Macrophage Phospholipid Transfer Protein Contributes Significantly to Total Plasma Phospholipid Transfer Activity and Its Deficiency Leads to Diminished Atherosclerotic Lesion Development

Atherosclerosis and Lipoproteins Macrophage Phospholipid Transfer Protein Contributes Significantly to Total Plasma Phospholipid Transfer Activity and Its Deficiency Leads to Diminished Atherosclerotic Lesion Development Riikka Vikstedt, Dan Ye, Jari Metso, Reeni B. Hildebrand, Theo J.C. Van Berkel, Christian Ehnholm, Matti Jauhiainen, Miranda Van Eck 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*103 ␮m2 in the PLTP⫺M/⫺M group (n⫽10), as compared with 539⫾35*103 ␮m2 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. (Arterioscler Thromb Vasc Biol. 2007;27:578-586.) Key Words: apolipoproteins 䡲 atherosclerosis 䡲 lipid transfer proteins 䡲 macrophages 䡲 mouse models

P

hospholipid 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 lipolysis4 and also participates in HDL conversion.5,6 PLTP modifies HDL3 particles and concomitantly generates large particles resembling HDL2 and a population of small poorly lipidated pre␤-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 hepatocytes19,20 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 pre␤-HDL particles;12,14,15 (2) determination of plasma HDL levels21,22 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

Original received June 13, 2006; final version accepted November 23, 2006. 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. Correspondence to Miranda Van Eck, Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail [email protected] © 2007 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org

DOI: 10.1161/01.ATV.0000254815.49414.be

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Vikstedt et al 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 A1mediated 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 Ro¨ntgen source (YXLON International, Copenhagen, Denmark) 1 day before transplantation. Irradiated recipients were transplanted by intravenous injection of 0.5⫻107 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. Pre␤-HDL and ␣-HDL levels,12 mouse apolipoprotein (apo)A-I12, PLTP activity,5,30 hepatic lipase activity,31 and lecithin-cholesterol acyltransferase activity32 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 mice33 using real time-quantitative polymerase chain reaction. Furthermore, PLTP protein levels were determined immu-

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nohistochemically 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 ⴚ/ⴚ

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 PLTP21 into LDLr⫺/⫺ mice (supplemental Figures I and II, available online at http://atvb.ahajournals.org).

Macrophage PLTP Deficiency Decreases Atherosclerosis in LDLrⴚ/ⴚ 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 1A, macrophage PLTP deficiency leads to a 29% (P⬍0.01) decrease in the mean atherosclerotic lesion area (PLTP⫹M/⫹M, 539⫾35⫻103 ␮m2, n⫽14 versus PLTP⫺M/⫺M, 384⫾36⫻103 ␮m2, n⫽10). The relative macrophage content of the lesions of WT 3 LDLr⫺/⫺ mice was 32⫾3%, whereas the collagen content was 7⫾1% (Figure 1B and 1C). 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 1C). 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 ␮m2, P⬍0.01) as compared with control transplanted animals (21⫾4 ␮m2). 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.

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 2A). However, HDL cholesterol was significantly higher in the PLTP⫺M/⫺M group (P⬍0.01). On challenging the mice with WTD, the concen-

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Arterioscler Thromb Vasc Biol.

A

B

Macrophages (%) Lesion size (x103 µm2)

580

March 2007

PLTP+M/+M

PLTP-M/-M

PLTP+M/+M

PLTP-M/-M

PLTP+M/+M

PLTP-M/-M

**

Collagen (%)

C

P=0.07

PLTP+M/+M PLTP-M/-M

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 (␮m2) 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 ⫻50.

trations 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 TABLE 1.

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 2B).

Effect of Macrophage PLTP Deficiency on Serum Lipids and ApoA-I Concentrations

Mice Group

Time (wk)

Diet

n

TC (mmol/L)

FC (mmol/L)

TG* (mmol/L)

PL (mmol/L)

PLTP⫹M/⫹M

0

Chow

14

9.86⫾0.42

1.78⫾0.11

ND

5.19⫾0.21

ND

8

Chow

14

7.14⫾0.26

1.44⫾0.05

1.44⫾0.07

6.41⫾0.15

1.44⫾0.07

17

WTD

14

29.13⫾1.66储

8.42⫾0.53储

2.08⫾0.23¶

9.33⫾0.42储

0.98⫾0.08储

0

Chow

13

9.53⫾0.65

1.84⫾0.12

ND

5.58⫾0.34

ND

8

Chow

11

7.59⫾0.14

1.64⫾0.03

1.67⫾0.14

7.28⫾0.08‡

1.30⫾0.11

17

WTD

11

21.67⫾1.24†储

6.71⫾0.62†储

1.16⫾0.14‡¶

8.02⫾0.50

1.63⫾0.15§

⫺M/⫺M

PLTP

⫹M/⫹M

⫺M/⫺M

ApoA-I* (g/L)

Serum lipids and apoliprotein A-I concentrations in PLTP and PLTP mice maintained on a chow diet for 8 weeks and on a high-cholesterol Western-type diet (WTD) for 9 weeks (17 weeks after bone marrow transplantation 关BMT兴). Data present means⫾SEM. ND indicates not determined. *Analyzed from plasma samples † P⬍0.05, ‡P⬍0.01, §P⬍0.001 difference between PLTP⫹M/⫹M and PLTP⫺M/⫺M mice at the indicated times posttransplantation. ¶ P⬍0.05, 储P⬍0.001difference between 8 and 17 weeks posttransplantation in the indicated mice.

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A

581

C 500

400

Chow

PLTP+M/+M

PLTP-M/-M

VLDL+LDL

Phospholipids (µm ol/L)

Cho lestero l (µ m o l/L )

400

HDL**

300 200

100

300

HDL

VLDL+LDL

200

100

0

0

1

5

9

13

17

1

21

Fraction number

B

5

9

13

17

21

Fraction number

D 200

1200 PLTP+M/+M PLTP-M/-M

1000

VLDL+LDL*

HDL

800 600 400

PLTP+M/+M

WTD Phospholipids (µmol/L)

WTD Ch olesterol (µ m o l/L )

PLTP+M/+M

Chow

PLTP-M/-M

PLTP-M/-M

VLDL+LDL*

150

HDL**

100

50

200 0

0 1

5

9

13

17

21

Fraction number

1

5

9

13

17

21

Fraction number

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.

Under these feeding conditions, HDL cholesterol was decreased in both groups but there was no significant difference between the groups. 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 2C). 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 2D). 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 pre␤-HDL particles, it was also of interest to study pre␤-HDL levels in this experimental setting. At 8 weeks posttransplantation, no significant effect of macrophage PLTP deficiency was observed on circulating pre␤-HDL levels. However, after 9 weeks of WTD feeding, the pre␤-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

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Arterioscler Thromb Vasc Biol. TABLE 2. Mice Group PLTP⫹M/⫹M

PLTP⫺M/⫺M

March 2007

Effect of Macrophage PLTP Deficiency on Plasma PLTP, HL, and LCAT Activities Time (wk)

Diet

n

PLTP (␮mol/mL per hour) ND

HL (␮mol/mL per hour)

LCAT (nmol/mL per hour)

0

Chow

14

ND

ND

8

Chow

14

19.51⫾1.33

17.20⫾0.82

ND

17

WTD

14

48.35⫾4.49‡

18.06⫾1.17

9.83⫾0.75

0

Chow

13

ND

ND

ND

8

Chow

11

14.29⫾0.85*

19.22⫾1.16

ND

17

WTD

11

24.03⫾2.38†‡

19.38⫾1.01

11.03⫾0.70

Plasma PLTP, HL, and LCAT activities in PLTP⫹M/⫹M and PLTP⫺M/⫺M mice maintained on a chow diet for 8 weeks and on a high-cholesterol WTD for 9 weeks (17 weeks after BMT). Data present means⫾SEM. *P⬍0.01, †P⬍0.001 difference between PLTP⫹M/⫹M and PLTP⫺M/⫺M mice at the indicated time posttransplantation. ‡ P⬍0.001 difference between 8 and 17 weeks posttransplantation in the indicated mice.

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 ␮mol/mL per hour versus PLTP⫺M/⫺M, 14.3 ␮mol/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 ␮mol/mL per hour versus PLTP⫺M/⫺M 24.0 ␮mol/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). 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 3A). 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. 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 deter-

mined in purified parenchymal cells, Kupffer cells, and endothelial cells isolated from livers of C57Bl/6J mice on normal chow diet (Figure 3B). 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 4A). 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 4B). 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. 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 pre␤-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 (␤-)VLDL, in arterial macrophages transforming them into lipid-laden foam cells. The opposite event,

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0.015

PLTP activity (nmol/mg protein)

PLTP mRNA expression (A.U.)

A

0.010

0.005

0.000

50

40 ** 30

20

10

0 PLTP+M/+M PLTP-M/-M

0.25 ** **

0.20

0.15

0.10 * 0.05

0.00 PC

EC

KC

Absolute PLTP mRNA expression (A.U.)

B

Relative PLTP mRNA expression (A.U.)

PLTP+M/+M PLTP-M/-M

0.035

** **

0.030 0.025 0.020 0.015

*

0.010 0.005 0.000 PC

EC

KC

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.

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 athero-

A PLTP staining

Macrophage F4/80 staining

PLTP-M/-M

Liver

PLTP+M/+M

B

PLTP staining

Lung

PLTP+M/+M

PLTP-M/-M

Macrophage F4/80 staining

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.

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sclerotic 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, ABCG138,39 and scavenger receptor BI40,41 mediate the efflux of cholesterol to mature HDL. Recently, we have shown that absence of endogenous PLTP impairs ATP-binding cassette transporter A1dependent 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 al46 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, pre␤-HDL levels were lower in mice lacking PLTP in macrophages. Pre␤-HDL is highly efficient in the removal of cholesterol from cells. However, despite the decreased levels of pre␤-HDL, lesion development was reduced, implicating that the pro-atherogenic effects of lower pre␤-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 al48 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

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Vikstedt et al 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 al48 used males. Recently, Yang et al50 reported that in mice many hepatic genes show sexual dimorphism (⬇70%). Furthermore, the largest changes (⬎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 (huPLTPtg/wt), or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors, and LDLr⫺/⫺ mice as recipients that Western-type diet-induced atherosclerosis was increased in the huPLTPtg/wt3 LDLr⫺/⫺ mice (2.3-fold) and in huPLTPtg/tg3 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 al48 postulated that the contribution of PLTP to atherosclerosis is determined by a balance between lesion PLTP activity (anti-atherogenic) and plasma PLTP activity (proatherogenic). 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

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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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32. Jauhiainen M, Dolphin PJ. Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism. J Biol Chem. 1986; 261:7032–7043. 33. Nagelkerke JF, Barto KP, van Berkel TJ. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells. J Biol Chem. 1983;258:12221–12227. 34. Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol. 1977;72:441– 455. 35. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res. 1995;36:211–228. 36. Lee-Rueckert M, Vikstedt R, Metso J, Ehnholm C, Kovanen PT, Jauhiainen M. Absence of endogenous phospholipid transfer protein (PLTP) impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells. J Lipid Res. 2006;47:1725–1732. 37. Oram JF. AT. P-binding cassette transporter A1 and cholesterol trafficking. Curr Opin Lipidol. 2002;13:373–381. 38. Kennedy MA, Barrera GC, Nakamura K, Baldan A, Tarr P, Fishbein MC, Frank J, Francone OL, Edwards PA. ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation. Cell Metab. 2005;1:121–131. 39. Wang N, Lan D, Chen W, Matsuura F, Tall AR. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins. Proc Natl Acad Sci U S A. 2004;101:9774 –9779. 40. Jian B, de la Llera-Moya M, Ji Y, Wang N, Phillips MC, Swaney JB, Tall AR, Rothblat GH. Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors. J Biol Chem. 1998;273:5599 –5606. 41. Yancey PG, de la Llera-Moya M, Swarnakar S, Monzo P, Klein SM, Connelly MA, Johnson WJ, Williams DL, Rothblat GH. High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI. J Biol Chem. 2000;275:36596 –36604. 42. Van Eck M, Herijgers N, Vidgeon-Hart M, Pearce NJ, Hoogerbrugge PM, Groot PH, Van Berkel TJ. Accelerated atherosclerosis in C57Bl/6 mice transplanted with ApoE-deficient bone marrow. Atherosclerosis. 2000; 150:71– 80. 43. Janis MT, Metso J, Lankinen H, Strandin T, Olkkonen VM, Rye KA, Jauhiainen M, Ehnholm C. Apolipoprotein E activates the low-activity form of human phospholipid transfer protein. Biochem Biophys Res Commun. 2005;331:333–340. 44. Laffitte BA, Repa JJ, Joseph SB, Wilpitz DC, Kast HR, Mangelsdorf DJ, Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci U S A. 2001;98:507–512. 45. Remaley AT, Stonik JA, Demosky SJ, Neufeld EB, Bocharov AV, Vishnyakova TG, Eggerman TL, Patterson AP, Duverger NJ, Santamarina-Fojo S, Brewer HB, Jr. Apolipoprotein specificity for lipid efflux by the human ABCAI transporter. Biochem Biophys Res Commun. 2001;280:818–823. 46. Paradis K, Sharp HL, Vallera DA, Blazar BR. Kupffer cell engraftment across the major histocompatibility barrier in mice: bone marrow origin, class II antigen expression, and antigen-presenting capacity. J Pediatr Gastroenterol Nutr. 1990;11:525–533. 47. Janis MT, Siggins S, Tahvanainen E, Vikstedt R, Silander K, Metso J, Aromaa A, Taskinen MR, Olkkonen VM, Jauhiainen M, Ehnholm C. Active and low-active forms of serum phospholipid transfer protein in a normal Finnish population sample. J Lipid Res. 2004;45:2303–2309. 48. Valenta DT, Ogier, Bradshaw G, Black AS, Bonnet DJ, Lagrost L, Curtiss LK, Desrumaux CM. Atheroprotective potential of macrophagederived phospholipid transfer protein in low-density lipoprotein receptordeficient mice is overcome by apolipoprotein A-I overproduction. Arterioscler Thromb Vasc Biol. 2006;26:1572–1578. 49. Curtiss LK. Is two out of three enough for ABCG1? Arterioscler Thromb Vacs Biol. 2006;26:2175–2177. 50. Yang X, Schadt EE, Wang S, Wang H, Arnold AP, Ingram-Drake L, Drake TA, Lusis AJ. Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Res. 2006;16:995–1004. 51. Van Haperen R, Samyn H, Moerland M, van Gent T, Peeters M, Grosveld F, Van Tol A, de Crom R. Elevated macrophage expression of phospholipid transfer protein causes atherosclerosis. Circulation. 2006; 114(suppl II):225.

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ONLINE SUPPLEMENT

Macrophage PLTP Contributes Significantly to Total Plasma Phospholipid Transfer Activity and its Deficiency Leads to Diminished Atherosclerotic Lesion Development Riikka Vikstedt, Dan Ye, Jari Metso, Reeni B. Hildebrand, Theo J.C. Van Berkel, Christian Ehnholm, Matti Jauhiainen, Miranda Van Eck

MATERIALS AND METHODS Mice PLTP knockout mice on the C57BL/6J background (PLTP-/-)1 and control C57Bl/6J mice (PLTP+/+) were obtained from Viikki Laboratory Animal Centre, University of Helsinki, Helsinki, Finland. Homozygous LDL receptor knockout (LDLr-/-) mice2 were obtained from The Jackson Laboratory (Bar Harbor, ME) as mating pairs and bred at the Gorlaeus Laboratory, Leiden, The Netherlands. Mice were housed in sterilised filter-top cages and given unlimited access to food and water. Mice were maintained on sterilized regular chow, containing 4.3 % (w/w) fat and no cholesterol (RM3, Special Diet Services, Witham, UK), or were fed a semi-synthetic Westerntype diet (WTD), containing 15 % (w/w) total fat and 0.25 % (w/w) cholesterol (Diet W, Special Diet Services, Witham, UK). Drinking water was supplied with antibiotics (83 mg/L ciprofloxacin and 67 mg/L polymyxin B sulphate) and 6.5 g/L sucrose. 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.

1

Bone marrow transplantation To induce bone marrow aplasia, female LDLr-/- mice were exposed to a single dose of 9 grays (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) with a 6-mm aluminium filter, one day before transplantation. Bone marrow was isolated by flushing the femurs and tibias from male wild-type PLTP+/+ mice or male PLTP-/- mice with phosphate-buffered saline (PBS). Single-cell suspensions were prepared by passing the cells through a 30-µm nylon gauze. Irradiated recipients received 0.5 x 107 bone marrow cells by intravenous injection into the tail vein. After that, mice (indicated as PLTP+M/+M and PLTP-M/-M mice, respectively) were maintained on sterilised regular chow diet, containing 4.3 % (w/w) fat and no cholesterol for 8 weeks to allow the mice to recover from the bone marrow transplantation. After 8 weeks, the diet was switched to a semi-synthetic Western-type diet (WTD) containing 15 % (w/w) total fat and 0.25 % (w/w) cholesterol. Serum lipid levels were monitored during the course of the experiment and after 9 weeks WTD, the mice were sacrificed and the size of the lesions in the aortic root was determined (see Figure I). BMT Lipid analyses

Weeks

0

Lipid analyses

8

Lesion quantification, lipid analyses

17

Chow diet (no cholesterol, 4.3 % fat)

WTD (0.25 % cholesterol, 15 % fat)

Figure I. Design of the experiment.

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Assesment of chimerism Successful reconstitution of recipient LDLr-/- mice with the donor cells was verified in genomic DNA from bone marrow by PCR at 17 weeks after bone marrow transplantation. For simultaneous PCR amplification of the wild-type and the null mutant PLTP gene, oIMR1494 (5’-AAAGGCTGCTGGACCCGCG-3’), oIMR1495 (5’-GCAGCGCATCGCCTTCTATC-3’),

and

oIMR1616

(5’-

TGGTCATGCAACTAGAACGGAGT-3’) (The Jackson Laboratory, Bar Harbor, ME) were used.

PLTP+M/+M

PLTP-M/-M 823 bp 613 bp

Figure II. Successful reconstitution of recipient LDLr-/- mice with cells of donor origin after bone marrow transplantation. At the experiment (17 weeks after bone marrow transplantation) the success of the transplantation was verified by PCR amplification of genomic DNA isolated from bone marrow of the transplanted animals. Genomic DNA isolated from bone marrow of PLTP+M/+M mice displayed only the wild-type specific band with the size of 613 bp. Instead, from bone marrow of PLTP-M/-M mice a band of 823 bp was visible demonstrating that the PLTP gene was successfully disrupted and that bone marrow in the recipient mice was largely replaced by donor PLTP-/- bone marrow.

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Histological analysis of the aortic root To induce the development of atherosclerosis at the aortic root, transplanted LDLr-/mice were fed Western-type diet for 9 weeks after which the mice were sacrificed. The arterial tree was perfused in situ with PBS (with the pressure of 100 mm Hg) for 20 minutes via a cannula in the left ventricular apex. The heart plus aortic root and descending aorta were excised and stored in 3.7 % neutral-buffered formalin (Formalfixx®, Shandon Scientific Ltd., England). The atherosclerotic lesion areas in oil red Ostained cryostat sections of the aortic root were quantified using the Leica image analysis system, consisting of a Leica DMRE microscope coupled to a video camera and Leica Qwin Imaging software (Leica Ltd., Cambridge, England). Mean lesion area (µm2) was calculated from 10 oil red O-stained sections, starting at the appearance of the tricuspid valves. Cap thickness was analysed at 30 different locations of the lesion per section. For the assessment of macrophage area sections were immunolabeled with MOMA-2 (polyclonal rat IgG2b, 1:50 dilution, Research Diagnostics Inc). The MOMA-2-positive lesion area was subsequently quantified using the Leica image analysis system. The amount of collagen in the lesions was determined using Masson’s Trichrome Accustain according to manufacturer’s instructions (Sigma Diagnostics). All quantifications were done blinded by computeraided morphometric analysis using the Leica image analysis system.

Serum lipid, lipoprotein and apolipoprotein analyses After an overnight fasting-period, approximately 100 µL blood was drawn from each individual mouse by tail bleeding. Concentrations of serum cholesterol (Roche Diagnostics, Mannheim, Germany), triglycerides (Roche) and choline containing phospholipids (Wako chemicals GmbH, Neuss, Germany) were determined using

4

enzymatic colorimetric assays. The distribution of lipids between the different lipoproteins in serum was determined by fractionation of 30 µL serum from each mouse by size-exclusion chromatography on a Superose 6 PC 3.2/30 precision column (Amersham Biosciences, Piscataway, NJ). Total cholesterol, triglycerides and phospholipids in the elution fractions were determined by enzymatic assays as above.

Preβ-HDL and α-HDL levels of plasma samples were quantified by crossed immunoelectrophoresis as previously described.3 Briefly, in the first dimension lipoproteins with preβ- and α-mobility were separated by agarose gel electrophoresis, followed by electrophoresis in the second dimension agarose gel containing 7.5 % (v/v) rabbit anti-mouse apoA-I antiserum. For visualization, dried agarose gels were stained using Coomassie brilliant blue R250. Preβ-HDL area is expressed as percentage of the sum of α-HDL and preβ-HDL areas.

Mouse apoA-I was quantified by a noncompetitive ELISA assay.3 Briefly, the wells of 96-well plates were coated with a polyclonal rabbit antibody, R196, against mouse apoA-I. After sample incubations, the bound apoA-I was detected using the same antibody conjugated with horseradish peroxidase.

Plasma PLTP, hepatic lipase and LCAT activities For the radiometric PLTP activity assay, phosphatidylcholine liposomes were prepared essentially as described.4 Each assay contained HDL3 acceptor (250 µg as protein), [14C]phosphatidylcholine-liposomes (150 nmol as phosphatidylcholine), sample (4 µL or 10 µL of 1:10 diluted mouse EDTA-plasma, 10 µL or 20 µL of fractions from the size-exclusion chromatography), and sample buffer (10 mM Tris–

5

HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA) in a final assay volume of 400 µL. Assay tubes were incubated for 45 min at +37°C after which the excess liposomes and plasma derived apolipoprotein B (apoB)-containing lipoproteins were precipitated with 300 µL of 215 mM MnCl2·4H2O, 500 mM NaCl containing 484 U/mL heparin, as described.4,5

2000 PLTP+M/+M

PLTP activity (nmol/mL/h)

PLTP-M/-M

VLDL+LDL

1600

HDL

1200

800

400

0 1

5

9

13

17

21

Fraction number

Figure III. Distribution of PLTP activity in mice sera fractionated by sizeexclusion chromatography. Serum samples for analyses were obtained at 17 weeks posttransplant after 9 weeks feeding of Western-type diet (WTD). Distribution of PLTP activity between different lipoprotein fractions in serum was determined by fractionation of 30 µL serum from PLTP+M/+M (n=14) and PLTP-M/-M (n=11) mice by size-exclusion chromatography on a Superose 6 PC 3.2/30 precision column (Amersham Biosciences, Piscataway, NJ). PLTP activity from the fractions was analyzed as described above.

6

Substrate for hepatic lipase (HL) assay was prepared as described by Ehnholm and Kuusi.6 In the hepatic lipase assay, 200 µL of the trioleate assay substrate was incubated with 10 µL of mouse EDTA-plasma, 100 µL of 5 M NaCl and 190 µL of 0.2 M Tris-HCl, pH 8.4 for 1 h at +28°C. For blank, corresponding volume of 0.2 M Tris-HCl, pH 8.4 was added to the reaction instead of a plasma sample. The reactions were stopped by addition of 3.25 mL of methanol:chloroform:heptane mixture (1.41/1.25/1.00, v/v/v). Thereafter, 750 µL of potassium borate-carbonate buffer (0.14 M K2CO3, 0.14 M H3BO3, pH 10.5) was added, tubes were mixed and centrifuged at 2000 rpm for 15 min and supernatants were assayed for radioactivity. The results were calculated as reported earlier6 and expressed as µmol free fatty acid released per milliliter of enzyme per hour (µmol FFA/mL/h).

Substrate for lecithin-cholesterol acyltransferase assay (LCAT) was prepared as described by Jauhiainen and Dolphin.7 For measuring LCAT activity the assay typically consisted of 250 µL of assay buffer (10mM Tris-HCl, pH 7.4 containing 140 mM NaCl and 1 mM EDTA), 125 µL of 2.0 % BSA and 50 µL of LCAT substrate. The mixtures were preincubated for 30 min at +37°C after which 25 µL of 0.1 M βmercaptoethanol and 25 µL of mouse EDTA-plasma sample were added. The mixtures were vortexed and incubated for 30 min at +37°C. The reactions were stopped by addition of 8 mL of chloroform:methanol (2:1, v/v) and 2 mL of 0.58 % NaCl and the lipids were extracted by the method of Folch et al.8 The organic phase of the extracts were dried under N2 and dissolved in chloroform, and the lipids were separated by thin layer chromatography on silica gel plates (Merck, Darmstadt, Germany) using n-heptane:isopropyl ether:acetic acid:methanol (70:30:4:2, v/v) as a plate running organic solvent. Radioactivity of cholesterol ester containing regions

7

were quantified by liquid scintillation counting (Wallac WinSpectral 1414, Wallac/PerkinElmer Life Sciences, Turku, Finland) using OptiPhase HiSafe 3 scintillation fluid (PerkinElmer Life Sciences, Boston, MA).

PLTP activity in liver samples Liver samples (snap-frozen and kept at -70oC before analysis) were homogenized in 1 mL of homogenizing buffer (10 mM HEPES, pH 7.4 containing 1 mM EDTA, 0.008 % G418 (analog of gentamicin B1) and protein inhibitor cocktail 1x, PIC, Roche, Germany). Homogenization was performed in Eppendorf tubes with Ultra-Turrax T8 (IKA-Werke, Staufen, Germany) on ice for 1 min with a power-set of 5. After homogenization the mixture was centrifuged at 13 000 rpm for 1 min at room temperature to sediment particulate matter. PLTP activity (10 µL and 25 µL used per assay) and total protein content were analyzed from the clear supernatant, and PLTP activity is expressed as nmol/h/mg protein. PLTP activity was analyzed as described above.

Hepatic cell separation Mice were anaesthetized and the vena cava inferior was cannulated. Subsequently, the vena porta was ligated and the liver was perfused for 10 min with oxygenated Hanks’ buffer pH 7.4, containing HEPES (1.6 g/L). The perfusion was continued for 10 minutes with Hanks’/HEPES buffer containing 0.05 % (w/v) collagenase (type IV, Sigma-Aldrich, St. Louis, MO) and 1 mM CaCl2. Parenchymal cells were isolated after mincing the liver in Hanks’ buffer containing 0.3 % BSA, filtering through nylon gauze and centrifugation (3x10 min, 50xg). The pellets contained pure (> 99 %) parenchymal cells (PC) as judged by light microscopy. The supernatants were

8

centrifuged for 10 min at 500xg in order to harvest the non-parenchymal cells. By means of centrifugal elutriation endothelial cells (EC) and Kupffer cells (KC) were separated.9 The purity of each cell fraction (> 95 % for both) was checked by light microscopy.

PLTP mRNA analysis using Real-Time Quantitative PCR Total RNA was extracted from whole liver or isolated hepatic parenchymal cells, Kupffer cells, and endothelial cells by the acid guanidium thiocyanate-phenol chloroform extraction method according to Chomczynski et al.10 cDNA was synthesised from 0.5–1 µg of total RNA using RevertAid™ M-MuLV Reverse Transcriptase. Levels of mRNA were quantitatively determined on an ABI Prism® 7700 Sequence Detection system (Applied Biosystems, Foster City, CA) using SYBR-green technology according to manufacturer’s instructions. For detection of PLTP

mRNA

5’-

ATCAATGCCTCGGCGGGA-3’

and

5’-

CGACCACTGGAATCCTGGG-3’ were used as forward and reverse primer, respectively. PLTP mRNA expression levels were calculated relative to the average of the housekeeping genes HPRT (primers 5’-TTGCTCGAGATGTCATGAAGGA-3’ and

5’-AGCAGGTCAGCAAAGAACTTATAG-3’),

AACCGTGAAAAGATGACCCAGAT-3’ CACAGCCTGGATGGCTACGTA-3’),

β-actin

(primers

and and

GAPDH

5’5’-

(primers

5’-

TCCATGACAACTTTGGCATTG-3’ and 5’- TCACGCCACAGCTTTCCA-3’) .

Immunohistochemical analysis of PLTP. Formalin-fixed cryostat sections (6 µm) of livers and lungs of PLTP+M/+M and PLTP-M/-M mice were incubated for 5 min with prewarmed (37oC) 0.025% trypsin at room temperature, blocked with 2% BSA in PBS/0.1% Triton, and incubated overnight with polyclonal rabbit-anti-recombinant 9

human PLTP antibody (Biovision, Mountain View, USA; dilution 1:50) that crossreacts with mouse PLTP. After rinsing with PBS, the sections were blocked with 10% normal goat serum in PBS/2% BSA and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes Europe Bv, Leiden, The Netherlands; dilution 1:200) for 1 hour in the dark. Nuclear staining was performed with DAPI (Serva, Heidelberg, Germany). For detection of macrophage localisation, sections were immunohistochemically stained with rat-anti-mouse F4/80 (BMA Biomedicals AG, Augst, Switzerland) as primary antibody and Cy3-goat-anti-rat IgG (Jackson ImmunoResearch Europe Soham, Cambridgeshire, UK) as secondary antibody. Fluorescence was analysed using a Bio-Rad Radiance 2100 MP confocal laser scanning system equipped with a Nikon Eclipse TE2000-U inverted fluorescence microscope (Melville, NY).

Statistical analyses Statistical analyses were performed utilising the unpaired Student’s t-test (Instat GraphPad software, San Diego, CA).

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Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497-509.

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Nagelkerke JF, Barto KP, van Berkel TJ. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells. J Biol Chem. 1983;258:12221-12227.

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thiocyanate-phenol-chloroform

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Macrophage Phospholipid Transfer Protein Contributes Significantly to Total Plasma Phospholipid Transfer Activity and Its Deficiency Leads to Diminished Atherosclerotic Lesion Development Riikka Vikstedt, Dan Ye, Jari Metso, Reeni B. Hildebrand, Theo J.C. Van Berkel, Christian Ehnholm, Matti Jauhiainen and Miranda Van Eck Arterioscler Thromb Vasc Biol. 2007;27:578-586; originally published online December 14, 2006; doi: 10.1161/01.ATV.0000254815.49414.be Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2006 American Heart Association, Inc. All rights reserved. Print ISSN: 1079-5642. Online ISSN: 1524-4636

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