogenic effects of lower preß-HDL levels are overruled by other factors. PLTP deficiency of macrophages also resulted on WTD in significantly higher plasma apoA-I levels and HDL phospholipids. Distribution of HDL subclasses and the roles of the different subclasses in reverse cholesterol transport are at present far from resolved. We assume that the elevated apoA-I and HDL-associated phospholipids may result in the formation of HDL subclasses that could contribute to enhanced cholesterol efflux, and provide an explanation for the reduced size of the lesions formed in PLTP -M/-M mice.
Consistent with the lower plasma PLTP activity, macrophage PLTP deficiency also resulted in substantially lower concentrations of cholesterol and triglycerides, mainly as a consequence of lower VLDL levels. Because Kupffer cells seem to contribute significantly to PLTP mRNA expression and PLTP activity in the liver, it is conceivable that Kupffer cell PLTP could directly or indirectly influence VLDL biosynthesis or secretion and could thus provide an explanation for the lower levels of apoB-containing lipoproteins in PLTP -M/-M mice. Plasma PLTP activity reportedly correlates positively with triglyceride levels. 47 Furthermore, in apoE -/- and human apoB transgenic mice, total PLTP deficiency decreased serum levels and production of apoB-containing lipoproteins. 20 However, total PLTP deficiency did not influence serum apoB-lipoprotein levels or their production in LDLr -/- mice. 20 In our chimeric mouse model, macrophage-specific PLTP deficiency in LDLr -/- mice did cause a reduction in the levels of apoB-containing lipoproteins, which is probably related to the reduced plasma PLTP activity and provides an important explanation for the reduced susceptibility of the PLTP -M/-M mice to atherosclerotic lesion development.
Recently, using a similar experimental setup as we have used, Valenta et al 48 showed that diet-induced atherosclerosis was increased in LDLr -/- mice on disruption of PLTP in hematopoietic cells. The complexity of atherosclerotic lesion formation in LDLr -/- mice was recently clearly illustrated in an editorial by Curtiss, 49 in which it was summarized that atherosclerosis can be influenced by: (1) the degree of hypercholesterolemia achieved by the use of different atherogenic diets; (2) the time dependency of the progression of atherosclerosis; (3) the genetic background and sex of the experimental mice; and (4) other factors that might influence the outcome of the different studies, including environmental factors as well as the time of recovery from irradiation.
In our studies, the irradiated recipient animals were allowed to recover for 8 weeks after the bone marrow transplantation before starting the atherogenic diet feeding as compared with 4 weeks in the study of Valenta et al. 48 This difference in time of recovery from irradiation might have influenced the level of replacement of resident tissue macrophages, including Kupffer cells in the liver.
Furthermore, under our experimental conditions in which the mice were fed a mild atherogenic WTD, containing 15% fat and 0.25% cholesterol for 9 weeks, macrophage PLTP production contributes to plasma PLTP activity, induces higher VLDL cholesterol levels, and thus is considered pro-atherogenic. In the study of Valenta et al, 48 the effect of macrophage PLTP production on atherosclerotic lesion formation was studied using a higher cholesterol-containing diet, composed of 15.8% fat and 1.25% cholesterol for 16 weeks. Under these high-cholesterol conditions, the macrophage PLTP deletion did not affect VLDL cholesterol levels and protected against the development of atherosclerosis. In both studies the evaluation of atherosclerotic lesion development was performed using only one time point.
The genetic background of the mice used in the two studies was identical, but we used female recipients, whereas Valenta et al 48 used males. Recently, Yang et al 50 reported that in mice many hepatic genes show sexual dimorphism ( 70%). Furthermore, 3-fold) in gene expression between females and males were observed in genes involved in steroid and lipid metabolism. In addition to the differences in the cholesterol content of the diets between our work and that published by Valenta et al, 48 this sexual dimorphism could contribute to the differential effects of macrophage-derived PLTP on serum VLDL levels.
Another way to study the role of macrophage PLTP was recently reported in an abstract by Van Haperen et al. 51 The authors demonstrated using bone marrow transplantation with wild-type mice, hemizygous transgenic mice (huPLTP tg/wt ), or homozygous PLTP transgenic mice (huPLTP tg/tg ) as donors, and LDLr -/- mice as recipients that Western-type diet-induced atherosclerosis was increased in the huPLTP tg/wt LDLr -/- mice (2.3-fold) and in huPLTP tg/tg LDLr -/- mice (4.5-fold) compared with control mice. The increase in lesion development coincided with increased VLDL cholesterol and decreased HDL cholesterol levels. Their conclusion that PLTP expression in macrophages results in increased atherosclerotic lesion formation is in line with our present data which show that absence of PLTP is atheroprotective.
Valenta et al 48 postulated that the contribution of PLTP to atherosclerosis is determined by a balance between lesion PLTP activity (anti-atherogenic) and plasma PLTP activity (pro-atherogenic). In our study and the study of Van Haperen et al, 51 the effect of macrophage PLTP production on plasma PLTP activity affected the VLDL cholesterol levels, resulting in a pro-atherogenic role of macrophage PLTP. In the study of Valenta et al the effect of macrophage-derived PLTP on plasma PLTP activity did not affect VLDL cholesterol levels and protected against the development of atherosclerosis probably as a result of the local anti-atherogenic properties of macrophage PLTP in the lesion. These studies thus strengthen the postulation that the balance between factors influencing the anti-atherogenic lesion PLTP activity and factors affecting the pro-atherogenic plasma PLTP activity is essential for the eventual outcome of PLTP modulation on atherosclerotic lesion development.
In conclusion, our study shows that macrophage PLTP significantly contributes to plasma PLTP activity and that deficiency of macrophage PLTP results in increased apoA-I and decreased VLDL/LDL levels, changes that may explain why deficiency of PLTP in macrophages leads to a decrease in atherosclerotic lesion development in LDLr -/- mice.
Acknowledgments
We thank Ritva Nurmi and Sari Nuutinen for their excellent technical assistance.
Sources of Funding
The study was supported by the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, the Netherlands Heart Foundation (2001T041), and the Netherlands Organisation for Scientific Research (917.66.301).
Disclosures
None.
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作者单位：From National Public Health Institute (R.V., J.M., C.E., M.J.), Department of Molecular Medicine, Biomedicum, Helsinki, Finland; Division of Biopharmaceutics (D.Y., R.B.H. Th.J.C.V.B., M.V.E.), Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands.